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Stem Cell Reports
Repor t Functional Vascular Endothelium Derived from Human Induced Pluripotent Stem Cells
William J. Adams,1,2,4 Yuzhi Zhang,1 Jennifer Cloutier,1 Pranati Kuchimanchi,1 Gail Newton,1 Seema Sehrawat,1 William C. Aird,3 Tanya N. Mayadas,1 Francis W. Luscinskas,1 a-Carden a1,2,5,* and Guillermo Garc
for Excellence in Vascular Biology, Department of Pathology, Brigham and Womens Hospital, Boston, MA 02115, USA in Developmental and Regenerative Biology, Harvard Medical School, Boston, MA 02115, USA 3Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA 4School of Engineering and Applied Sciences 5Harvard Stem Cell Institute Harvard University, Cambridge, MA 02138, USA *Correspondence: guillermo_garcia-cardena@hms.harvard.edu http://dx.doi.org/10.1016/j.stemcr.2013.06.007 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
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SUMMARY
Vascular endothelium is a dynamic cellular interface that displays a unique phenotypic plasticity. This plasticity is critical for vascular function and when dysregulated is pathogenic in several diseases. Human genotype-phenotype studies of endothelium are limited by the unavailability of patient-specic endothelial cells. To establish a cellular platform for studying endothelial biology, we have generated vascular endothelium from human induced pluripotent stem cells (iPSCs) exhibiting the rich functional phenotypic plasticity of mature primary vascular endothelium. These endothelial cells respond to diverse proinammatory stimuli, adopting an activated phenotype including leukocyte adhesion molecule expression, cytokine production, and support for leukocyte transmigration. They maintain dynamic barrier properties responsive to multiple vascular permeability factors. Importantly, biomechanical or pharmacological stimuli can induce pathophysiologically relevant atheroprotective or atheroprone phenotypes. Our results demonstrate that iPSC-derived endothelium possesses a repertoire of functional phenotypic plasticity and is amenable to cell-based assays probing endothelial contributions to inammatory and cardiovascular diseases.
INTRODUCTION
The vascular endothelium, the single-cell layer lining blood vessels, is a multifunctional interface that displays a striking phenotypic plasticity necessary for maintaining vascular homeostasis. In this context, the vascular endothelium is critical to initiate an inammatory response, trigger thrombosis, regulate vasomotor tone, and control vascular permeability. Dysfunction of the endothelium plays a signicant pathogenic role in cardiovascular diseases, namely, atherosclerosis and its consequences: heart attacks and strokes (Gimbrone et al., 2000; Hansson, 2005). Notably, studies at the genetic and molecular level of human endothelium have been limited by the availability of relevant tissue derived from cadaveric, discarded surgical, or umbilical vasculature sources. Recent developments in stem cell biology promise new resources for modeling genetic diseases. In particular, induced pluripotent stem cells (iPSCs) offer the ability to study the effects of genetic alterations and mechanisms of genetic diseases in currently inaccessible cell types (Takahashi and Yamanaka, 2006). Although iPSCs have been differentiated into many cell types including endothelium (Choi et al., 2009; Homma et al., 2010; Li et al., 2011; Park et al., 2010; Rufaihah et al., 2011, 2013; Taura et al., 2009;
White et al., 2013), the delity and functional mimicry of stem cell-derived tissues and their relevance to human disease remain poorly characterized. This functionality must be carefully assessed before their scientic and therapeutic potential can be realized (Soldner and Jaenisch, 2012). The goals of this study were to reproducibly generate human iPSC-derived vascular endothelial cells (iPSC-ECs) and to then assess whether they could acquire specic functions critical for vascular homeostasis displayed by primary cultures of human vascular endothelium. To this end, we differentiated human iPSCs as embryoid bodies (EBs) and isolated the endothelial population for detailed functional characterization. Signicantly, in addition to displaying characteristic endothelial molecular and structural features, these ECs display phenotypic plasticity that allows them to mediate leukocyte transmigration and maintain a dynamic barrier. Furthermore, we have documented that the iPSC-ECs can be directed to acquire an atheroprotective or atheroprone phenotype in response to distinct biomechanical or pharmacological stimuli. Collectively, our results demonstrate that human iPSCECs support a spectrum of physiological endothelial functions and possess the relevant phenotypic plasticity to probe important features of human cardiovascular pathophysiology in a patient-specic manner.
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Functional Endothelium Derived from Human iPSC
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106 Stem Cell Reports j Vol. 1 j 105113 j August 6, 2013 j 2013 The Authors
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RESULTS
Human iPSC Differentiation into Vascular Endothelium To generate ECs, we differentiated iPSCs of the BJ1 cell line as EBs in suspension by replacing iPSC medium with differentiation medium containing fetal calf serum. The specic serum chosen was selected from a screen optimizing proliferation and morphology of cultured human ECs (data not shown). To carefully characterize the timescale of EC differentiation, we performed quantitative real-time TaqMan PCR using RNA harvested daily from EBs from the time of their generation from iPSC colonies, day 0, through 18 days of differentiation. Under these conditions, we observed increasing expression of EC markers CDH5 (VE-cadherin), KDR (VEGFR2), and CD31 (PECAM1) (Figure 1A). To better dene the EC population, we measured the expression of VE-cadherin, CD31, and KDR by ow cytometry within dissociated EBs after different durations of differentiation. As seen in Figure 1B, the VE-cadherin+jCD31+ EC fraction peaked at 18% 4% (mean SD) of the EBs after 10 days of differentiation. The EBs grew in large cystic tissues containing cord-like structures (Figure 1C). Two-photon confocal microscopy visualized uorescently labeled VE-cadherin within the intact 1 mm diameter EBs indicating that most VE-cadherin+ cells reside in networked structures (Figure 1D). Because the proportion of VE-cadherin+jCD31+ cells peaks at day 10, we isolated the EC population (iPSC-EC) from dissociated day 10 EBs for further analysis. Due to its endothelial specicity and to the presence of CD31+jVE-cadherin, CD31+KDR, and CD31jKDR+ cells within the EBs (Figure 1E, left and center panels) and due to the fact that a subpopulation of CD31+ cells expresses CD45 (Figure S1A available online), we chose to isolate ECs based on VE-cadherin expression. Magnetic bead sorting produced a population with greater than 95% VE-cadherin+ cells (Figure 1E, right panel). To ensure that this endothelial differentiation protocol is generalizable, we
tested additional human iPSC lines generated with different technologies. Importantly, each cell line was reproducibly able to generate ECs, although with different yields (Figure 1F). Next, we assessed whether isolated iPSC-ECs exhibit characteristic EC molecular markers and cellular behavior after in vitro culture. As seen in Figures 1G1M, iPSC-ECs displayed typical cobblestone morphology, expressed VE-cadherin and CD31 at cell junctions, expressed VE-cadherin, KDR, and thrombomodulin on their cell surface, expressed endothelial nitric oxide synthase (eNOS) at the perinuclear region and plasma membrane, endocytosed uorescent acetylated LDL, and could form networks on Matrigel in the presence of VEGF. We also observed that iPSC-ECs displayed punctate von Willebrand factor (vWF) and angiopoietin-2 staining (Figure S1B). Because vWF and angiopoietin-2 are normally stored in endothelia-restricted organelles termed WeibelPalade bodies (WPBs), we investigated via electron microscopy the presence of these organelles in iPSC-ECs. We identied electron-dense striated structures typical of WPBs that stained for vWF by immunogold labeling (Figure S1B). Functionally, WPBs store and rapidly exocytose vWF and P-selectin to initiate a hemostatic response (Wagner and Frenette, 2008). In order to verify that the WPBs were competent for rapid exocytosis, we treated the iPSCECs with histamine and observed rapid and transient presentation of P-selectin to the cell surface (Figure S1B). Proinammatory Endothelial Activation After documenting essential molecular features of vascular endothelium, we investigated whether iPSC-ECs can display multiple functional phenotypes characteristic of mature endothelium. A pathophysiologically essential property of endothelium is the response to proinammatory stimuli and participation in inammatory responses. To assess this activated functional phenotype, we exposed iPSC-ECs to the proinammatory stimuli, interleukin-1 beta (IL-1b), tumor necrosis factor alpha (TNF-a), and
Figure 1. Derivation of iPSC-ECs (A) Gene expression time proles of CDH5 (VE-cadherin), KDR, and CD31 measured by quantitative real-time PCR (n = 3) with the asterisks (*) denoting values different than day 0 (p < 0.05). (B) CD31, VE-cadherin, and KDR expression in EB cells measured by ow cytometry (n = 3). (C) Phase-contrast image of an EB after 10 days of differentiation. (D) Reconstruction of two-photon confocal microscopy images of VE-cadherin (green) and nuclei (blue) within an EB after 10 days of differentiation. (E) The isolation of VE-cadherin+ cells from the EB with magnetic bead sorting. (FM) Fraction of EB cells expressing VE-cadherin from different iPSC lines (F; n = 4). Isolated iPSC-ECs characterized by phase contrast (G), immunouorescence for VE-cadherin (H) and CD31 (I), ow cytometry (J) for VE-cadherin, KDR, and thrombomodulin (sample in solid line; isotype control in dotted), immunouorescence for eNOS (K) and endocytosed acetylated-LDL (L), and a phase-contrast image of the network formation on Matrigel (M). All pooled data are represented as mean SD. See also Figure S1.
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lipopolysaccharide (LPS), and assessed the cell surface expression of the adhesion molecules E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1), critical for endothelial-leukocyte interactions (Figure 2A; for uorescence intensity values, see Figure S2A). After 6 hr, IL-1b, TNF-a, and LPS induced the expression of E-selectin and ICAM-1 in the majority of the cell population, and of VCAM-1 in a fraction. After 24 hr of IL-1b, TNF-a, or LPS stimulation, the iPSC-ECs expressed E-selectin to a lesser degree, and ICAM-1 and VCAM-1. Additionally, treatment with interferon-gamma (IFNg) induced the ECs to upregulate ICAM-1 expression synergistically with TNF-a in a timedependent manner as reported in studies of primary EC cultures (Doukas and Pober, 1990) (Figure S2B). In addition to presenting adhesion molecules, the activated endothelial phenotype includes the secretion of proinammatory cytokines. Therefore, we examined whether iPSC-ECs have this capability by measuring the concentration of several proinammatory cytokines in the culture supernatant. Treatment for 24 hr with IL-1b, TNF-a, or LPS induced the iPSC-ECs to secrete monocyte chemotactic protein-1 (MCP1), IL-8, RANTES, and IFNginduced protein 10 (IP10), potent chemoattractants that act on T cells, neutrophils, and monocytes (Figure 2B), whereas treatment with IFNg induced the ECs to secrete IP10 and monokine induced by gamma interferon (MIG). Importantly, the coordinated expression of adhesion molecules and secretion of proinammatory cytokines by the vascular endothelium orchestrates the transendothelial migration of leukocytes. The iPSC-ECs, after treatment for 4 hr with 10 ng/ml TNF-a, were able to coordinate the integrated behavior of inducing human neutrophil and T lymphocyte rolling, arrest, and transmigration under laminar uid ow (Figure 2C; Movies S1 and S2). The fractions of transmigrating leukocytes were similar to those seen with primary HUVECs, the human model currently most commonly used for leukocyte transmigration studies (Figure 2D). It is important to note that this transmigration was dependent on T lymphocyte and neutrophil b2-integrin (CD18) and on endothelial ICAM-1 as demonstrated by the diminished transmigration in the presence of relevant blocking antibodies (Figures 2E2H) similar to primary ECs (Rao et al., 2007). Maintenance of a Dynamic Barrier A critical function of the vascular endothelium is the maintenance of a tight dynamic barrier to contain the bloods plasma and cellular constituents. ECs actively rearrange junctional and cytoskeletal proteins to modulate their barrier function in response to multiple physiological stimuli (Dejana et al., 2001; Mehta and Malik, 2006). To characterize the barrier phenotype of iPSC-ECs, we examined
the role of known EC permeability factors on the electrical resistance across a monolayer of iPSC-ECs, a real time measure of permeability. As seen in Figure 3A, histamine induced a transient increase in permeability, whereas VEGF induced a sustained increase in permeability, which was partially abrogated by pretreatment with a FAK inhibitor (Figure S3A), as demonstrated in primary endothelium (Chen et al., 2012). Prostaglandin E2 produced a decrease in permeability as did sphingosine-1-phosphate. Treatment of iPSC-ECs with the cAMP analog, 8-pCPT-20 O-MecAMP (O-Me) that selectively activates Epacs (Bos, 2006), induced a sustained decrease in permeability as reported in primary ECs (Cullere et al., 2005). Compared to HUVECs, the iPSC-ECs display a lower VEGF-induced permeability and slower response to O-Me (Figure S3B). We next documented the rearrangements of structural proteins that modulate barrier properties (Mehta and Malik, 2006). As seen in Figure 3B, we found that treatment with O-Me resulted in redistribution of VE-cadherin from a jagged to a more linear geometry and favored the generation of actin laments at the cell periphery over longitudinal actin laments, as seen in primary ECs (Cullere et al., 2005). Directing Phenotypic Plasticity to Disease-Protective and -Prone States A promising application of iPSC-derived endothelium is the ability to study endothelium in pathophysiologically relevant states in a patient-specic manner. Therefore, we evaluated whether iPSC-ECs are able to adopt atherosclerosis-protective and atherosclerosis-prone phenotypes in vitro, which are critical to enable their use in studies of endothelial contributions to cardiovascular disease. Distinct local hemodynamic environments have been linked to the nonrandom predictable distribution of atherosclerotic lesions in human and animal studies a-Carden a, (Chiu and Chien, 2011; Gimbrone and Garc 2013). In fact, local biomechanical forces can strongly inuence endothelial phenotype and induce a functional phenotype or one that is dysfunctional, an early step in atherogenesis (Davies, 2009; Gimbrone, 1999; Hahn and Schwartz, 2009). In particular, the specic shear stress patterns exerted on ECs at the sites of atheroprotection and atherosusceptibility in the human carotid artery induce atheroprotective and atheroprone phenotypes respectively in cultured ECs (Dai et al., 2004). In order to generate these biomechanically induced phenotypes in iPSC-ECs, we applied atheroprotective or atheroprone shear stress waveforms for 72 hr (Figure 4A). As seen in Figures 4B and 4C, the iPSC-ECs selectively responded to atheroprotective ow by aligning in the direction of ow, whereas those exposed to atheroprone ow did not. A key molecular descriptor of the endothelial atheroprotective versus
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Figure 2. Assessment of Endothelial Activation in iPSC-ECs (A) Surface expression of E-selectin, ICAM-1, and VCAM-1 after 6 or 24 hr of 10 U/ml IL-1b, 10 ng/ml TNF-a, or 1 mg/ml LPS treatment measured by ow cytometry. (B) Culture supernatant concentration of chemokines after 24 hr treatment of TNF-a, IL-1b, IFNg, LPS, or TNF-a+IFNg with the asterisks (*) indicating difference to vehicle (veh) (p < 0.05; n = 34). (C) Time prole of transmigrated neutrophils and T cells after 4 hr TNF-a treatment. (D) Fraction of neutrophils or T cells transmigrated across TNF-a- stimulated iPSC-ECs and HUVECs (n = 3; n.s., not signicant). (EH) Transmigration of neutrophils after pretreatment of neutrophils with anti-CD18 antibody (E) or pretreatment of iPSC-ECs with antiICAM-1 antibody (F). Transmigration of T cells after pretreatment of T cells with anti-CD18 antibody (G) or pretreatment of iPSC-ECs with anti- ICAM-1 antibody (H). A binding nonblocking anti-MHC class I antibody was used as a control (n = 2). All pooled data are represented as mean SD. See also Figure S2 and Movies S1 and S2. atheroprone phenotype is expression of the transcription factors Kruppel-like factors 2 (KLF2) and 4 (KLF4), integrators of the ow-mediated vasoprotective phenotype (Dekker et al., 2006; Parmar et al., 2006; Villarreal et al., 2010). Thus, we measured the expression of these genes in iPSCECs exposed to atheroprotective or atheroprone shear
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Figure 3. Barrier Properties of iPSC-ECs (A) Changes in transendothelial electrical resistance after treatment with 10 mM histamine, 100 ng/ml VEGF, 200 ng/ml prostaglandin E2, 0.5 mg/ml sphingosine-1-phosphate, or 100 mM O-Me. Data are represented as mean SD (n = 3) with the asterisks (*) denoting different from control (p < 0.05). Arrows indicate time of addition of compound. (B) VE-cadherin and actin after 24 hr treatment with 100 mM O-Me seen by immunouorescence microscopy. See also Figure S3.
stress waveforms and observed that the atheroprotective waveform differentially induced the expression of KLF2 and KLF4 (Figure 4D). Furthermore, we measured the expression of several other mechano-activated genes and downstream effectors of KLF2 and KLF4 and observed that atheroprotective shear stress upregulates NOS3 (eNOS), argininosuccinate synthase 1 (ASS1), and downregulates vWF. In contrast, atheroprone ow upregulated endothelin-1 (EDN1), a potent vasoconstrictor known to be differentially regulated by ow (Figure 4D). In addition to biomechanical forces, ECs are responsive to pharmacological agents. In particular, our laboratory and others have documented that the endothelial atheroprotective phenotype and KLF2 expression can be evoked by statins, a class of HMG-CoA reductase inhibitors (Parmar et al., 2005; Sen-Banerjee et al., 2005). To illustrate that iPSC-ECs are a suitable substrate for personalized pharmacological studies, we examined whether iPSC-EC phenotype can be similarly modulated by statin treatment.
As seen in Figure 4E, simvastatin upregulated KLF2 expression as well as downstream effectors NOS3, ASS1, thrombomodulin (THBD), integrin beta 4 (ITGB4), and prostaglandinH2 D-isomerase (PTGDS). Simvastatin also downregulated proinammatory ANGPT2 and EDN1. The induction of KLF2 and NOS3 in iPSC-ECs showed similar sensitivity to simvastatin concentration as primary ECs (Figure S4).
DISCUSSION
In this study, we have generated vascular ECs from human iPSCs and characterized their humoral-, pharmacological-, and biomechanical-induced functional phenotypes, advancing iPSC-ECs as an experimental platform to study endothelial biology. The methodology to differentiate iPSCs into vascular endothelium was reproducible and robust across several iPSC lines created with different technologies. Previous studies have differentiated iPSCs to
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Figure 4. Acquisition of Atheroprotective and Atheroprone Endothelial Phenotypes (AC) A single period of atheroprotective and atheroprone shear stress waveforms (A). iPSC-ECs imaged by phase contrast after 72 hr of atheroprone (B) or atheroprotective ow (C; arrow indicates direction of ow). (D) Relative (Rel.) gene expression after 72 hr of atheroprotective or atheroprone shear stress. (E) Relative gene expression after 24 hr treatment with 10 mM simvastatin. The asterisks (*) indicate statistical signicance with p < 0.05 (n = 4). All pooled data are represented as mean SD. See also Figure S4.
isolate ECs based on expression of CD31 or KDR (Choi et al., 2009; Homma et al., 2010; Li et al., 2011; Park et al., 2010; Rufaihah et al., 2011, 2013; Taura et al., 2009; White et al., 2013). In contrast, we isolated populations of ECs from the EBs based on VE-cadherin expression, a strong marker of endothelial identity, as we observed CD31+, and KDR+ cells subpopulations lacking VE-cadherin, and a CD31+ subpopulation expressing CD45. After isolation and culture, iPSC-ECs displayed molecular markers of vascular endothelium and contained vWF-positive WPBs that can be rapidly exocytosed, a critical function for hemostasis. It is possible that the perinuclear localization observed in many of the WPBs may suggest an immature stage in WPB biogenesis (Valentijn et al.,
2011; Zenner et al., 2007). iPSC-ECs are competent for the functional repertoire displayed by vascular endothelium critical in pathophysiological settings. Specically, we demonstrated that proinammatory stimuli induce an activated proinammatory phenotype that included expression of leukocyte adhesion molecules, secretion of proinammatory cytokines, and support for human leukocyte transmigration. We also observed that iPSC-EC monolayers display a dynamic permeability in response to a panel of physiological stimuli. Human iPSC-ECs are a promising platform for studying endothelial contributions to cardiovascular diseases in the context of specic patients genetic backgrounds. We demonstrated that iPSC-ECs have the plasticity to acquire
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distinct ow-dependent phenotypes, namely atheroprotective and atheroprone phenotypes critical for atherosclerosis resistance and susceptibility. We observed that iPSC-ECs respond to atheroprotective shear stress by activating an atheroprotective gene expression program transcriptionally mediated by KLF2 and KLF4 expression, shown to integrate the ow-mediated endothelial atheroprotective functional phenotype by regulating leukocyte adhesion, redox state, and thrombotic function in cultured human ECs (Dekker et al., 2006; Lin et al., 2005; Parmar et al., 2006; SenBanerjee et al., 2004; Villarreal et al., 2010). The ability to direct the acquisition of ow-dependent functional and dysfunctional phenotypes using patient-specic endothelium should allow the study of specic genetic contributions to endothelial function and dysfunction in the context of human cardiovascular disease. Furthermore, we demonstrated that simvastatin activated an atheroprotective gene expression program and downregulated genes associated with an atheroprone phenotype in iPSC-ECs. This suggests that iPSC-ECs may be a suitable surrogate to assess the effects of drugs on the endothelia of specic patients. The ability to generate human vascular endothelium from iPSCs is an enabling platform. Cell-based assays of endothelial function can be combined with ECs derived from iPSC lines produced from patients with genetic diseases, creating powerful model systems of human disease or associations where the pathological molecular mechanisms and their functional consequences are poorly undera-Carden a, 2012; Grskovic et al., stood (Adams and Garc 2011; Kiskinis and Eggan, 2010). Extension of these disease iPSC-based assays to high throughput may enable the new therapeutic discovery for genetic diseases and permit the personalization of drug toxicity testing, an unmet need because vascular toxicity is a signicant cause of attrition in drug development.
bead-conjugated anti-PE antibody (Miltenyi). Isolated iPSC-ECs were plated at 20,000 cells/cm2 on 50 mg/ml bronectin (Sigma-Aldrich)-coated dishes in EC medium. Experiments were performed on passage one. Please see Table S1 for a list of TaqMan probes used in this study and Table S2 for clones and vendors of primary antibodies.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures, four gures, and two movies and can be found with this article online at http://dx.doi.org/10.1016/j.stemcr.2013.06. 007.
ACKNOWLEDGMENTS
We wish to thank Kay Case and Vannessa Davis for the isolation of human umbilical cord ECs and Odelya Hartung for instruction in human iPSC culture. This work was supported by grants RO1AG032443, PO1HL076540, PO1HL036028, and RO1HL089940 from the National Institutes of Health. W.J.A. is a recipient of a Ruth L. Kirchstein National Research Service Award F31AG037249 from the National Institutes of Health. Received: March 20, 2013 Revised: June 23, 2013 Accepted: June 26, 2013 Published: July 25, 2013
REFERENCES
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EXPERIMENTAL PROCEDURES
Cell Culture
Four human iPSC lines were used in this study. Lines BJ1 and MSC1 were received from the laboratory of Dr. George Q. Daley (Childrens Hospital Boston), line BMC1 from the laboratory of Dr. Darrell N. Kotton (Boston University Medical Center), and line DH1F from the Harvard Stem Cell Institute.
Differentiation and Isolation of iPSC-ECs

Colonies of iPSCs were differentiated as EBs in suspension in ultralow adhesion plates (Corning) in differentiation medium for 10 days. EBs were dissociated into single cells with 2 mg/ml collagenase B (Roche) for 2 hr then Cell Dissociation Buffer (Invitrogen) for 15 min at 37 C shaking at 1,100 rpm. VE-cadherin+ cells were isolated by magnetic bead sorting (Miltenyi Biotec) using PE-conjugated anti-VE-cadherin antibody (BD Biosciences) and magnetic
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Davies, P.F. (2009). Hemodynamic shear stress and the endothelium in cardiovascular pathophysiology. Nat. Clin. Pract. Cardiovasc. Med. 6, 1626. Dejana, E., Spagnuolo, R., and Bazzoni, G. (2001). Interendothelial junctions and their role in the control of angiogenesis, vascular permeability and leukocyte transmigration. Thromb. Haemost. 86, 308315. Dekker, R.J., Boon, R.A., Rondaij, M.G., Kragt, A., Volger, O.L., Elderkamp, Y.W., Meijers, J.C., Voorberg, J., Pannekoek, H., and Horrevoets, A.J. (2006). KLF2 provokes a gene expression pattern that establishes functional quiescent differentiation of the endothelium. Blood 107, 43544363. Doukas, J., and Pober, J.S. (1990). IFN-gamma enhances endothelial activation induced by tumor necrosis factor but not IL-1. J. Immunol. 145, 17271733. Gimbrone, M.A., Jr. (1999). Endothelial dysfunction, hemodynamic forces, and atherosclerosis. Thromb. Haemost. 82, 722726. a-Carden a, G. (2013). Vascular Gimbrone, M.A., Jr., and Garc endothelium, hemodynamics, and the pathobiology of atherosclerosis. Cardiovasc. Pathol. 22, 915. Gimbrone, M.A., Jr., Topper, J.N., Nagel, T., Anderson, K.R., and a, G. (2000). Endothelial dysfunction, hemodyGarcia-Carden namic forces, and atherogenesis. Ann. N Y Acad. Sci. 902, 230239. Grskovic, M., Javaherian, A., Strulovici, B., and Daley, G.Q. (2011). Induced pluripotent stem cellsopportunities for disease modelling and drug discovery. Nat. Rev. Drug Discov. 10, 915929. Hahn, C., and Schwartz, M.A. (2009). Mechanotransduction in vascular physiology and atherogenesis. Nat. Rev. Mol. Cell Biol. 10, 5362. Hansson, G.K. (2005). Inammation, atherosclerosis, and coronary artery disease. N. Engl. J. Med. 352, 16851695. Homma, K., Sone, M., Taura, D., Yamahara, K., Suzuki, Y., Takahashi, K., Sonoyama, T., Inuzuka, M., Fukunaga, Y., Tamura, N., et al. (2010). Sirt1 plays an important role in mediating greater functionality of human ES/iPS-derived vascular endothelial cells. Atherosclerosis 212, 4247. Kiskinis, E., and Eggan, K. (2010). Progress toward the clinical application of patient-specic pluripotent stem cells. J. Clin. Invest. 120, 5159. Li, Z., Hu, S., Ghosh, Z., Han, Z., and Wu, J.C. (2011). Functional characterization and expression proling of human induced pluripotent stem cell- and embryonic stem cell-derived endothelial cells. Stem Cells Dev. 20, 17011710. Lin, Z., Kumar, A., SenBanerjee, S., Staniszewski, K., Parmar, K., aVaughan, D.E., Gimbrone, M.A., Jr., Balasubramanian, V., Garc a, G., and Jain, M.K. (2005). Kruppel-like factor 2 (KLF2) Carden regulates endothelial thrombotic function. Circ. Res. 96, e48e57. Mehta, D., and Malik, A.B. (2006). Signaling mechanisms regulating endothelial permeability. Physiol. Rev. 86, 279367. Park, S.W., Jun Koh, Y., Jeon, J., Cho, Y.H., Jang, M.J., Kang, Y., Kim, M.J., Choi, C., Sook Cho, Y., Chung, H.M., et al. (2010). Efcient differentiation of human pluripotent stem cells into functional CD34+ progenitor cells by combined modulation of the MEK/ ERK and BMP4 signaling pathways. Blood 116, 57625772.
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Stem Cell Reports

Repor t Temporal Control of Retroviral Transgene Expression in Newborn Cells in the Adult Brain
Simon M.G. Braun,1,2,3 Raquel A.C. Machado,1,2 and Sebastian Jessberger1,2,3,*
Research Institute, Faculty of Medicine, University of Zurich, 8057 Zurich, Switzerland of Molecular Health Sciences, Department of Biology, Swiss Federal Institute of Technology (ETH) Zurich, 8093 Zurich, Switzerland 3Neuroscience Center Zurich, University of Zurich and ETH Zurich, 8057 Zurich, Switzerland *Correspondence: jessberger@hifo.uzh.ch http://dx.doi.org/10.1016/j.stemcr.2013.06.003 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
2Institute 1Brain
SUMMARY
Neural stem/progenitor cells (NSPCs) generate new neurons throughout life in distinct areas of the adult mammalian brain. Besides classical transgenesis-based approaches, retrovirus-mediated genetic manipulation is frequently used to study mechanisms that regulate neurogenesis in the nervous system. Here, we show that fusion of a tamoxifen-regulatable estrogen receptor (ERT2) motif to transcription factors (i.e., ASCL1 and NEUROD1) enables temporal control of transgene expression in adult mouse NSPCs in vitro and in vivo. Thus, the approach described here represents a versatile strategy for regulating gene expression to study gene function in dividing cells and their progeny.
INTRODUCTION
Neural stem/progenitor cells (NSPCs) generate new neurons throughout life in distinct areas of the adult mammalian brain, including the subventricular zone (SVZ) lining the lateral ventricles from which newborn cells migrate toward the olfactory bulb and the hippocampal dentate gyrus (DG) (Zhao et al., 2008). Due to the relative sparseness of NSPCs and their progeny in relation to pre-existing neural structures, there is a need to selectively manipulate gene activity in NSPCs and their progeny to address their functional signicance during the course of development from a dividing NSPC to a fully mature and synaptically integrated neuron (Dhaliwal and Lagace, 2011). One approach to test the functional signicance of genes/pathways is to use transgenic mice carrying oxed alleles of genes of interest, together with Tamoxifen (TAM)-regulatable Cre-recombinase controlled by NSPCor immature neuron-selective promoters, to genetically recombine and delete genes (Ihrie et al., 2011; Sahay et al., 2011). In addition, retroviral vectors derived from Moloney murine leukemia viruses have proved to be an important tool to visualize newborn cells through the expression of uorescent proteins, as well as to manipulate gene expression using both gain- and loss-of-function strategies (Ge et al., 2006; van Praag et al., 2002). The use of retroviruses has the advantage that it is highly selective for dividing cells (i.e., neurogenic cells when injected into the SVZ or DG) and is a very fast method because extensive breeding to obtain correct genotypes, as is the case for classical transgenesis, is not required (Zhao and Gage, 2008). However, retroviral vectors do not target bona de NSPCs that are largely quiescent but integrate into highly prolifer-
ative neural progenitors, which restrict virus-mediated genetic manipulations to later steps of neurogenesis. Furthermore, current vectors do not allow inducible or temporally controlled expression of the virus-driven transgene. Temporal control of transgene expression would be advantageous for studying gene function during distinct steps in the course of neuronal development. This is especially true for transcription factors (TFs) that may exert stage-specic functions depending on the age of a given newborn cell (Iwano et al., 2012). We reasoned that fusion of a TAM-regulatable estrogen receptor (ERT2) motif (Indra et al., 1999; Jiang et al., 2010) to expression constructs of TFs involved in neurogenesis would enable TAM-induced transgene expression, allowing for temporal control of virus-mediated gene expression (Figures 1A and 1B; Supplemental Experimental Procedures available online).
RESULTS
It was previously shown that Ascl1 overexpression in cultured adult NSPCs results in robust neuronal differentiation (Jessberger et al., 2008). To analyze whether fusion of Ascl1 to the ERT2 motif leads to functional ASCL1 expression upon TAM treatment, we transduced NSPCs isolated from adult mice with Ascl1-ERT2-IRES-GFP (hereafter called Ascl1-ERT2)-expressing retroviruses in vitro. To induce translocation of the ASCL1-ERT2 fusion protein, we treated the cells with hydroxy-TAM (OH-TAM) and analyzed them 7 days after the onset of differentiation. Whereas Ascl1ERT2-expressing cells cultured without OH-TAM did not show any difference in their rate of neuronal differentiation compared with cells transduced with a control
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Inducible Retroviral Gene Expression in NSPCs
Figure 1. Temporal Control of Retrovirus-Mediated Ascl1-ERT2 Activity In Vitro (A) Two-step PCR cloning strategy to fuse an ERT2 motif to TFs expressed from a retroviral vector. Upon binding of TAM to the ERT2 motif, the TF translocates to the nucleus and regulates target gene transcription. (B) Western blot analysis reveals the fusion of an ERT2 motif to ASCL1, using an ASCL1 antibody to visualize the WT and fusion proteins. (C) Differentiation of adult NSPCs transduced with Ascl1-ERT2-expressing virus (green) without OH-TAM yields only a minority of neuronally differentiated cells expressing MAP2AB (red), whereas most cells differentiate into glial cells expressing GFAP (blue). Right bars show quantications. (D) Exposure of Ascl1-ERT2-expressing cells (green) to OH-TAM for 4 days leads to the dramatic induction of neuronal differentiation as measured with MAP2AB (red) and a reduction in GFAP-positive cells (blue). Right bars show quantications. TAM: 8.04% 1.53% neurons; +TAM: 91.58% 2.14% neurons; *p < 0.001; n = 3, biological replicates. (E) NSPCs transduced with Ascl1-ERT2-expressing virus (green) can be properly propagated as monolayers expressing SOX2 (red, left panels) or oating neurospheres in the absence of OH-TAM (right panel), indicating the tight control of virus-mediated gene expression with TAM. Images were taken 10 days after viral transduction. Error bars represent mean SEM. Scale bars represent 40 mm (C, D, and E, left panel) and 100 mm (E, right panel). Nuclei were stained with DAPI (gray). See also Figures S1 and S3.
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Figure 2. Inducible NeuroD1 Expression Using TAM in Cultured NSPCs (A) Differentiation of adult NSPCs transduced with NeuroD1-ERT2-expressing virus (green) without OH-TAM does not alter neural differentiation as measured by MAP2AB expression (red) for neuronal cells and GFAP (blue) for glial cells. (B) Cells transduced with NeuroD1-ERT2expressing virus almost exclusively differentiate into neuronal cells expressing MAP2AB (red) upon OH-TAM exposure. (C) Quantication of neuronal differentiation of NeuroD1-ERT2-expressing cells in vitro with (right graphs) and without OH-TAM exposure (left graphs), indicating the robust control of TAM-regulated gene expression. TAM: 8.08% 1.85% neurons; +TAM: 93.59% 1.46% neurons; *p < 0.001; n = 3, biological replicates. (D) Western blot analysis reveals the fusion of an ERT2 motif to a NeuroD1 expression construct, using NEUROD1 antibody to visualize the WT and fusion proteins. Error bars represent mean SEM. Scale bars represent 40 mm. Nuclei were stained with DAPI (gray). See also Figure S2.
retrovirus (expressing IRES-GFP), treatment of Ascl1-ERT2expressing cells with OH-TAM resulted in a dramatic increase in neuronal differentiation (Figures 1C, 1D, S1A, and S1B). These ndings suggest that OH-TAM treatment resulted in the robust and efcient transcriptional activation of ASCL1 target genes to induce neuronal differentiation in vitro. To conrm that this approach is applicable to other TFs and not restricted to ASCL1, we fused the basic helix-loop-helix TF NEUROD1 to an ERT2 motif (Figure 2D) and infected NSPC cultures with NeuroD1-ERT2-IRES-GFPexpressing retroviruses. As before, we observed the almost complete neuronal differentiation of NSPCs upon OHTAM treatment (Figures 2A2C). This nding is consistent with the induction of NEUROD1 transcriptional activity, as it was previously shown that NEUROD1 overexpression promotes neuronal differentiation of NSPCs (Gao et al., 2009). Furthermore, we were able to conrm that the subcellular localization of ASCL1-ERT2 and NEUROD1-ERT2 was indeed controlled by OH-TAM-dependent translocation of the fusion proteins from the cytoplasm to the nucleus of transduced NSPCs (Figures S2A and S2B). A current limitation in studying neuronal differentiation using cultured NSPCs is that the rate of neuronal differentiation of adult NSPCs is rather incomplete: when standard
protocols of growth-factor withdrawal are used, only 8% 30% of NSPC progeny differentiate toward the neuronal lineage (as measured by MAP2AB or TUJ1 expression), whereas the majority of NSPC-derived cells differentiate into glial cells (Babu et al., 2007; Seaberg et al., 2005). Therefore, an inducible system that allows complete and reproducible conversion of undifferentiated NSPCs into neurons would represent a major advantage compared with existing protocols. Thus, we next analyzed whether NSPCs expressing ASCL1-ERT2 fusion proteins can be propagated as proliferating neurospheres or monolayer cultures when OH-TAM is absent, and be committed to neuronal differentiation when OH-TAM is added. NSPCs were transduced with Ascl1-ERT2 expressing retroviruses and cultured for several weeks thereafter. In the absence of OH-TAM, Ascl1-ERT2-expressing NSPCs continued to express the NSPC marker SOX2, showed very similar rates of proliferation compared with control cells labeled with a GFPexpressing retrovirus, and could be repeatedly passaged as neurospheres (Figure 1E). Strikingly, the pulse of OHTAM induced the near-complete differentiation of Ascl1ERT2-expressing NSPCs into neurons (as measured by MAP2AB labeling), indicating that controllable transgene expression is tightly regulated and that transient induction
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of Ascl1 is sufcient to promote neuronal differentiation of cultured NSPCs. Therefore, after an initial induction of ASCL1 transcriptional activity to trigger neuronal differentiation, NSPCs differentiate under normal conditions, as ASCL1-ERT2 is no longer active in the absence of OH-TAM. Again, similar results were obtained when we used NeuroD1-ERT2-expressing cells, further supporting the feasibility of this inducible approach (data not shown). To analyze the dynamics of Ascl1-ERT2-mediated neuronal differentiation in greater detail, we rst shortened the time of OH-TAM treatment from 4 days to 2 days, which also resulted in a strong increase in neuronal differentiation (Figures S3A and S3B). In addition, we performed luciferase assays using a basic helix-loop-helix (bHLH) reporter construct as described previously (Castro et al., 2006) to monitor ASCL1 transcriptional activity. We measured a rapid induction of luciferase activity upon OH-TAM treatment (Figure S3C), further conrming the efcacy and tight temporal control of the ERT2-based system. Next, we sought to analyze whether TAM-inducible Ascl1-ERT2 gene expression is sufcient to convert NSPCderived astrocytes toward a neuronal lineage. As shown above, the majority of NSPCs differentiated into glial brillary acidic protein (GFAP)-expressing astrocytes after growth-factor withdrawal (Figure 1C). To test whether these astroglia could be converted toward a neuronal fate (Heinrich et al., 2010) using the Ascl1-ERT2 system, NSPCs were differentiated for 7 days, exposed to OH-TAM for 4 days, and then analyzed for neuronal differentiation. Strikingly, treatment of differentiated cells with OH-TAM led to a robust increase in MAP2AB-expressing neurons (Figures 3A and 3B). These ndings indicate that ERT2based control of proneural gene expression is effective for driving neuronal differentiation in nondividing (i.e., ethynyl-deoxy-uridine [EdU] negative), differentiated astroglial cells (Figures 3A3C). After showing that fusion of TFs to an ERT2 motif is suitable for controlling gene expression in vitro, we next tested the system in vivo within the neurogenic niche of the adult hippocampus. We previously showed that retrovirusmediated ASCL1 overexpression within the adult DG redirects the progeny of NSPCs away from a neuronal fate and toward the oligodendrocytic lineage (Jessberger et al., 2008). Therefore, NSPCs respond differently to ASCL1 overexpression in vivo (i.e., oligodendrocytic differentiation) and in vitro (i.e., neuronal differentiation). At this time, it remains unclear whether this is due to intrinsic differences or niche-derived cues that are responsible for the context-dependent behavior of adult hippocampal NSPCs (Jessberger et al., 2008). Be that as it may, we made use of this robust cellular phenotype and tested whether the system of retrovirus-mediated, TAM-controlled gene
expression also functions in vivo. Animals were injected with Ascl1-ERT2-expressing retroviruses and treated for 5 consecutive days with i.p. TAM injections (to switch the transgene on). Notably, 3 weeks after viral injection, the vast majority of newborn cells in the TAM-treated mice displayed an oligodendrocytic phenotype and expressed OLIG2, NG2, and SOX10 (Figures 4B4D). These results phenocopied previous results obtained using noninducible retroviruses with constitutively active chicken beta-actin promoter driving Ascl1, although the phenotypic switch was not as complete (Ascl1-ERT2: +TAM 73.96% 6.72% oligodendrocytes, constitutively active Ascl1 overexpression: 87.52% 2.08% oligodendrocytes, resulting in an efciency of 84.50% to redirect newborn cells toward the oligodendrocytic lineage using the ERT2-based approach compared with constitutively active Ascl1 overexpression; see also Jessberger et al., 2008). These data clearly indicate the functional induction of ASCL1 target gene expression using the Ascl1-ERT2 system within the adult hippocampus. In striking contrast, we observed almost no fate change of newborn cells toward the oligodendrocytic lineage in mice that were injected with Ascl1-ERT2-expressing retroviruses but had not received TAM injections (Figures 4A and 4C). Notably, delayed onset of TAM administration, starting 4 weeks after stereotactic injections of Ascl1-ERT2-expressing viruses, did not affect the fate-choice decisions of newborn cells in the adult DG, in contrast to the in vitro situation where glial cells could be redirected toward a neuronal fate upon TAMinduced ASCL1-ERT2 activation (Figures 3A3C and S4BS4E). In summary, these data further underline the tightness of the ERT2-based system to drive gene expression in newborn cells.
DISCUSSION
We have described an approach that achieves inducible, retrovirus-mediated gene expression by using the coding sequence of TFs fused to an ERT2 motif. Fusion of the ERT2 motif to two TFs that regulate fate-choice decisions in the course of adult neurogenesis (i.e., ASCL1 and NEUROD1) allowed tight temporal control of functional gene expression in cultured NSPCs as well as within a neurogenic niche of the adult brain. The approach described here extends the range of applications for retrovirus-mediated gene manipulation. This highly selective and extremely fast strategy to test gene function in the context of mammalian neurogenesis can be combined with an ERT2-mediated system for temporal control of gene expression, allowing one to switch gene expression on (and off) with the use of TAM. Thus, with this simple two-step cloning protocol, it is now possible
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Figure 3. TAM Treatment Converts Astroglia toward a Neuronal Fate in Differentiated Ascl1-ERT2-Expressing NSPCs (A) Ascl1-ERT2-expressing cells were differentiated for 7 days in the absence of OH-TAM and mainly gave rise to MAP2AB-negative differentiated astroglia. Right bars show quantications. Notably, virtually all of the NSPCs (>99.5%) are negative for EdU (blue), indicating that the cells have exited the cell cycle and become postmitotic. The arrow points toward one of the rare EdU-labeled cells (n = 3, biological replicates). (B) The vast majority of Ascl1-ERT2-expressing cells (green) continue to differentiate into glial cells expressing GFAP (blue) and are negative for MAP2AB (red) in the absence of OH-TAM. In striking contrast, OH-TAM exposure 7 days after initiation of differentiation results in a robust increase in MAP2AB-expressing, neuronally differentiated cells, suggesting that OH-TAM-induced ASCL1 activity is also sufcient to drive neuronal differentiation in NSPC-derived cells that have adopted an astroglial fate. Right bars show quantications. TAM: 15.24% 4.84% neurons; +TAM: 57.80% 3.07% neurons; *p < 0.001; n = 3, biological replicates. (C) Experimental design of delayed OH-TAM treatment. Error bars represent mean SEM. Scale bars represent 40 mm. Nuclei were stained with DAPI (gray). See also Figure S3. to test the function of genes in distinct stages or for different durations during neural development both in vitro and in vivo. Using this inducible system, we were able to dramatically increase the neuronal differentiation of NSPCs, with >90% of NSPC-derived cells differentiating into neurons in vitro, which is similar to the rate of neuronal differentiation observed within the hippocampal niche. Thus, the approach described here overcomes the problems associated with mixed neuronal and glial cultures. For example, proteomics or metabolomics studies comparing NSPCs with their neuronally differentiated progeny, which require large amounts of cells, can now be performed with reproducible
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Figure 4. Inducible and Controllable Gene Expression in Newborn Cells within the Adult Hippocampus (A) Injection of Ascl1-ERT2-expressing retroviruses (green) into the adult DG without TAM treatment leads to the labeling of newborn granule cells (NEUN positive, red) 3 weeks after viral injection, similar to transduction of dividing cells with a control virus (data not shown), indicating that ASCL1 expression is not active without TAM-induced nuclear translocation. (B) TAM treatment for 5 days after injection of Ascl1-ERT2-expressing retroviruses induces oligodendrocytic differentiation (OLIG2 positive, red) of transduced cells within the adult DG, indicating the tight temporal control of gene expression in adult-born cells. (C) Quantications of the phenotypic shift induced by TAM-mediated activation of ASCL1 transcriptional activation in Ascl1-ERT2expressing cells. TAM: 13.98% 1.92% oligodendrocytes; +TAM: 73.96% 6.72% oligodendrocytes; *p < 0.001; n = 4, biological replicates. (D) TAM-induced gene expression in Ascl1-ERT2-transduced cells (green) results in expression of NG2 (red, upper panels) and SOX10 (red, lower panels), indicating a fate switch from the neuronal toward the oligodendrocytic lineage. Error bars represent mean SEM. Scale bar represents 20 mm. Nuclei were stained with DAPI (blue). See also Figure S4. and almost pure neuronal populations. We were also able to use inducible ASCL1 overexpression to convert NSPCderived astrocytes into neurons. Furthermore, there is increasing evidence that newborn neurons of different ages have specic functions within the hippocampus; therefore, the newly developed inducible strategy may help improve our understanding of the molecular mechanisms that regulate distinct steps of newborn neuron maturation. Clearly, the approach described here is not limited to studying genes in the context of adult neurogenesis. A large number of experiments aiming to manipulate TFs will benet from an ERT2-based temporal control of gene expression (in retroviral or plasmid DNA format) in both adult and embryonic tissues (e.g., using in utero electroporation of plasmids encoding for TF-ERT2-fusion proteins). In addition, the approach described here will be useful for studying the temporal requirements of selected TFs in the
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context of cellular reprogramming of somatic cells toward distinct lineages or pluripotency (Takahashi and Yamanaka, 2006; Vierbuchen et al., 2010).
Cloning
The complementary DNA (cDNA) sequence of Ascl1 and NeuroD1 TFs was fused to the ERT2 motif by fusion PCR as described in Figure 1A and Supplemental Experimental Procedures. TF and ERT2 cDNA fragments were PCR amplied individually (PCR 1: primers P1 and P2, TF cDNA as template; PCR 2: primers P3 and P4, ERT2 cDNA as template). The PCR products were then used as templates for the fusion PCR reaction (PCR 3: primers P1 and P4, products of PCR 1 and 2 as template). Note: Phusion polymerase (NEB) was used for the PCR reactions (cycling parameters: 98 C 30 s, 60 C 45 s, 72 C 1 min [30 cycles]). Primers P2 and P3 were designed so that 20 bases would be complementary to each other. To improve PCR amplication, all primers should be designed within a similar Tm range (see Supplemental Experimental Procedures for information regarding primer design). The fusion PCR product, TF-ERT2, was then cloned into a replication-incompetent Moloney murine leukemia virus construct, pCAG-IRES-GFP (mCherry or CFP). The vector was digested with BamHI restriction enzymes (NEB), blunted with Klenow polymerase (NEB), dephosphorylated with alkaline-phosphatase (Roche), and ligated with the fusion PCR product using T4 DNA ligase (Roche). Positive clones were evaluated by NheI restriction digest and their sequence was conrmed by DNA sequencing. The product of these reactions was the retroviral vector pCAG-TF-ERT2-IRES-GFP (mCherry or CFP).
glass coverslips coated with poly-L-ornithine (Sigma) and laminin (Invitrogen), and heparin (5 mg/mL) was added to the medium to obtain adherent cultures. All experiments were done in triplicates. To analyze the efcacy of the TF-ERT2 system in vitro, WT mDG NSPCs were infected with Ascl1-ERT2-IRES-GFP retrovirus, NeuroD1-ERT2-IRES-GFP retrovirus, or CAG-IRES-GFP retrovirus as a control. They were expanded for 24 hr for acute experiments and passaged for several weeks for passaged cell experiments. Differentiation was induced by growth factor withdrawal in the presence and absence of hydroxy-TAM (OH-TAM; 0.5 mM). OHTAM was present in the differentiating medium for 2 days or a maximum of 4 days. The medium was changed every 23 days. Cells were xed 7 days after the onset of differentiation or exposed to OH-TAM for 4 days and further differentiated for an additional 4 days. NSPC proliferation was measured using a 1 hr pulse with EdU (10 mM; Sigma) at 37 C followed by paraformaldehyde (PFA) xation. EdU stainings were performed before antibody incubation using the Click-iT EdU Imaging Kit (Invitrogen) according to the manufacturers protocol.
Animals
All animal experiments were approved by the veterinary ofce of the Canton of Zurich, Switzerland. Mice were kept with littermates under a 12 hr dark/light cycle in single ventilated cages and with ad libitum access to food and water. Virus injections were performed as previously described (Knobloch et al., 2013). To test Ascl1-ERT2expressing retroviruses in vivo, 6- to 8-week-old WT mice were stereotactically injected in the DG with 1.5 ml Ascl1-ERT2-IRESGFP-expressing retroviruses (Karalay et al., 2011). In the rst set of experiments, the day after retrovirus injection, animals received the rst of ve consecutive daily i.p. injections of TAM dissolved in sunower oil (100 mg/kg, both reagents from Sigma). As a control, animals injected with the retroviruses did not receive TAM injections. The animals were killed 3 weeks after the last TAM injection (n = 4 for each condition). In the second set of experiments, mice were injected with Ascl1-ERT2-IRES-GFP-expressing retroviruses and red uorescent protein (RFP)-expressing retroviruses (Vadodaria et al., 2013) as a control. These mice received 5 daily TAM injections 4 weeks after viral injections and were killed 1 week after the last TAM injection (n = 4). To test for directed differentiation efciency of the ERT2-based approached compared with constitutive ASCL1 overexpression, we injected Ascl1-IRESGFP-expressing retroviruses into WT mice and killed the animals 3 weeks later (n = 4). For tissue collection, mice were given a lethal dose of Esconarkon (Streuli) and ushed transcardially with 0.9% sterile NaCl, followed by xation with 4% PFA/0.1 M phosphate buffer, pH 7.4. The brains were postxed in 4% PFA/0.1 M phosphate buffer overnight at 4 C, followed by 30% sucrose/0.1 M phosphate buffer, and stored at 4 C.
Virus Production
Viruses were produced as previously described (Zhao et al., 2006). In brief, ten 10 cm plates of conuent human embryonic kidney (HEK293T) cells were transfected with retroviral constructs and packaging plasmids (pCMV-vsvg and pCMV-gp) using Lipofectamine 2000 (Invitrogen). Two days after transfection, the virus containing cell culture media (100 mL) was collected, ltered with a 0.22 mm Steritop lter (Millipore), and centrifuged at 19,400 rpm in an ultracentrifuge for 2 hr at 4 C. The viral pellet was resuspended in 4 ml PBS and centrifuged a second time through a 20% sucrose cushion at 19,400 rpm for 2 hr at 4 C. The nal viral pellet was resuspended in 40 ml of PBS and used for subsequent infections.
Cell Culture
Wild-type mouse DG NSPCs (WT mDG NSPCs) were isolated as previously described (Bracko et al., 2012). The resulting singlecell suspension was cultured as a sphere culture in Dulbeccos modied Eagles medium (DMEM)/F12 medium supplemented with B27 (Invitrogen), human epidermal growth factor (20 ng/ml), and human basic broblast growth factor 2 (20 ng/ml; Peprotech). The medium contained an antibiotic/ antimycotic (Anti-Anti; Invitrogen). Cells were then plated on
Immunohistochemistry
We cut the brains into 40-mm-thick free-oating sections by snapfreezing them with dry ice after mounting them on a microtome as described previously (Karalay et al., 2011). For blocking, we used 13 TBS containing 3% donkey serum and 0.25% Triton X-100 for 30 min. Primary antibody incubation was done overnight at
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4 C in the concentrations specied below. Secondary antibodies (Jackson ImmunoResearch) were applied at 1:250 at room temperature for 1 hr. Cell nuclei were counterstained with DAPI (1:5,000; Sigma). For cell stainings, immunocytochemistry was performed as described above on cells xed with 4% PFA for 15 min at 37 C. The antibodies used were chicken a-GFP (1:500; Aves), mouse a-MAP2AB (1:500; Sigma), rabbit a-GFAP (1:500; Dako), goat a-SOX2 (1:250; Santa Cruz), mouse a-NEUN (1:200; Millipore), rabbit a-OLIG2 (1:250; Millipore), rabbit a-NG2 (1:500; Millipore), goat a-SOX10 (1:100; Santa Cruz), goat a-NEUROD1 (1:250; Santa Cruz), and rabbit a-DsRed/RFP (Clontech; 1:500).
Statistical Analyses
Statistical analyses were performed using Excel (Microsoft). Two sample t tests were used for all comparisons. Differences were considered signicant at p < 0.001.
Supplemental Information includes four gures and Supplemental Experimental Procedures and can be found with this article online at http://dx.doi.org/10.1016/j.stemcr.2013.06.003.
ACKNOWLEDGMENTS
We thank Burkhard Becher for conceptual input, Fred H. Gage for critical comments on the manuscript, Franc ois Guillemot for bHLH luciferase constructs, and the Center for Microscopy and Image Analysis (ZMB) of the University of Zurich and the Light Microscopy and Screening Center (LMSC) of ETH Zurich for help with imaging. This work was supported by the NCCR Neural Plasticity and Repair, Swiss National Science Foundation, Zurich Neuroscience Center (ZNZ), and the EMBO Young Investigator Program (to S.J.). Received: January 24, 2013 Revised: June 4, 2013 Accepted: June 5, 2013 Published: July 11, 2013
Luciferase Assays
We electroporated Ascl1-ERT2-expressing NSPCs with pE3x6 Luciferase (Castro et al., 2006) and pTk Renilla constructs using a nucleofector device (Amaxa). The NSPCs were then treated with OH-TAM for 0, 5, 10, and 24 hr. The cells were then lysed in PLB lysis buffer (Promega). We determined Luciferase and Renilla activity using the Dual Luciferase reporter assay (Promega). Luminescence was measured with a Novostar plate reader. Transcriptional activity was determined as the ratio of Luciferase activity over Renilla activity.
Image Analysis
For colocalization experiments and cell quantications, we performed confocal microscopy (AOBS-SP2; Leica) followed by colocalization analyses using ImageJ with the Cell Counter plugin. Photoshop (CS5) was used for contrast enhancements and color adjustments.
REFERENCES
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Western Blot
HEK293T cells were transfected with retroviral constructs expressing Ascl1, Ascl1-ERT2, NeuroD1, or NeuroD1-ERT2 using Lipofectamine (Invitrogen). Cells were lysed and extracted protein concentrations were determined by Bradford assay (BioRad). Proteins were separated by SDS-PAGE electrophoresis, transferred to a nitrocellulose membrane (BioRad), and probed with primary antibodies, goat a-NEUROD1 (1:1,000; Santa Cruz), mouse a-ASCL1 (1:1,000; BD), mouse a-GAPDH (1:10,000; HyTest), and rabbit a-MATRIN3 (1:2,000; Bethyl) followed by horseradishperoxidase-conjugated secondary antibodies (Jackson ImmunoResearch), and bands were detected by chemiluminescence (Thermo Scientic).
Fractionation of Cytoplasmic and Nuclear Proteins

Fractionations were performed as previously described (Knobloch et al., 2013). Ascl1-ERT2-expressing NSPCs were lysed in 0.5% Triton buffer. Lysates were centrifuged at 13 krpm for 15 min at 4 C to separate insoluble nuclei. The supernatant containing the membrane proteins was removed and stored on ice. The nuclear pellet was rinsed twice with lysis buffer and then resuspended in lysis buffer containing 0.5% SDS and sonicated. The samples were precleared by centrifugation at 13 krpm for 15 min at 4 C and the supernatant containing the nuclear proteins was stored on ice. The samples were then used for western blot analysis.
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(2010). Directing astroglia from the cerebral cortex into subtype specic functional neurons. PLoS Biol. 8, e1000373. Ihrie, R.A., Shah, J.K., Harwell, C.C., Levine, J.H., Guinto, C.D., Lezameta, M., Kriegstein, A.R., and Alvarez-Buylla, A. (2011). Persistent sonic hedgehog signaling in adult brain determines neural stem cell positional identity. Neuron 71, 250262. Indra, A.K., Warot, X., Brocard, J., Bornert, J.M., Xiao, J.H., Chambon, P., and Metzger, D. (1999). Temporally-controlled site-specic mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases. Nucleic Acids Res. 27, 43244327. Iwano, T., Masuda, A., Kiyonari, H., Enomoto, H., and Matsuzaki, F. (2012). Prox1 postmitotically denes dentate gyrus cells by specifying granule cell identity over CA3 pyramidal cell fate in the hippocampus. Development 139, 30513062. Jessberger, S., Toni, N., Clemenson, G.D., Jr., Ray, J., and Gage, F.H. (2008). Directed differentiation of hippocampal stem/progenitor cells in the adult brain. Nat. Neurosci. 11, 888893. Jiang, J., Yu, H., Shou, Y., Neale, G., Zhou, S., Lu, T., and Sorrentino, B.P. (2010). Hemgn is a direct transcriptional target of HOXB4 and induces expansion of murine myeloid progenitor cells. Blood 116, 711719. Karalay, O., Doberauer, K., Vadodaria, K.C., Knobloch, M., Berti, L., Miquelajauregui, A., Schwark, M., Jagasia, R., Taketo, M.M., Tarabykin, V., et al. (2011). Prospero-related homeobox 1 gene (Prox1) is regulated by canonical Wnt signaling and has a stagespecic role in adult hippocampal neurogenesis. Proc. Natl. Acad. Sci. USA 108, 58075812. Knobloch, M., Braun, S.M., Zurkirchen, L., von Schoultz, C., zo-Bravo, M.J., Kovacs, W.J., Karalay, O., Suter, Zamboni, N., Arau U., Machado, R.A., et al. (2013). Metabolic control of adult neural
stem cell activity by Fasn-dependent lipogenesis. Nature 493, 226230. Sahay, A., Scobie, K.N., Hill, A.S., OCarroll, C.M., Kheirbek, M.A., Burghardt, N.S., Fenton, A.A., Dranovsky, A., and Hen, R. (2011). Increasing adult hippocampal neurogenesis is sufcient to improve pattern separation. Nature 472, 466470. Seaberg, R.M., Smukler, S.R., and van der Kooy, D. (2005). Intrinsic differences distinguish transiently neurogenic progenitors from neural stem cells in the early postnatal brain. Dev. Biol. 278, 7185. Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult broblast cultures by dened factors. Cell 126, 663676. Vadodaria, K.C., Brakebusch, C., Suter, U., and Jessberger, S. (2013). Stage-specic functions of the small rho GTPases cdc42 and rac1 for adult hippocampal neurogenesis. J. Neurosci. 33, 11791189. van Praag, H., Schinder, A.F., Christie, B.R., Toni, N., Palmer, T.D., and Gage, F.H. (2002). Functional neurogenesis in the adult hippocampus. Nature 415, 10301034. dhof, T.C., Vierbuchen, T., Ostermeier, A., Pang, Z.P., Kokubu, Y., Su and Wernig, M. (2010). Direct conversion of broblasts to functional neurons by dened factors. Nature 463, 10351041. Zhao, C., and Gage, F.H. (2008). Retrovirus-mediated cell labeling. In Adult Neurogenesis, F.H. Gage, ed. (Long Island, NY: CSHL Press), pp. 111117. Zhao, C., Teng, E.M., Summers, R.G., Jr., Ming, G.L., and Gage, F.H. (2006). Distinct morphological stages of dentate granule neuron maturation in the adult mouse hippocampus. J. Neurosci. 26, 311. Zhao, C., Deng, W., and Gage, F.H. (2008). Mechanisms and functional implications of adult neurogenesis. Cell 132, 645660.
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Ar ticle DNA Damage in Mammalian Neural Stem Cells Leads to Astrocytic Differentiation Mediated by BMP2 Signaling through JAK-STAT
Leonid Schneider,1,7,* Serena Pellegatta,2,3 Rebecca Favaro,4 Federica Pisati,2 Paola Roncaglia,5,8 Giuseppe Testa,3 Silvia K. Nicolis,4 Gaetano Finocchiaro,2,3 and Fabrizio dAdda di Fagagna1,6,*
FoundationThe FIRC Institute of Molecular Oncology Foundation, Via Adamello 16, 20139 Milan, Italy I.R.C.C.S Istituto Neurologico C. Besta, Via Celoria 11, 20133 Milan, Italy 3European Institute of Oncology, Via Adamello 16, 20139 Milan, Italy 4Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy 5Neurobiology Sector, SISSA, Via Bonomea 265, 34136 Trieste, Italy 6Istituto di Genetica Molecolare CNR, Via Abbiategrasso 207, 27100 Pavia, Italy 7Present address: Technische Universita t Darmstadt, Schnittspahnstrasse 13, 64287 Darmstadt, Germany 8Present address: EMBL-EBI, Hinxton, Cambridge CB10 1SD, UK *Correspondence: schneider@bio.tu-darmstadt.de (L.S.), fabrizio.dadda@ifom.eu (F.dA.d.F.) http://dx.doi.org/10.1016/j.stemcr.2013.06.004 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
2Fondazione 1IFOM
SUMMARY
The consequences of DNA damage generation in mammalian somatic stem cells, including neural stem cells (NSCs), are poorly understood despite their potential relevance for tissue homeostasis. Here, we show that, following ionizing radiation-induced DNA damage, NSCs enter irreversible proliferative arrest with features of cellular senescence. This is characterized by increased cytokine secretion, loss of stem cell markers, and astrocytic differentiation. We demonstrate that BMP2 is necessary to induce expression of the astrocyte marker GFAP in irradiated NSCs via a noncanonical signaling pathway engaging JAK-STAT. This is promoted by ATM and antagonized by p53. Using a SOX2-Cre reporter mouse model for cell-lineage tracing, we demonstrate irradiation-induced NSC differentiation in vivo. Furthermore, glioblastoma assays reveal that irradiation therapy affects the tumorigenic potential of cancer stem cells by ablating selfrenewal and inducing astroglial differentiation.
INTRODUCTION
The relationship between cell-cycle control and regulation of differentiation is a major question in stem cell biology. Neural stem cells (NSCs) are among the best characterized mammalian stem cells; they generate the central nervous system during development and support adult neurogenesis throughout life in the subventricular zone (SVZ) and subgranular layer of the hippocampus (Bonfanti and Peretto, 2007; Doetsch, 2003). NSCs were the rst somatic stem cell type shown to grow indenitely in vitro under self-renewing conditions as neurospheres (Reynolds and Weiss, 1992). NSC cultures can be derived ex vivo from both the developing and adult brain or from embryonic stem (ES) cells and can differentiate into the three brain lineages: neurons, astrocytes, and oligodendrocytes (Conti et al., 2005; Pollard et al., 2006). This differentiation is governed by extracellular ligands and cytokines (Gangemi et al., 2004) and is associated with the downregulation of NSC markers such as Nestin, SOX2, and PAX6 (Conti mez-Lo pez et al., 2011). Self-renewing et al., 2005; Go cells with gene expression patterns similar to normal NSCs can also be found in glioblastoma multiforme (GBM), supporting the concept of cancer stem cells (Nicolis, 2007).
We recently showed that the canonical DNA damage response (DDR) signaling pathways (Figure S1A available online) are functional in NSCs (Schneider et al., 2012). Generation of DNA double-strand breaks (DSBs), e.g., by ionizing radiation, leads to activation and focal recruitment of the apical PI3K-like serine/threonine kinase (ATM), which labels chromatin at DNA lesions through phosphorylation of the histone H2A variant H2AX (gH2AX). ATM also phosphorylates the serine/threonineglutamine (S/TQ) motif of many downstream effectors, some of which are focally recruited at DSBs (e.g., 53BP1), whereas kinases and transcription factors like CHK2 and p53 further relay DDR signaling, causing transient cellcycle arrest to allow DNA repair or, depending on the nature of the DNA damage, apoptosis or cellular senescence (dAdda di Fagagna, 2008; Jackson and Bartek, 2009; Shiloh, 2006).
RESULTS
DNA Damage in NSCs Leads to Cellular Senescence Despite Transcriptional Downregulation of DDR Signaling We induced DSBs in proliferating self-renewing NSCs, which uniformly display all key features of radial glia
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DNA-Damage-Induced Astrocytic Differentiation
Figure 1. X-Ray Irradiation of NSC Leads to a Senescence-like Cell-Cycle Arrest, Associated with Progressive Downregulation of DDR Signaling (A) Time-course study of DNA replication and cellular senescence in nonirradiated (non-irr) and irradiated NSCs (irr) by wide-eld microscopy. BrdU was supplied 24 hr prior to xation and BrdU-incorporating cells were detected by immunouorescence (IF). Senescent NSCs were analyzed by senescence-associated b-galactosidase (SA-b-Gal) activity assay and scored as positive for the characteristic blue signal. Error bars show SD. (B) Representative confocal images showing changes in cell morphology in irr NSCs; cytoskeleton was visualized by a-tubulin IF. Bar: 15 mm. (legend continued on next page)
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(Conti et al., 2005), by acute exposure to 10 Gy X-ray irradiation (irr). Many cells survived and exited the cell cycle, as indicated by reduced bromodeoxyuridine (BrdU) incorporation (Figure 1A) and expression of cell-cycle arrest markers such as p21CIP, p27KIP, and Rb-dephosphorylation (Figure S1B). Within 24 hr of irr, most and, after 3 days, all NSCs became enlarged with attened morphology and expressed senescence-associated b-galactosidase (SA-b-gal) activity (Figures 1B and S1C). Upon DNA damage, such dramatic changes are usually associated with cellular senescence, commonly requiring continuous DDR signaling (dAdda di Fagagna, 2008). Unexpectedly, whereas DDR signaling was promptly activated in NSCs immediately after irr (Figures 1C and S1D), it was progressively lost in the majority of cells entering senescence, as determined by DDR foci detection at the single-cell level for the DDR markers pS/TQ, gH2AX (Figure 1C), phosphoATM, and 53BP1 (Figure S1D). Progressive reduction in DDR signaling was conrmed by immunoblotting for gH2AX, phospho-ATM, phospho-Chk2, and phosphop53 (Figure 1D). Reduction in DDR foci is usually interpreted as accomplished DNA repair, including in NSCs (Acharya et al., 2010), and indeed we conrmed DSB repair prociency in irr NSCs (Figure S1E). Yet, we noticed that the progressive loss in detectable phospho-ATM and its target phospho-CHK2 correlated with reduced expression of total ATM and CHK2 proteins in irr cells (Figure 1D). We then performed microarray analyses on control NSCs and NSCs 7 days after irr. In irr NSCs, we detected gene expression changes associated with cell-cycle arrest (p21CIP, PTEN, GADD45a, CDC6, CDC25B) but also a widespread downregulation of DDR genes (Figure 1E). The transcriptional downregulation of selected key DDR genes (ATM; CHK2, p53, MDC1, H2AX) was conrmed by quantitative realtime PCR (Figure 1F). Our study thus revealed a form of DNA-damage-induced cellular senescence maintained in the absence of a functional DDR signaling. DNA Damage Induces Astrocytic Differentiation According to our previous report (Schneider et al., 2012), these ndings may indicate that irr NSCs had lost their stem cell features and committed to an astrocytic fate. When we investigated the expression of the self-
renewal-associated cell membrane marker SSEA1/LeX (Capela and Temple, 2006), only a small fraction of irr NSCs at day 7 stained positive, whereas all control cells did (Figure 2A). We also detected a progressive loss of NSC-associated cytoskeletal lament Nestin in individual senescent NSCs and the appearance of cells expressing the astrocyte-associated intermediate lament GFAP (Figures 2B, 2C, and S2A). Next, we quantied the nuclear signal of the key mez-Lo pez et al., stem cell transcription factor SOX2 (Go 2011) and SOX9, another SOX-family member relevant for NSC self-renewal (Scott et al., 2010). Neither of these markers, clearly expressed in non-irr NSCs, were detected in most nuclei analyzed at day 7 after irr (Figures 2D, S2B, and S2C). Progressive differentiation toward the astrocytic lineage was indicated by an increasing fraction of cells positive for the astrocyte-typical membrane channel Aquaporin 4 (Yoneda et al., 2001) at day 7 post-irr (Figures 2D and S2D). Western blotting conrmed reduction of Nestin, the self-renewal-promoting transcription factors PAX6 (Go pez et al., 2011) and OLIG2 (Ligon et al., 2007), mez-Lo and upregulation of GFAP in irr NSCs (Figures 2E and S2E). We consistently detected downregulation of Nestin, SOX2, and PAX6 mRNA by quantitative real-time PCR in several independent irr experiments (Figure 2F). Moreover, we observed widespread reduction in expression of genes associated with pathways typical of NSC biology and selfrenewal (Figure 2G): transcription factors SOX9 and OLIG2, RNA-binding proteins Musashi-1 (Okano et al., 2002) and ARS2 (Andreu-Agullo et al., 2012), nuclear receptor TLX (Qu et al., 2010), and the intermediate lament Vimentin (Conti et al., 2005). We extended this to genome-wide analysis in irr NSCs using cDNA microarrays. Using data sets from brain-derived astrocytes (Cahoy et al., 2008) or astrocytes differentiated in vitro from NSCs by serum stimulation (Obayashi et al., 2009) as references, we observed that numerous genes upregulated or downregulated specically during astrocytic differentiation showed a similar pattern in irr NSCs at day 7 (Figure 2H). The shift toward the expression of astrocytic markers was not associated with augmented expression of neuronal genes (detected either by microarrays or quantitative realtime PCR), even at later time points post-irr (Figure S2F).
(C) One hour after irr, NSCs uniformly display ATM/ATR kinase activity as demonstrated by the detection of foci of the typical phosphoepitope (pS/TQ, upper panel, red) and the phosphorylated histone H2AX (gH2AX, lower panel, green), as analyzed by confocal microscopy of IF stainings. Bar: 15 mm. Quantications of DDR-positive cells are provided on the right-hand side. (D) Western blot (WB) analysis of DDR signaling in NSCs after irr. DDR activation was detected by autophosporylated ATM (phospho-serine 1981), mobility shift of CHK2, p53 phosphorylation at ATM-dependent site (serine 15), and gH2AX. (E) Microarray analysis and heatmap of DDR and cell-cycle control genes from gene sets obtained from Gene Ontology classes and literature (Jackson and Bartek, 2009). Relevant genes are highlighted. (F) Quantitative real-time PCR analysis of NSCs on day 7 post-irr for the expression of key DDR and DNA repair genes. Error bars show SD. See also Figure S1.
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Figure 2. Irradiation Leads to Downregulation of Stem Cell Markers and Differentiation toward the Astrocytic Lineage (A) Quantication of the expression of NSC-typical membrane marker SSEA-1 in non-irr and irr cells at day 7 as detected by immunostaining and ow cytometry. Error bars show SD. (B) Representative confocal images showing the NSC-typical lament Nestin and the astrocyte lament GFAP prior and 7 days after irr, as detected by double IF analysis. Bar: 15 mm (C) Quantication of a time-course study of Nestin and GFAP expression in irr NSCs as detected by IF and wide-eld microscopy. Error bars show SEM. (legend continued on next page)
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This phenotype of DNA-damage-induced differentiation became increasingly robust over time to include 14 days post-irr the expression of S100b (Figures S2F and S2G), a marker of more mature astrocytes (Raponi et al., 2007). Importantly, NSCs were still tripotent shortly after irr, as they could efciently produce TUJ1-positive neurons under appropriate differentiation conditions (Figure S2H). DNA Damage Leads to Cytokine Secretion and GFAP Gene Induction through BMP2 and JAK-STAT Signaling in Senescent NSCs Interestingly, the efciency of GFAP induction correlated directly with the initial cell density at the time of irr and inversely with the volume of cell culture medium (Figure 2I), implying a potential role for soluble factors secreted from irr NSCs. It is generally understood that GFAP gene expression during astrocytic differentiation is induced by the synergic action of cytokines of the BMP (BMP2 and 4) and interleukin (IL)-6 families (i.e., LIF), which signal, respectively, through SMAD1-SMAD4 and JAK-STAT (Fukuda et al., 2007; Nakashima et al., 1999). Other IL-6-type cytokines (CNTF, OsM, and CT-1) are also known to signal through LIF receptor (Turnley and Bartlett, 2000). Among the candidate cytokines analyzed (Table S1), we observed that BMP2, BMP4, LIF, and IL-6 were consistently expressed in irr NSCs at day 7. A short time-course analysis of their expression showed a strong induction of BMP2 and LIF within 8 hr of irr, preceding GFAP induction and SOX2 downregulation, with BMP4 or IL-6 being induced only after the GFAP increase (Figure 3A). BMP2 and LIF signal canonically through downstream SMAD1 and STAT3 mediators, respectively (Fukuda et al., 2007; Nakashima et al., 1999). We studied the activation of these pathways in irr NSCs by probing for the phosphorylated (activated) forms of STAT3 and SMAD1. Remarkably, the phospho-STAT3 signal increased and persisted from 8 hr after irr onward, whereas SMAD1, poorly expressed in NSCs, accumulated and became phosphorylated only 3 days post-irr (Figure 3B). Gene Ontology
analysis of microarray data suggested activation of JAKSTAT pathway, whereas SMAD cascade proling showed a very poor induction of canonical downstream genes (Figure S3A). Next, we probed these signaling pathways using a set of specic inhibitors. The polypeptide Noggin prevents BMP2/4 signaling through the BMPR1a receptor (Lim et al., 2000). Noggin treatment of NSCs after irr prevented GFAP upregulation, despite robust induction of BMP2; a similar effect was observed upon suppression of JAK-STAT signaling with a pharmacological pan-JAK-kinase inhibitor (JAKi; Figure 3C and Figure S3B). Moreover, the use of a small-molecule inhibitor of BMPR1a kinase activity (BMPR1i) as well as JAKi both abolished GFAP induction, separately and in combination (Figure 3D). Interestingly, irr NSCs still showed STAT3 phosphorylation when BMP2/4 signaling was suppressed, and phospho-SMAD signal was detectable when JAKi was applied; this was nevertheless not sufcient to trigger GFAP induction (Figures 3D and S3B). Suppression of glial differentiation by JAKi in irr NSCs did not result in the expression of neuronal genes (Figure S3C). Remarkably, these inhibitors did not restore the expression of self-renewal genes (Figure 3C) or that of proliferation markers (Figure S3C), or the DNA replication activity in irr NSCs (Figure 3E). Instead, cells retained the expression of cellular senescence markers (Figures 3F and S3D), thus ruling out the possibility of cellular quiescence, reported elsewhere as associated with GFAP upregulation and controlled by BMP2/4 (Mira et al., 2010; Sun et al., 2011). Consistent with the ablation of DDR-suppressing astrocytic differentiation (Schneider et al., 2012), we detected a higher residual DDR activity in JAKi-treated irr cells compared to DMSO-treated irr cells (Figures S3E and S3F). Next, we collected conditioned medium (CM) from non-irr and irr cells and tested its effect on non-irr NSCs (Figure 3G). Strikingly, CM from irr cells, unlike that from non-irr cells, was sufcient to induce robust STAT3 phosphorylation and GFAP induction in non-irr cells Importantly, this effect was suppressed by supplementing
(D) Quantication of the nuclear signal of NSC transcription factors SOX2 and SOX9 as well as the expression of astrocyte-typical membrane channel Aquaporin4 (AQP4) in non-irr and irr NSCs as detected by IF and wide-eld microscopy. Error bars show SD. (E) WB analysis of stem cell (Nestin, PAX6) and astrocyte (GFAP) relevant protein expression upon irr of NSCs. (F) Median values of six or more experiments, analyzed by quantitative real-time PCR for the expression of NSC markers Nestin, SOX2, and PAX6 and the astrocyte marker GFAP. Error bars show SEM. (G) Quantitative real-time PCR analysis of NSCs at day 7 post-irr showing downregulation of typical NSC markers: OLIG2, SOX9, Musashi1 (MSI1), Vimentin (VIM), TLX, and ARS2. Error bars show SD. (H) Microarray analysis and heat map of genes reported as upregulated in brain-derived astrocytes and astrocytes produced from NSCs through serum exposure (Cahoy et al., 2008; Obayashi et al., 2009). Relevant astrocyte genes as mentioned in Cahoy et al. (2008) are highlighted. (I) NSCs were irradiated at different cell densities and medium replaced with 13 volume (low) or 23 volume (high). The expression of GFAP was analyzed by quantitative real-time PCR. Error bars show SD. See also Figure S2.
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Figure 3. GFAP Induction in Irradiated Senescent NSC Depends on BMP2 and JAK/STAT Signaling (A) Time-course study of the cytokine expression in irr NSCs by quantitative real-time PCR. SOX2 and GFAP expression reect self-renewal and differentiation, respectively. Error bars show SD. (B) WB analysis of the time course of STAT and SMAD signaling pathway activation in irr NSCs. GFAP signal reects the onset of differentiation. (C) Quantitative real-time PCR analysis of NSCs on day 7 post-irr. Note that continuous Noggin (left panel) or JAKi (right panel) treatment impaired GFAP induction, despite the ongoing expression of BMP2 and BMP4. Error bars show SD. (legend continued on next page)
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the CM with the JAKi prior to application (Figure 3G). Thus, growth factors secreted by irr NSCs mediate their astrocytic differentiation by activating the JAK-STAT signaling pathway. GFAP Induction and Astrocytic Differentiation Rely on Noncanonical BMP2 Signaling through JAK-STAT NSCs upregulated BMP2 as well as LIF immediately after irr (Figure 3A); however, only BMP2 expression remained stable even 1 month after irr (Figure S2G). To investigate the individual roles of these two cytokines, we treated non-irr NSCs with either BMP2 or LIF in the presence or absence of JAKi. LIF has been reported to stimulate GFAP expression by upregulating BMP2 (Fukuda et al., 2007). Predictably, LIF activated JAK-STAT signaling and induced GFAP; both events were prevented by JAKi (Figure 4A). Surprisingly, BMP2 not only proved a more potent GFAP inducer than LIF, that alone was sufcient to activate JAK-STAT signaling, both effects also were fully abolished by JAKi (Figure 4A). Importantly, BMP2 treatment did not stimulate transcriptional induction of LIF (Figure 4B). Moreover, whereas BMP2 exposure resulted in astrocytetypical morphology change in NSCs and profound GFAP upregulation, such effects were much less pronounced in LIF-treated and completely absent in IL-6-treated NSCs (Figure 4C). At 20 ng/ml, about 25% of LIF-treated NSCs and nearly all IL-6-treated cells were Nestin positive, whereas virtually all BMP2-exposed cells ceased expressing Nestin (Figure 4D). IL-6 reduced Nestin only at very high concentrations (100 ng/ml). Finally, we took advantage of wild-type and isogenic BMP2-knockout murine ES cells to derive NSCs through established methods (Conti et al., 2005; Ying et al., 2003). Although irr wild-type NSCs downregulated stem cell markers Nestin, SOX2, and PAX6 and upregulated GFAP, we could not detect any GFAP gene expression even by sensitive quantitative real-time PCR techniques in irr BMP2/ cells, despite downregulated stem cell markers (Figure 4E). Interestingly, BMP4 was also undetectable in BMP2/ cells (Figure 4E), indicating that its expression is controlled by BMP2, as previously suggested (Castranio and Mishina, 2009). Yet irr BMP2/ NSCs proved to be fully procient in inducing GFAP when exposed to recombinant BMP2 (Figure 4F).
Thus, BMP2 can signal noncanonically through JAKSTAT and induce GFAP expression independently from LIF or other IL-6-type cytokines. DNA-Damage-Induced Differentiation Requires ATM and Is Opposed by p53 Previous studies established a mechanistic link between the DNA-damage-induced permanent proliferative arrest and cytokine secretion, described as senescence-associated secretory phenotype or SASP, as secretion of IL-6/8 re et al., 2008; quires ATM and is opposed by p53 (Coppe Rodier et al., 2009). We investigated these molecular pathways in the context of DNA-damage-induced differentiation. First, we employed a previously characterized strain of ATM/ ES cells to generate ATM/ NSCs. Upon irr, both wild-type and ATM/ NSCs arrested proliferation (Figure S4A) and downregulated stem cell markers Nestin, SOX2, and PAX6, but ATM/ cells were less efcient in increasing BMP2 and GFAP levels (Figures 5A and 5B). p53 is a central downstream effector of the DDR cascade (Jackson and Bartek, 2009) and has been reported to regulate self-renewal and proliferation in NSCs (Meletis et al., et al., 2008). We used 2006) and suppress SASP (Coppe p53-decient ES cells to derive p53/ NSCs. When irradiated, these cells exited the cell cycle (Figure S4B) and underwent irr-induced glial differentiation even more readily than isogenic wild-type cells, as suggested by a signicantly stronger induction of GFAP (Figures 5C, 5D, and S4C). We next asked whether this was the consequence of stronger cytokine secretion: we irradiated p53-decient and isogenic wild-type cells and transferred CM from irr p53/ cells onto irr isogenic wild-type NSCs. We observed a much stronger induction of phosphoSTAT3 and GFAP in CM-treated irr wild-type NSCs than in CM-untreated cells (Figure 5F). DNA Damage In Vivo Leads to Astrocytic Differentiation of NSCs in the SVZ NSCs used in this study so far were derived from ES cells and resemble more closely embryonal radial glia than type B cells residing in adult brain (Conti et al., 2005; Doetsch, 2003; Pollard et al., 2006). Thus, we next employed cell lines derived from adult mouse forebrain (Conti
(D) WB analysis of the effects of JAK/STAT signaling inhibition (JAKi) and BMP2/4-receptor inhibition (BMPR1i) on GFAP induction. (E) BrdU incorporation-based assessment of DNA replication in irr NSCs treated continuously with JAKi or Noggin. BrdU was supplied for 24 hr prior to xation and detected by IF and wide-eld microscopy. Error bars show SD. (F) Representative wide-eld microscopy images of persistent senescence-associated b-galactosidase activity in irr NSCs continuously treated with JAKi or Noggin, magnication: 203. (G) WB analysis for the effect of conditioned medium (CM) from irr or not irr NSCs, daily collected for 3 days, and transferred onto separate irr or non-irr NSCs. Lane 7: CM from irr NSCs was supplemented with JAKi. See also Figure S3.
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Figure 4. BMP2 Signals Noncanonically through JAK/STAT to Induce GFAP Independently of LIF (A) WB analysis of the effects of the cytokines LIF and BMP2 at 20 ng/ml on GFAP induction and the role of JAK/STAT signaling in non-irr NSCs. (B) Quantitative real-time PCR analysis of GFAP and LIF expression levels in NSCs exposed for 8 hr to 20 ng/ml recombinant BMP2. Error bars show SD. (C) Representative wide-eld microscopy images of the effect recombinant cytokines (20 ng/ml) on NSCs after 6 days. GFAP detected by IF analysis, magnication: 403. (D) Quantication of Nestin and GFAP-positive cells after cytokine exposure for 6 days at 20 ng/ml or 100 ng/ml. (legend continued on next page)
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Figure 5. GFAP Induction Is Impaired in ATM-Decient Irradiated NSCs, whereas p53 Deciency Promotes DNADamage-Induced Differentiation through Increased Cytokine Secretion (A) WB analysis for Nestin and GFAP in irr wild-type and isogenic ATM-decient NSCs. Membrane was also probed for phospho- and total ATM protein. (B) Quantitative real-time PCR analysis of wild-type and isogenic ATM-decient NSCs for the expression of BMP2, Nestin, SOX2, PAX6, and GFAP. Error bars show SD. (C) Representative confocal images of wild-type and isogenic p53/ NSCs, GFAP detected by IF analysis. Bar: 15 mm. (D) Median values of two or more experiments of wild-type and isogenic p53/ NSCs, analyzed by quantitative real-time PCR for the expression of Nestin, SOX2, PAX6, and GFAP. Error bars show SEM. (E) WB analysis for the effect of conditioned medium (CM) from irr p53/ NSCs transferred on irr wild-types (promoting STAT3 phosphorylation and GFAP induction) and vice versa. See also Figure S4.
et al., 2005; Pollard et al., 2006). Consistent with our results with ES-derived NSCs, nearly all adult NSCs entered cellular senescence upon irr (Figures S5A and S5B). Compared to ES cell-derived NSCs, these cells also displayed a striking increase in GFAP expression upon irr (Figures 6A and S5C), likely reecting the more primed state of the GFAP promoter (Doetsch, 2003). Astroglial differentiation of adult NSCs was also demonstrated by upregulation of S100b mRNA and protein (Figures 6B and 6C) and a strong reduction of Nestin protein and mRNA, as well as other stem cell markers (SOX2, SOX9, PAX6) and DDR genes (ATM, CHK2) (Figures 6A6C).
Next, we extended our study to living animals and took advantage of a mouse strain in which NSCs can be labeled in vivo noninvasively and permanently (Favaro et al., 2009). These mice express Cre endonuclease fused to estrogen receptor (ERT2), under a SOX2 50 telencephalic enhancer/promoter. The animals were crossed with a strain carrying R26-eYFP-fLox-STOP transgene (Srinivas et al., 2001). In this way, treatment with tamoxifen allowed permanent labeling of SOX2-expressing cells with YFP, which is retained independently of SOX2 expression afterward.
(E) Quantitative real-time PCR analysis of wild-type and isogenic BMP2-decient NSCs for the expression of BMP2/4, NSC markers Nestin, SOX2, PAX6, and the astrocyte marker GFAP. Error bars show SD. zGFAP, BMP2, and BMP4 gene expression were not detectable in irr BMP2/ NSCs. (F) Quantitative real-time PCR analysis of BMP2-decient NSCs for the expression of Nestin and GFAP. Error bars show SD. zAs in (E), GFAP not detectable.
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Figure 6. NSCs from Adult Forebrain Undergo Astrocytic Differentiation upon Irradiation In Vitro as Well as in a Mouse Model of Cell-Fate Tracing (A) Representative confocal images of non-irr adult NSCs and cells at day 7 post-irr; Nestin and astrocyte markers GFAP and S100b were detected by IF analysis. Bar: 20 mm. (B) Quantication of adult NSCs at day 7 post-irr for the Nestin and GFAP expression, nuclear SOX9 signal, and the highly S100b-positive cells (s100b-HI) as analyzed by IF and wide-eld microscopy. (C) Quantitative real-time PCR analysis of adult NSCs on day 7 post-irr for the expression of self-renewal (Nestin, SOX2, PAX6) and astrocyte markers (GFAP, S100b), and DDR genes (ATM, CHK2). Error bars show SD. (legend continued on next page)
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We exposed mice with in-vivo-labeled SOX2-expressing NSCs to cranial irradiation with 10 Gy (or mock irradiation), sacriced them on day 3 after irr, and analyzed their brains by immunouorescence and confocal microscopy. In mock-irradiated (non-irr) animals, YFP-positive cells in the SVZ rarely expressed detectable GFAP or S100b (Figures 6D and S5D). Strikingly, in irr brains, YFP-expressing cells were often positive for both GFAP and S100b as detected by triple immunouorescence, indicating astrocytic differentiation of SOX2-expressing cells in SVZ within 3 days after irr (Figures 6E and S5E). Importantly, as an internal control, immunostaining for YFP showed similar intensities in both irr and non-irr brains. Because the astrocyte markers GFAP and S100b are both detectable in the cytoplasm, we could calculate the ratio with which the cytoplasmatic YFP signal overlapped quantitatively with those, in experimental animal triplicates. Quiescent radial glia in the SVZ (type B cells) express GFAP (Bonfanti and Peretto, 2007), and indeed we detected some overlap of YFP and GFAP signals in non-irr brains (20%). Moreover, due to prolonged tamoxifen treatment (10 days), a degree of homeostatic astroglial differentiation of previously labeled NSCs was expected. Nevertheless, in irr brains at day 3 we measured a statistically signicant 2-fold increase in the overlap of YFP signal with GFAP signal as well as with S100b signal, when compared to non-irr controls (Figure 6F). DNA Damage in Glioblastoma Stem Cells In Vitro and in Mouse Glioblastoma Model In Vivo Leads to Astrocytic Differentiation and Loss of Tumorigenic Potential Finally, we extended our study to brain cancer cells, which share key functional characteristics with untransformed NSCs, such as self-renewal and the gene expression signature (Nicolis, 2007). We exposed to 10 Gy irr a murine glioblastoma (GBM) cancer stem cell line (GL261-CSC), which ri et al., carries point mutations in K-Ras and p53 (Szatma 2006), expresses Nestin but not GFAP, and can be grown as adherent cultures as well as tumorspheres (Pellegatta et al., 2006). Here also, irradiation triggered downregulation
of Nestin and induction of GFAP, both in tumorspheres and in adherent cells (Figures 7A, 7B, S6A, and S6B). Although the clonal frequency of non-irr GBM cells was 25%, after irr it dropped to only 1.5% of the total number of cells plated (Figure 7C). Correspondingly, key proliferation genes were downregulated in irr GBM cells (Figure S6C). GL261-CSCs are widely used to simulate human GBMs and initiate aggressive and lethal brain tumors in rodent assays (Jacobs et al., 2011). We tested the in vivo tumorigenicity of irr versus non-irr GBM cells by grafting them into mouse brains. Three days after in vitro irr of GBM cells, ten animals each were injected into the nucleus caudatum with 103, 104, or 105 irr GL261-CSC or with equal numbers of non-irr cells. Non-irr GBM cells formed aggressive tumors and proved lethal to all host animals in about 2040 days (Figure S6D; Table S2). By contrast, when 103 irr GBM cells were injected, no tumor-induced mortality was observed during the entire observation period of 100 days, whereas 104 or 105 injected irr cells led to markedly reduced mortality (Figure S6D; Table S3). Finally, we probed DNA-damage-induced differentiation in murine glioblastoma in vivo by injecting 105 non-irr GL261-GLS and 10 days later exposing the glioma-bearing mice to focused cranial irradiation of 10 Gy. Radiation therapy led to a signicant increase in survival as compared to mock-irradiated glioma-bearing mice (Figure 7D). We examined irr and non-irr gliomas from mice sacriced at two time points. Ten days after irr, the tumor mass was small and localized near the injection site with necrotic areas, whereas the non-irr glioma was much larger and inltrated the contralateral hemisphere, showing high cellularity (Figure S6E). The majority of GBM cells in nonirr tumor reected their stem cell characteristics by a strong Nestin signal and a low GFAP presence. Upon irr, the majority of GBM cells lost their Nestin expression, whereas a large fraction of GBM cells near central tumor mass strongly upregulated the astrocytic differentiation marker GFAP (Figures 7E and 7F). Gliomas from mice sacriced 20 days after irr, however, displayed Nestin-positive cells preferentially located at tumor borders (34.6% 9.1% in the periphery and
(D) Mice were treated with tamoxifen to label SOX2 expressing cells, mock irradiated, and sacriced 3 days later. Brain sections containing the SVZ were stained by triple IF with an anti-YFP antibody in order to detect labeled cells and antibodies against astrocyte markers GFAP and S100b to address differentiation status. A collapsed confocal microscopy z stack for each channel is shown. Bar: 15 mm. The merged collapsed z stack of all channels is provided in Figure S5D. (E) Mice were treated as above, but subjected to cranial irradiation. Brain sections containing the SVZ were analyzed as above. Bar: 15 mm. The merged collapsed z stacks of all channels is provided in Figure S5E. (F) Three non-irr and irr brains each from two irradiation experiments were analyzed to obtain approximately ten confocal z stack series from several physical sections of each brains SVZ (<30 z stacks for each condition in total). The colocalization ratio of the YFP signal with astrocyte markers GFAP and S100b was calculated for each layer of the z stack as the Manders coefcient of YFP overlap with GFAP or S100b; median values are shown. p values were calculated by Mann-Whitney rank sum test. Error bars: SEM. See also Figure S5.
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Figure 7. Murine Glioblastoma Stem Cells Undergo Astrocytic Differentiation In Vitro and in a Mouse In Vivo Xenograft Model (A) Representative confocal images of murine GBM cell line GL261-CSC, irradiated in adherent conditions, Nestin and GFAP detected by IF analysis. Bar: 20 mm. (B) Quantitative real-time PCR analysis (TaqMan assay) of GL261-CSC grown in serum-free tumorsphere and adherent cultures on day 3 post-irr for the expression of Nestin and GFAP. Error bars show SD. (C) In vitro cloning analysis performed by serial dilution in 96-well plates on non-irr and irr GL261-CSC. (D) GBM tumors were induced in mice by injection of 105 GL261 cells. Radiation therapy was applied at day 10 (arrow). Kaplan-Meier curves (ve animals each) show signicantly prolonged survival of GBM-tumor-bearing mice after cranial irr. (E) Representative immunohistochemistry (IHC) analysis of Nestin and GFAP expression in GBMs from tumor-bearing animals, sacriced 10 days after radiation therapy. Upper panel: large areas of GBM tumors became negative for Nestin after irr. Lower (legend continued on next page)
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10.6% 1.3% in the center, Figure S6F). Correspondingly, GFAP-positive cells were located in the central tumor mass and not in the periphery (15.6% 1.8% center versus 1.2 1.2, periphery; (Figures 7G and S6G), where the tumor did progress. In summary, tumor growth requires a stem-like state of glioblastoma cells, and radiation therapy induces their differentiation and decreases their oncogenic potential.
DISCUSSION
This study demonstrates that DNA damage in NSCs leads to cellular senescence, depriving them of their self-renewal potential and promoting astrocytic differentiation. This process is niche independent; i.e., it occurs even under self-renewal-promoting culture conditions and relies on the cell-autologous DNA-damage-induced secretion of soluble factors. Our results also highlight a noncanonical BMP2 signaling pathway through JAK-STAT, which is responsible for promoting astrocytic differentiation of senescent cells. Moreover, our conclusions apply both in vitro and in vivo, including adult brain NSCs and GBM stem cells. Terminal differentiation of stem and progenitor cells is dened by an irreversible cell-cycle arrest, loss of expression of stem/progenitor cell markers, and upregulation of differentiation-associated genes. We observed this in both ES-derived and adult forebrain NSCs after irr. Moreover, we also observed loss of DDR signaling and DDR gene expression in irr NSCs, which is consistent with their differentiation toward the astrocytic lineage (Schneider et al., 2012). The differentiation bias of irr NSCs toward astrocytes may be explained by their glial nature (Doetsch, 2003). Indeed, NSCs sustaining mitochondrial DNA damage were reported to be more prone to astroglial fate when stimulated to differentiate (Wang et al., 2011). In our model, DNA damage forces cells into cellular senescence, whereas ATM-dependent and p53-antagonized cytokine secretion activates BMP2/JAK-STAT signaling and stimulates the differentiation process in a progressive feed-forward manner. This senescent state is very different from the GFAP-associated quiescence described elsewhere (Mira et al., 2010; Sun et al., 2011),
because quiescent NSCs are characterized by retention of their self-renewal prole. Moreover, this NSC-specic cellular senescence takes place in the absence of persistent DDR signaling, which is commonly required for senescence maintenance in non-stem cell types (dAdda di Fagagna, 2008; Jackson and Bartek, 2009). Hence, these cellular senescence and ablation of self-renewal are likely to involve epigenetic mechanisms that persist after initial DNA-damage-induced cues. Telomere-attrition-induced DNA damage in hematopoietic stem cells activates STAT3 and, in turn, BATF in a G-CSF-dependent manner, leading to their differentiation (Wang et al., 2012). Although our microarray data do not indicate this particular signaling activity in irr NSCs, STAT3 seems an important differentiation pathway as suggested by this and other studies (Fukuda et al., 2007; Lee et al., 2010). BMP2 and BMP4, which bind to the same receptor BMPR1, were shown to induce differentiation of glioblastoma-initiating cells (Piccirillo et al., 2006). In the nervous system, BMP2/4 is thought to act in concert with LIF (or another relevant IL-6 family member like CNTF), signaling through SMAD1 and JAK2-STAT3, respectively, to induce GFAP (Fukuda et al., 2007; Nakashima et al., 1999). Others have suggested that BMP2/4 may directly activate STAT3 signaling (Jeanpierre et al., 2008), and BMP4 alone was reported to induce GFAP (Obayashi et al., 2009). Our study provides evidence that BMP2 can activate JAK-STAT signaling and induce GFAP independently of LIF, whereas still requiring binding to its receptor and stimulating its kinase activity. We also demonstrate here that radiation therapy forces GBM cells to lose self-renewal and commit to terminal differentiation. Whether tumor progression and mortality are caused by the escape of some GBM cells from irr-induced differentiation and the ensuing expansion of clones retaining stem cell properties remains to be dened. Future therapeutic strategies might combine radiation therapy and treatment with differentiation-promoting cytokines to ensure the permanent ablation of self-renewal in GBM. Previous studies have indicated that NSCs in irradiated rodent brain activate cell-cycle checkpoints and lose their neurogenic capacity (Acharya et al., 2010; Monje et al., 2002). Other reports extended the concept of DNAdamage-induced differentiation to other types of somatic
panel: whereas non-irr GBM are generally GFAP negative, radiation therapy results in a tremendous increase in the GFAP signal. Magnication: 403. (F) Quantication of Nestin and GFAP-positive cells in the GBM tumor mass of non-irr mice or those subjected to radiation therapy. Two animals were studied for each condition. Error bars show SD. (G) Representative IHC analysis of GFAP expression in irr GBM tumors at day 30 (=day 20 after radiation therapy at day 10). Note that in advanced tumors the differentiation marker GFAP could was prominent only in the central glioma mass (top panel), but not in the expanding tumor periphery (bottom panel; *healthy tissue, **tumor, separated by dashed line). Magnication: 403. See also Figure S6.
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stem cells in vivo, such as hair bulge melanocyte stem cells (Inomata et al., 2009) and hematopoietic stem cells (Wang et al., 2012). Together with our ndings, these convergent lines of evidence suggest that DNA-damage-induced differentiation may have been selected during evolution to disarm the oncogenic potential of damaged stem cells without the side effects associated with their physical elimination.
Microscopy Analyses
Details of microscopy analyses are provided in Supplemental Experimental Procedures. Cells were xed and used for senescence assays or stained with primary antibodies for immunouorescence. Brain sections were treated for immunouorescence (IF) with 50 mM NH4Cl for epitope recovery and permeabilized/ blocked with 0.2% Triton X-100 and 1% BSA in PBS. DNA was stained with DAPI (Sigma-Aldrich). For immunohistochemistry (IHC), parafn-embedded tumor sections were probed and acquired using a Leica MDLB microscope. The percentages of Nestin and GFAP cells were calculated in in triplicate by two observers (F.P. and S.P.), indiscriminately for tumor center and periphery.
ES-Derived Neural Stem Cell Lines
NSC culture and derivation from murine embryonic stem cells (ESCs) of various genetic background, based on protocols established by A. Smiths laboratory (Conti et al., 2005; Ying et al., 2003) are described in detail in Supplemental Experimental Procedures. Predominantly, NSCs derived from E14Tg2a ES background (Burgold et al., 2008) were used. Gene-decient ESC lines were used together with isogenic wild-types to derive genedecient NSCs and kindly provided as follows: BMP2/, Trisha Castranio and Yuji Mishina (NIEHS-NIH, USA and U. of Michigan, respectively); ATM/, Yang Xu (UCSD); and p53/, JeanChristophe Marine (VIB, Belgium). References for the original ES cell strains are available in Supplemental Experimental Procedures.
Immunoblotting
Cells were lysed and analyzed by western blotting using primary antibodies as described in detail in Supplemental Experimental Procedures. Membrane equal loading was assessed with probing for a-tubulin or vinculin.
Gene Expression Analysis

RNA extraction and SYBR-Green-based real-time quantitative PCR gene expression analyses were performed using primers designed with Roche UniversalProbe Library online software against Mus musculus as described in detail in Supplemental Experimental Procedures. In all experiments, b2-microglobulin (B2M) was used as housekeeping gene.
Cell Treatments
X-ray irradiation of cells was performed in a Faxitron RX-650 device at 2 Gy/min for 5 min (total of 10 Gy). Cells were not passaged after irr and medium change was performed on day 1 after irr and then every other day. BrdU was applied at 3.3 mM for 24 hr; JAKi I (Calbiochem) and LDN193189 (BMPR1 inhibitor; Axon Medchem) at 1 mM, with DMSO as control. Recombinant murine Noggin, LIF, IL-6, and human BMP2 (Prospec) were applied at 200 ng/ml (Noggin) and 20 ng/ml (unless stated otherwise). CM supernatants were collected daily, ltered with 0.45 mm lters and supplemented with one-third of fresh medium. In vitro cloning dilution assays on GL261-CSC were performed by dissociation of 10 Gy irr tumorspheres into single cells, plated after serial dilution as 1 cell/well in 96-well plates (n = 10/condition) and scored after 10 days for clonally derived secondary spheres.
Microarray Analysis
Irradiation experiments on NSCs were performed in a quadruplicate, four of each control (C14), and day 7 post-irr (I1-4) RNA extractions were performed as above. Labeled complementary RNA was hybridized on Affymetrix GeneChip Mouse Genome 430 2.0 Arrays, containing 45,101 probe sets corresponding to over 39,000 transcripts. Analyses and calculations were performed as described in detail in Supplemental Experimental Procedures.
Flow Cytometry
Cells were stained live in suspension on ice with SSEA-1 antibody (#3063-25 BioVision) and then with Alexa-Fluor-488-labeled secondary antibody (Invitrogen). Cells stained with secondary antibodies only were used as negative controls. Immediately after staining, data were acquired and quantied by uorescenceactivated cell sorting on Becton Dickinson FACScalibur.
Animal Treatments
For in vivo cell-fate tracing, SOX2-CreERT2 mice (Favaro et al., 2009) were crossed onto R26::loxP-stop-loxP::YFP background (Srinivas et al., 2001), treated with tamoxifen, irradiated with a RADGIL irradiator, sacriced 3 days later, and processed as described in detail in Supplemental Experimental Procedures. For in vivo irradiation, C57BL/6N mice received brain injection of 105 GL261 cells, 10 days after tumor implantation mice were cranially irradiated using a 6 MeV Varian linear accelerator at a dose of 10 Gy. The eyes were covered using a protective lead band. Ten animals each were evaluated for survival analysis; two of each group for histological analysis when moribund. Two mice (n = 1/group) were sacriced 20 days after tumor implantation to evaluate the glioma engraftment.
ACCESSION NUMBERS
The microarray data discussed here have been deposited in the GEO database with accession number GSE38031.
Supplemental Information includes Supplemental Experimental Procedures, six gures, and four tables and can be found with this article online at http://dx.doi.org/10.1016/j.stemcr. 2013.06.004.
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ACKNOWLEDGMENTS
We thank Mario P. Colombo, Ivano Arioli, Alfonso Passafaro, and Francesco Ghielmetti for help and advice with animal irradiation experiments; Stefano Gustincich and Luca Ferrarini for microarray analysis; Thomas Burgold for help with establishing of NSC cultures; Luciano Conti for critical manuscript reading and advice; Luca Bonfanti and Elena Cattaneo for advice and discussions; Marzia Fumagalli for help with projects early experiments; IFOM Imaging and other facilities and IFOM and IEO members for reagents and technical advice; and all F.dA.d.F. lab members for help and feedback throughout this work. F.dA.d.F laboratory was supported by FIRC (Fondazione Italiana per la Ricerca sul Cancro), AIRC (Associazione Italiana per la Ricerca sul Cancro; #12971), Cariplo Foundation (#2010.0818 to F.dA.d.F. and G.F.), Human Frontier Science Program (#RGP 0014/2012), Marie Curie Initial Training Network CodAge, European Research Council (#322726), and TELETHON (#GGP12059). G.T.s laboratory was supported by AIRC, Italian Ministry of Health, and CNR (EPIGEN Flagship Project). R.F. and S.K.N. were supported by grants from AIRC (IG-5801), Fondazione Cariplo (#20100673), ASTIL regione Lombardia (SAL-19, Prpt FL 16874), and Telethon (GGP12152). Received: May 22, 2013 Revised: June 11, 2013 Accepted: June 12, 2013 Published: July 25, 2013
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Ar ticle Multipotent Human Mesenchymal Stromal Cells Mediate Expansion of Myeloid-Derived Suppressor Cells via Hepatocyte Growth Factor/c-Met and STAT3
B. Linju Yen,1,2,* Men-Luh Yen,3,4 Pei-Ju Hsu,1 Ko-Jiunn Liu,5,6 Chia-Jen Wang,8 Chyi-Huey Bai,7 and Huey-Kang Sytwu8
1Regenerative Medicine Research Group, Institute of Cellular and System Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 35053, Taiwan 2Department of Obstetrics/Gynecology, Cathay General Hospital Shiji, No. 2 Lane 59 Chien-chen Road, Shiji, New Taipei City 221, Taiwan 3Department of Primary Care Medicine 4Department of Obstetrics/Gynecology National Taiwan University Hospital and College of Medicine, National Taiwan University, No. 1 Section 1, Jen-Ai Road, Taipei 10617, Taiwan 5National Institute of Cancer Research, National Health Research Institutes, 367 Shen-Li Road, Tainan 704, Taiwan 6School of Medical Laboratory Science and Biotechnology 7Department of Public Health, College of Medicine Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan 8Graduate Institute of Microbiology and Immunology, National Defense Medical Center, No. 161 Section 6, Ming-Chuan E. Road, Taipei 114, Taiwan *Correspondence: blyen@nhri.org.tw http://dx.doi.org/10.1016/j.stemcr.2013.06.006 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
SUMMARY
Mesenchymal stromal cells (MSCs) are multilineage progenitors with immunomodulatory properties, including expansion of immunomodulatory leukocytes such as regulatory T lymphocytes (Tregs) and tolerogenic dendritic cells. We report that human MSCs can expand CD14CD11b+CD33+ human myeloid-derived suppressor cells (MDSCs). MSC-expanded MDSCs suppress allogeneic lymphocyte proliferation, express arginase-1 and inducible nitric oxide synthase, and increase the number of Tregs. This expansion occurs through the secretion of hepatocyte growth factor (HGF), with effects replicated by adding HGF singly and abrogated by HGF knockdown in MSCs. In wild-type mice, the liver, which secretes high levels of HGF, contains high numbers of Gr-1+CD11b+ MDSCs, and injection of HGF into mice signicantly increases the number of MDSCs. Expansion of MDSCs by MSC-secreted HGF involves c-Met (its receptor) and downstream phosphorylation of STAT3, a key factor in MDSC expansion. Our data further support the strong immunomodulatory nature of MSCs and demonstrate the role of HGF, a mitogenic molecule, in the expansion of MDSCs.
INTRODUCTION
Multipotent mesenchymal stromal cells (MSCs) are a population of multilineage progenitor cells that were rst isolated from the bone marrow (Friedenstein, 1976; Pittenger et al., 1999). These somatic progenitor cells harbor the capacity to differentiate into adipocytes, osteoblasts, and chondrocytes, as well as a number of extramesodermal lineages (Prockop, 1997). Recent studies have demonstrated that MSCs exert strong immunomodulatory effects on multiple populations of leukocytes via various mechanisms, including suppression of CD4 and CD8 lymphocyte proliferation and responses, induction of T regulatory lymphocytes (Tregs; a population of immunomodulatory T cells), and secretion of immunosuppressive molecules such as transforming growth factor-b (TGF-b) and indoleamine-2,3-dioxygenase (IDO) (Uccelli et al., 2008). MSCs also strongly suppress natural killer lymphocyte cytotoxicity and affect dendritic cell (DC) maturation, e.g., by inhibiting the differentiation of monocytes to immature myeloid DCs and decreasing the effector functions of
plasmacytoid DCs (Le Blanc and Mougiakakos, 2012; Uccelli et al., 2008). Many of these components are similar to the immunomodulatory armamentarium of the immune system, which is important for preventing autoimmunity and establishing tolerance (Guleria and Sayegh, 2007; Wing and Sakaguchi, 2010), with mechanisms ranging from anti-inammatory molecules such as TGF-b, IDO, and interleukin-10 (IL-10) to leukocyte subpopulations such as Tregs and tolerogenic DCs (Mellor and Munn, 2004; Sakaguchi et al., 2006; Swiecki and Colonna, 2010). As with many biological phenomena, immunomodulation is a double-edged sword, and many of these tolerogenic mechanisms appear to be manipulated by cancer cells to create an immunoprivileged niche to further their own growth (Rabinovich et al., 2007). One of the most prominent immunomodulatory leukocyte subpopulations in cancer consists of myeloid-derived suppressor cells (MDSCs) (Ostrand-Rosenberg and Sinha, 2009). Derived from myeloid precursors, MDSCs suppress immune response by a number of mechanisms, such as suppressing
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MSC-Secreted HGF Expand MDSCs via c-Met and STAT3
cytotoxic lymphocyte effector functions and targeting T cells by expressing the enzymes arginase 1 (ARG1) and inducible nitric oxide synthase (iNOS), both of which block the production of the T cell CD3-z chain by metabolizing L-arginine (Gabrilovich and Nagaraj, 2009; Gabrilovich et al., 2012). Human and mouse studies have revealed that chronic inammation and proinammatory mediators such granulocyte macrophage colony-stimulating factor (GM-CSF), IL-1b, IL-6, and prostaglandin E2 (PGE2) are involved in the induction of these suppressor leukocytes (Bunt et al., 2007; Serani et al., 2004; Sinha et al., 2007; Young and Wright, 1992). Although it is clear that the tumor microenvironment is maintained by diverse cell types, the role of secreted factors other than cytokines and proinammatory factors in the expansion of MDSCs has largely been unexplored, with the exception of vascular endothelial growth factor (Fricke et al., 2007; Shojaei et al., 2007). We report that MDSCs can be expanded by MSCsecreted hepatocyte growth factor (HGF), a potent mitogenic growth factor.
RESULTS
MSCs Can Expand High Numbers of Functional CD14CD11b+CD33+ MDSCs from Peripheral Blood Leukocytes We hypothesized that the strong immunosuppressive properties of diverse sources of MSCs extend to involve the expansion of MDSCs. We rst isolated and cultured MSCs from placenta and bone marrow, and then characterized the cells for surface marker expression and multilineage differentiation potential. Both bone marrow and placental MSCs are positive for surface expression of CD73, CD105, and CD90, but negative for hematopoietic markers such as the costimulatory molecules CD80 and 86 (Figure 1A; Chang et al., 2006; Uccelli et al., 2008; Yen et al., 2005). Both populations of MSCs can differentiate into osteoblastic, chondrogenic, and adipocytic lineages, and thus meet the criteria for multipotent MSCs (Figure 1B; Dominici et al., 2006; Liu et al., 2011; Pittenger et al., 1999). To test whether MSCs can expand MDSCs, we cocultured MSCs with human peripheral blood leukocytes (PBLs) and assayed for MDSCs, which in the human system are most commonly characterized as CD14CD11b+CD33+ leukocytes with a strong suppressor function (Ostrand-Rosenberg and Sinha, 2009; Poschke and Kiessling, 2012). We found that both bone marrow and placental MSCs could increase the number of CD14CD11b+CD33+ cells from PBLs (Figure 2A). Both cell percentage and numbers were increased signicantly after PBL coculture with MSCs, with CD11b+ cells showing an increase from a baseline of 8.9% 0.6% (10,179 926 cells; averages SEM) to
19.2% 1.2% (19,508 1,258 cells) after coculture with MSCs, and CD14CD11b+CD33+ cells increasing from 1.3% 0.1% (1,052 90 cells) at baseline to 2.4% 0.2% (2,343 147 cells) after coculture with MSCs (Figure 2B). To assess whether these MSC-expanded MDSCs were functional, we sorted MSC-induced CD14CD11b+CD33+ cells and cocultured these cells with allogeneic proliferating lymphocytes to assess for suppressive capacity. We found that the MSC-expanded CD14CD11b+CD33+ cells could suppress lymphocyte proliferation in a dose-dependent manner (Figure 2C). Human MDSCs are known to express guez and Ochoa, ARG1 and iNOS (Ochoa et al., 2007; Rodr 2008), and we found that MSC-induced MDSCs also expressed these enzymes and at signicantly higher cell numbers compared with baseline (Figures 2D and 2E, respectively). MDSCs have also been shown to express IL-10 and TGFb in some reports (Poschke and Kiessling, 2012), but we did not nd any expression of these molecules at baseline or after MSC coculture (Figure S1A available online). CD14CD11b+CD33+ MDSCs have been reported to induce Tregs (Dugast et al., 2008; Huang et al., 2006), and we also found that MSC-induced CD14CD11b+CD33+ cells were able to induce high numbers of CD4+CD25highCD127low Tregs from stimulated PBL (Figures 2F and S1B). Thus, MSC-expanded CD14CD11b+CD33+ cells have multiple immunomodulatory functions. Expansion of MDSCs by MSCs Is Mediated by Secreted HGF We next investigated the mechanisms involved in MSC expansion of MDSCs. We found that the expansion of MDSCs by MSCs was not affected by transwell separation of cells, indicatig that cell-cell contact was not needed and implicating secreted factors in this process (Figure 3A). We therefore analyzed the conditioned medium of MSCs to assess for relevant secreted molecules. MSCs secrete a number of stromal-related factors, such as RANTES/CCL5, HGF, and IL-6, the latter of which has been implicated in the expansion of MDSCs (Bunt et al., 2007; Figure 3B). However, using both blocking antibodies and small interfering RNA (siRNA) knockdown studies, we did not nd that IL-6 contributed to the expansion of MDSCs by MSCs (Figure S2). It was previously reported that STAT3 is critical for the expansion of MDSCs (Gabrilovich and Nagaraj, 2009). STAT3 is also an important molecule in the signal transduction pathway of HGF (Trusolino et al., 2010), a molecule that is known to be highly secreted by many cell types, including MSCs (Di Nicola et al., 2002; Takai et al., 1997; Trusolino et al., 2010). Since we also found that HGF was highly secreted by MSCs (Figure 3B), we assessed whether this molecule is involved in MSC expansion of MDSCs.
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Figure 1. Characterization of Bone Marrow and Placental Multipotent MSCs (A and B) Surface marker prole (A) and trilineage differentiation phenotype (B) for bone marrow (BM) multipotent MSCs and placental MSCs (P-MSCs). Adipo, adipogenic lineage (stained with oil red O to assess for oil droplet formation); chondro, chondrogenic lineage (stained with Alcian blue to assess for the presence of glucosaminoglycans); osteo, osteogenic lineage (stained with alizarin red to assess calcium deposition). Scale bar: 200 mm.
We found that the addition of recombinant HGF alone could result in the expansion of MDSCs, and this effect was dose dependent up to a concentration of 30 ng/ml (Figure 3C), which is approximately the upper limit found in MSC-conditioned medium (Figure 3B). HGF-expanded MDSCs also expressed iNOS and ARG1, and at higher cell
numbers compared with baseline (Figure 3D). Moreover, HGF-expanded MDSCs can suppress allogeneic lymphocytes proliferation as well (Figure 3E). To further ascertain the involvement of HGF in MSC expansion of MDSCs, we suppressed the secretion of HGF by MSCs with siRNA. Using siRNA specic for HGF, we were able to effectively
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Figure 2. Human MSCs Expand the Number of Functional CD14CD11b+CD33+ MDSCs in Allogeneic PBLs (A) Expansion of CD14CD11b+CD33+ cells from PBLs by MSCs. Allogeneic PBLs (40 donors) were cocultured alone (top panel) or with MSCs (lower panel; three donors of BM-MSCs and four donors of P-MSCs; representative dot plots/ histogram shown). PBLs were gated on forward (legend continued on next page)
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suppress the secretion of the molecule by MSCs, and this abrogated the expansion of MDSCs by MSCs (Figure 3F). Thus, our data support that HGF secreted by MSCs is involved in the expansion of MDSCs. HGF is a well-known and important mitogen for cancer cell growth (Mueller and Fusenig, 2004). Because MDSCs play a critical role in creating the immunoprivileged niche of tumors, we assessed whether cancer cell lines that secrete high levels of HGF could expand higher numbers of MDSCs. We found that, indeed, the level of HGF secreted by several cell lines correlated with the number of MDSCs expanded (Figures 4A and 4B). We then assessed the role of MSC-secreted HGF in the in vivo setting of the tumor microenvironment. When mice were inoculated with tumor cells admixed with MSCs silenced for HGF expression, there was a signicant reduction in tumor-associated Gr-1+CD11b+ MDSCs (Figure 4C). Moreover, in both wild-type C57BL/6 and BALB/c mice, we found that the liver, an organ that is known to secrete high levels of HGF, contained a signicantly higher proportion of Gr-1+CD11b+ MDSCs than the spleen (Figure 4D). Furthermore, when we inhibited the HGF/c-Met pathway in wildtype mice by injecting PHA-665752 (a second-generation c-Met inhibitor; Christensen et al., 2003), we observed a signicant decrease in hepatic, but not splenic, MDSCs, even after we excluded other myeloid cells, such as macrophages and Kupffer cells (resident hepatic macrophages), using the surface marker F4/80 (Kinoshita et al., 2010; Lee et al., 1986; Figures 4E and S3). Critically, the HGF/ c-Met pathway appears to be responsible for the high numbers and vast majority of hepatic Gr-1+CD11b+F4/ 80 MDSCs, since c-Met inhibition resulted in a signicant
loss of 70% of this population (Figure 4E), which was not observed for splenic MDSCs. These results are highly suggestive of a critical role for HGF in maintaining the high numbers of MDSCs in the liver. To further validate the in vivo relevance of our ndings, we injected recombinant HGF intravenously into wild-type C57BL/6 mice. After 3 days, we found a signicant increase of Gr-1+CD11b+ MDSCs in the bone marrow (where myeloid cells are most abundant; Gabrilovich et al., 2012) of these mice (Figure 4F), indicating that HGF can expand MDSCs in an in vivo setting. Collectively, these results indicate the relevance of HGF in tumor-associated MDSCs and in vivo settings. Expansion of MDSCs by HGF Is Mediated by the HGF Receptor c-Met and Increased Phosphorylation of STAT3 To investigate the mechanism behind HGF-mediated expansion of MDSCs, we checked for expression of c-Met, the cognate receptor for HGF (Cecchi et al., 2010; Trusolino et al., 2010), on MDSCs. We found that CD14 leukocytes and MDSCs constitutively expressed low levels of c-Met (Figures 5A and S4), and when c-Met on PBLs was blocked with neutralizing antibodies, the expansion of MDSCs by HGF was abrogated (Figure 5B). To investigate whether the effects of the HGF/c-Met axis were mediated through STAT3, we checked for HGF-induced STAT3 phosphorylation, which indicates activation of the pathway, in MDSCs. We found that exogenous addition of HGF induced an increase over baseline levels of phosphorylated STAT3 (pSTAT3), most consistently and signicantly at 1 hr after treatment (Figures 5C and S5). Moreover, when the
scatter (FSC)/side scatter (SSC) (R1, 100,000 events for all analyses), analyzed for CD14 and CD11b+ (R2), and further analyzed for CD33+ (R3). (B) Quantication of percentage (left-side charts) and cell number (right-side charts) of PBL CD11b+ (upper graphs) or CD14CD11b+CD33+ MDSCs (lower graphs) before and after coculture with MSCs. Averages SEM; 57 independent experiments; ***p < 0.001 compared with PBL only. (C) Suppressive function of MSC-induced MDSCs. Allogeneic PBLs (T, target cells; eight donors) were stained with CFSE for assessment of cell division and stimulated with anti-CD3/28 (a-CD3/28) without or with the addition of MSC-expanded MDSCs (from three donors) at various E:T ratios. Flow-cytometric analysis was performed to assess PBL cell proliferation/division as evidenced by decreasing CFSE staining. The chart on the right is a quantitative summary of experimental results; x axis: ratio of MSC-expanded MDSCs to activated PBL; y axis: percentage suppression of PBL proliferation by MSC-expanded MDSCs; eight independent experiments; *p < 0.05 for trend. E, effector cells. (D and E) Expression of iNOS (D) and ARG1 (E) by MSC-expanded MDSCs (ve donors of MSCs; representative histograms shown). Negatively selected CD14 cells from PBLs (eight donors) were cultured alone (top panels) or with MSCs (lower panels). CD14 cells were gated on FSC/ SSC (R1), analyzed for CD11b+ and CD33+ (R2), and further analyzed for either (D) iNOS or (E) ARG1 (shaded gray areas in histogram; unlled black line: isotype control). Average cell numbers SEM are indicated in the upper-right corner of the histogram; *p < 0.05 comparing CD14 + MSCs with CD14 only. (F) Expansion of CD4+CD25high Tregs by MSC-expanded MDSCs. CD14CD11b+CD33+ MSC-expanded MDSCs (three donors) were FACS sorted and cocultured with anti-CD3/28-stimulated allogeneic PBLs (three donors) at various ratios and assessed for induction of CD4+CD25high T cells. The chart on the right is a quantitative summary of the experimental results of three independent experiments. *p < 0.05 compared with anti-CD3/28-activated PBL (leftmost bar). See also Figure S1.
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STAT3 inhibitor cpd188 was added in the presence of exogenous HGF, the expansion of MDSCs was abrogated in a dose-dependent fashion (Figure 5D). Thus, HGF mediates the expansion of MDSCs by binding to its receptor, c-Met, which leads to increased phosphorylation of STAT3 (Fig. 6).
DISCUSSION
Recent reports have highlighted MDSCs as a prominent leukocyte subpopulation involved not only in tumor-associated immune suppression but also in regulation of the immune system at large (Almand et al., 2001; Gabrilovich guez and Ochoa, 2008). Previous and Nagaraj, 2009; Rodr data have shown that a number of proinammatory mediators are important inducers of these cells, but there has been no report regarding the involvement of tumor-associated mitogenic growth factors in the process. Our data link HGF secreted by MSCs to the expansion of MDSCs. Our ndings can help to explain the strong association of MDSCs with tumors, since it is well established that in the tumor microenvironment, HGF is highly secreted by both the cancer cells themselves and the supporting stromal cells, promoting cell survival and tumor growth (Cecchi et al., 2010; Mueller and Fusenig, 2004). While HGF has been implicated in immunoregulatory responses (Benkhoucha et al., 2010; Di Nicola et al., 2002), and the liver, which naturally secretes high levels of HGF, was reported to have some immunological functions (Sheth and Bankey, 2001), the specic molecular mechanisms underlying these observations have been largely unexplored and the reported effects have not been consistently replicated (Le Blanc et al., 2003). Our nding that high numbers
of hepatic MDSCs in mice can be signicantly altered by disruption of the HGF/c-Met pathway sheds some mechanistic light on this issue, and, overall, our data demonstrate that HGF mediates the expansion of functional MDSCs by engaging c-Met, its receptor, and increasing the phosphorylation of STAT3, one of its downstream molecules. The immunomodulatory properties of MSCs have been highlighted as being therapeutic for autoimmune diseases and other immune-related diseases such as graft-versushost disease (Abdi et al., 2008; Djouad et al., 2009; Keating, 2012; Le Blanc et al., 2008). As exciting as these ndings are, some researchers have noted that the same immunomodulatory effects of MSCs can allow for the growth of tumors (Djouad et al., 2003). However, whether MSCs denitively enhance or inhibit tumor growth and cancer progression is still unresolved, since differences in the experimental design, cancer histologic cell type, and MSC isolation technique used can all affect the experimental outcome (Klopp et al., 2011; Yen and Yen, 2008). Moreover, to date, studies on MSC and cancer interactions have largely focused on the homing of MSCs to tumors, rather than the immunological aspects of MSCs (Elzaouk et al., 2006; Studeny et al., 2002; Xin et al., 2007). Although a considerable amount of data support MSC expansion of Tregs (Uccelli et al., 2008), and these immunosuppressive T lymphocytes are also found in tumors, MDSCs appear to play a more crucial role in maintaining the profound immune suppression of the tumor niche (Almand et al., 2001; Ostrand-Rosenberg and Sinha, 2009; Sinha et al., 2005; Young and Wright, 1992). Our data suggest that through HGF and the consequent expansion of MDSCs, MSCs may play a role in maintaining tumor growth. While the association of MDSCs in a wide range of cancers has been known for some time, the mechanisms
Figure 3. MSC Expansion of MDSCs Is Mediated by Secreted HGF (A) MSC expansion of MDSCs is mediated by secreted factors. Allogeneic PBLs (ve donors) were cocultured with MSCs either in direct contact (MSC; three donors) or separated by transwell (TW) and assessed for expansion of CD14CD11b+CD33+ MDSCs; ve independent experiments; *p < 0.05 compared with PBL only; n.s., not signicant. (B) Highly secreted factors of MSCs (four donors) as assessed by quantitative cytokine array. LAP, latency-associated peptide; SDF, stromal-derived factor. (C and D) Exogenous addition of recombinant HGF to PBLs (ve donors) increases the (C) percentage of MDSCs (*p < 0.05 for trend) and (D) expression of iNOS (top panel) and ARG1 (lower panel) in HGF-expanded MDSCs (ve donors; representative charts shown) as assessed by using negatively selected CD14 cells that were rst gated on FSC/SSC (R1), analyzed for CD11b+ and CD33+ (R2), and then further analyzed for either iNOS (top histogram, shaded gray area) or ARG1 expression (bottom histogram, shaded gray area). Unlled black line: isotype control; p = 0.051 for comparison of average number of CD14CD11b+CD33+ cells (SEM) expressing iNOS (367 39 without HGF treatment compared with 409 46 with HGF treatment); p < 0.05 for ARG1 expression (312 53 without HGF treatment compared with 396 56 with HGF treatment). (E) Suppression of anti-CD3/28-stimulated PBL proliferation by HGF-expanded MDSCs as assessed by ow-cytometric analysis of CFSE staining for cell division (n = 3; a representative chart is shown). (F) Knockdown of HGF secretion in MSCs with HGF-specic siRNA abrogates the expansion of MDSCs. Allogeneic PBLs (seven donors) were cocultured with MSCs (three donors) transfected with either NT siRNA or siHGF and assessed for CD14CD11b+CD33+ cells. *p < 0.05 compared with MSCs only (leftmost bar) or PBLs only (rightmost bar); n.s., not signicant (NT siRNA compared with siHGF). See also Figure S2.
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Figure 4. Cancer Cell-Secreted HGF and In Vivo Manipulation of HGF Expression in Mice Signicantly Alter MDSC Numbers (A and B) Level of HGF secretion by MSCs, MG63 (osteosarcoma cell line), human embryonic stem cell-derived mesenchymal progenitors (EMPs), and JEG-3 (choriocarcinoma cell line) (A), and fold expansion of MDSCs (B) after coculture of the four cell types with PBLs (three donors; three independent experiments; *p < 0.05 for trend). (C) Knockdown of HGF expression in MSCs signicantly decreases tumor-associated Gr-1+CD11b+ cells. Tumor growth was induced in nude mice by inoculating human colon cancer cells admixed with MSCs transfected with either control NT siRNA (n = 5 mice) or siHGF (n = 5 mice). Tumorassociated leukocytes were assessed for Gr1+CD11b+ cells by ow-cytometric analysis (see Experimental Proceduresfor detailed description); *p < 0.05, NT siRNA compared with siHGF. (D) Gr-1+CD11b+ cells (%) in the spleen and liver of C57BL/6 (B6) and BALB/c mice (n = 3 mice for each group); *p < 0.05, % of cells in liver compared with spleen. (E) Treatment of C57BL/6 mice with the c-Met inhibitor PHA-665752 (c-Met inh) signicantly decreases hepatic but not splenic Gr-1+CD11b+F4/80 cells (shown as a percentage of Gr-1+CD11b+ cells; average SEM; n = 3 mice for each group). *p < 0.05 for c-Met inhibitor treatment versus no treatment (see Experimental Procedures for a detailed description). (F) Tail-vein injection of recombinant HGF (100 ng) into C57BL/6 mice and assessment of Gr-1+CD11b+ cells (fold change) in the peripheral blood, spleen, and bone marrow 1 day and 3 days after injection (n = 6 mice for each group); *p < 0.05 compared with day 0. See also Figure S3.
involved in expansion of these immunomodulatory cells are just beginning to be unraveled. IL-6 has been reported to mediate the expansion of MDSCs, and this cytokine is known to be secreted by both MSCs and stromal cells (Hung et al., 2007; Nemunaitis et al., 1989). However, in this work, we did not nd IL-6 to be involved. This may be due to the lower levels of this cytokine and the masking of its effects by the much higher levels of HGF in our system (see Figure 3B). Moreover, the strong association of MDSCs with cancer suggests that multiple and redundant pathways are likely involved (Ostrand-Rosenberg and Sinha,
2009; Gabrilovich and Nagaraj, 2009). There is consensus, however, that STAT3 may be the nal transcription factor involved in the expansion of MDSCs. Our nding that STAT3 activation is involved in HGF-mediated MDSC expansion further supports the importance of this molecule in inammation and cancer (Yu et al., 2009). In summary, we found that HGF secreted by MSCs can lead to the expansion of MDSCs. MDSCs expanded by coculture with MSCs or addition of HGF are functional, expressing iNOS and ARG1, as well as harboring suppressive function and inducing Tregs. Mechanistically, the
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Stem Cell Reports

Figure 6. Summary of MSC-Mediated Expansion of Functional MDSCs HGF secreted by MSCs binds to its cognate receptor, c-Met, expressed on CD14 PBLs. This leads to increased pSTAT3, a critical transcription factor in MDSC expansion. Both MSC- and HGF-expanded MDSCs are functional, expressing ARG1 and iNOS, and inhibit allogeneic lymphocyte proliferation while increasing the number of immunomodulatory Tregs.
MSC-mediated expansion of MDSCs occurs via the HGF/ c-Met axis, with the downstream involvement of STAT3. Our ndings not only further demonstrate the strong immunoregulatory nature of MSCs, but also show the involvement of a mitogenic, noninammatory molecule in the expansion of MDSCs.
Cell Culture
Human MSCs from bone marrow and placenta were cultured and expanded as we previously described (Chang et al., 2006; Yen et al., 2005). Human bone marrow MSCs were purchased from Cambrex and cultured according to manufacturers instructions. For human placental MSCs, term placentas (3840 weeks of gestation) from
healthy donor mothers were obtained with informed consent according to the procedures of the institutional review board. The cells were isolated as previously described (Yen et al., 2005). Differentiation studies were carried out as previously described (Chang et al., 2006; Liu et al., 2011; Yen et al., 2005). Human PBLs were isolated from the buffy coat of healthy donor blood samples (Taiwan Blood Services Foundation, Taipei Blood Center, Taipei, Taiwan) obtained with informed consent according to the procedures of the institutional review board, and cultured as previously described (Chang et al., 2006; Liu et al., 2011).
Immunophenotyping
Flow-cytometric analyses of cell surface markers were performed as previously described (Chang et al., 2006; Liu et al., 2011; Yen et al., 2005). All antibodies were purchased from BD Biosciences, except for CD33 and CD11b (BioLegend), ARG1 (R&D Systems), iNOS
Figure 5. Expansion of MDSCs by HGF Is Mediated via c-Met, Its Receptor, and Increased Phosphorylation of STAT3 (A) Expression of c-Met on MDSCs as assessed by ow-cytometric analysis. PBLs (six donors) were rst gated on FSC/SSC (R1), analyzed for CD11b+ and CD33+ (R2), and then further analyzed for c-Met expression (shaded gray area in histogram; unlled black line: isotype control; the ow-cytometric analysis diagrams shown are representative). Right chart: quantication of mean uorescent intensity (MFI); *p < 0.05 compared with isotype control (Ctrl). (B) Involvement of c-Met in HGF-mediated expansion of MDSCs. PBLs (six donors) were blocked with isotype control antibodies (IsoAb) or c-Met blocking antibodies (anti-c-Met), treated with recombinant HGF (20 ng/ml), and assessed for expansion of CD14CD11b+CD33+ cells. PBLs were rst gated on FSC/SSC (R1), analyzed for CD14 and CD11b+ (R2), and then further analyzed for CD33+ (R3). Average percentages and cell numbers SEM are indicated in dot plot diagrams (1.54% 0.20% and 1,544 201 cells for isotype control). Charts on the right are quantitative summaries of the percentages (top right) and cell numbers (bottom right) of six independent experiments; *p < 0.05 compared with PBL only or anti-c-Met. (C) Involvement of STAT3 in HGF-mediated expansion of MDSCs. Recombinant HGF (20 ng/ml) was added to negatively selected CD14 leukocytes (eight donors) that were stained for CD11b, CD33, and pSTAT3. Control cells (no HGF treatment; black unlled line in histogram) and HGF-treated cells (red unlled line) were collected for assessment by ow cytometry at the indicated time points (shaded gray area: isotype control); average cell numbers SEM for control (C) and HGF treatment (H) are indicated below the histograms. The chart on the right is a quantitative summary of eight independent experiments; *p < 0.05 and **p < 0.005. (D) STAT3 inhibition abrogates HGF-mediated expansion of MDSCs. Recombinant HGF (20 ng/ml) was added to PBLs (six donors) with and without addition of cpd188 (STAT3 inhibitor) at the indicated doses and assessed by ow cytometry for MDSCs, with PBLs rst gated on FSC/SSC (R1), analyzed for CD14 and CD11b+ (R2), and then further analyzed for CD33+ (R3). Average percentages and cell numbers SEM are indicated in dot plot diagrams (1.37% 0.22% and 717 108 cells for 5 mM cpd188). Charts on the right are quantitative summaries of the percentages (top right) and cell numbers (bottom right) of CD14CD11b+CD33+ cells for six independent experiments. a, p < 0.05 compared with PBL only; b, p < 0.05 for trend; n.s., not signicant for 10 mM cpd188 compared with PBL only. See also Figures S4 and S5.
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(Abcam), and c-Met (eBiosciences). All analyses were done on a BD FACSCalibur ow-cytometric system (BD Biosciences).
Expansion of MDSCs
PBLs were cocultured with MSCs or cancer cell lines (10:1 ratio) for 3 days and then assessed for CD14CD11b+CD33+ cells, which also served as the cell surface markers for uorescence-activated cell sorting (FACS) of MDSCs with the BD Aria Cell Sorter (BD Biosciences). Instead of PBLs, some experiments were performed with CD14 cells negatively selected with the use of magnetic beads (Miltenyi Biotec) according to the manufacturers instructions. Transwell studies were performed as previously described using 24-well transwell inserts (0.4 mm pores; BD Falcon) with MSCs cultured on the culture plates below and PBLs cultured in the inserts (Liu et al., 2011). Recombinant human HGF (R&D Systems) was added to PBLs at the indicated doses.
for Gr-1+CD11b+ cells by ow-cytometric analysis. For c-Met inhibition studies, wild-type C57BL/6 mice were intraperitoneally injected with vehicle (DMSO, n = 3) or PHA-665752 (Tocris Bioscience), a c-Met inhibitor (0.15 mg, n = 3) as previously described (Gordin et al., 2010). The mice were sacriced 5 days later and their livers and spleens were collected for ow-cytometric assessment of Gr-1+CD11b+ and Gr-1+CD11b+F4/80 cells.
Statistical Analysis
Statistical analysis was performed with SPSS 18.0 software (SPSS), with statistical signicance dened as p < 0.05. Values were expressed as the mean SEM. Students t test was used for comparisons between two groups, and ANOVA was used for comparisons of multiple groups. For further details regarding the materials and methods used in this work, see Supplemental Experimental Procedures.
MDSC Suppression Analysis

Assessment of PBL cell division was performed as previously described (Chang et al., 2006). Briey, allogeneic PBLs were labeled with 2.5 mmol/l of the uorescent dye carboxyuorescein succinimidyl ester (CFSE; Molecular Probes/GIBCO-Invitrogen) for 10 min and then stimulated with anti-CD3/CD28 beads (GIBCOInvitrogen). MSC-expanded MDSCs were FACS sorted for homogeneity and then cocultured with stimulated allogeneic PBL for 3 days at various effector-to-target (E:T) ratios. Flow-cytometric analysis was performed to assess for PBL cell division in terms of CFSE dye intensity.
Supplemental Information includes ve gures and Supplemental Experimental Procedures and can be found with this article online at http://dx.doi.org/10.1016/j.stemcr.2013.06.006.
ACKNOWLEDGMENTS
This work was supported in part by funding from the National Health Research Institutes (102A1-CSPP06-014 to B.L.Y.) and the National Science Council of Taiwan (NSC101-2321-B-400-009 to B.L.Y.). Received: December 20, 2012 Revised: June 21, 2013 Accepted: June 22, 2013 Published: July 25, 2013
Quantication of HGF Secretion

Supernatants were collected from cell cultures for detection of HGF with the use of a commercially available ELISA kit (R&D Systems) according to the manufacturers instructions.
REFERENCES RNAi Experiments

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All animal work was performed in accordance with protocols approved by the Animal Care and Use Committee of the National Health Research Institutes. Mice (48 weeks old) were obtained from the National Laboratory Animal Center of Taiwan (Taipei, Taiwan). Recombinant mouse HGF (100 ng; R&D Systems) was injected via the tail vein, and C57BL/6 mice were sacriced 3 days after injection. Peripheral blood, bone marrow, and spleen were collected for ow-cytometric analysis of Gr-1+CD11b+ cells. For in vivo tumor studies, nude mice (BALB/c nu/nu) were inoculated subcutaneously with tumor cells (human colon cancer cell line HCT-116; 1 3 106 cells) admixed with human MSCs (5 3 105 cells at a 1:2 ratio to cancer cells) transfected with either control siRNA (NT siRNA) or HGF-specic siRNA (siHGF). The mice were sacriced 3 weeks after inoculation, and tumors were excised and minced with scissors to obtain leukocytes, which were assessed
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Ar ticle LNGFR+THY-1+VCAM-1hi+ Cells Reveal Functionally Distinct Subpopulations in Mesenchymal Stem Cells
Yo Mabuchi,1,3 Satoru Morikawa,1,2 Seiko Harada,1 Kunimichi Niibe,1,2 Sadafumi Suzuki,1 Francois Renault-Mihara,1 Diarmaid D. Houlihan,4 Chihiro Akazawa,3 Hideyuki Okano,1 and Yumi Matsuzaki1,5,*
of Physiology of Dentistry and Oral Surgery Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan 3Department of Biochemistry and Biophysics, Graduate School of Health Care Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan 4Centre for Liver Research, NIHR Biomedical Research Unit, University of Birmingham, Birmingham B15 2TT, UK 5Institute of Medical Science, Tokyo Medical University, Shinjuku-ku, Tokyo 160-8402, Japan *Correspondence: ymatsuzak@gmail.com http://dx.doi.org/10.1016/j.stemcr.2013.06.001 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
2Department 1Department
SUMMARY
Human mesenchymal stem cells (hMSCs), which conventionally are isolated based on their adherence to plastic, are heterogeneous and have poor growth and differentiation, limiting our ability to investigate their intrinsic characteristics. We report an improved prospective clonal isolation technique and reveal that the combination of three cell-surface markers (LNGFR, THY-1, and VCAM-1) allows for the selection of highly enriched clonogenic cells (one out of three isolated cells). Clonal characterization of LNGFR+THY-1+ cells demonstrated cellular heterogeneity among the clones. Rapidly expanding clones (RECs) exhibited robust multilineage differentiation and self-renewal potency, whereas the other clones tended to acquire cellular senescence via P16INK4a and exhibited frequent genomic errors. Furthermore, RECs exhibited unique expression of VCAM-1 and higher cellular motility compared with the other clones. The combination marker LNGFR+THY-1+VCAM-1hi+ (LTV) can be used selectively to isolate the most potent and genetically stable MSCs.
INTRODUCTION
Mesenchymal stem/stromal cells (MSCs) are dened as nonhematopoietic, plastic-adherent, self-renewing cells that are capable of in vitro trilineage differentiation into fat, bone, and cartilage (Pittenger et al., 1999). Additional plasticity of MSCs has been suggested by experiments demonstrating their in vitro differentiation into myocytes, neuron-like cells, and hepatocytes (Drost et al., 2009; Galvin and Jones, 2002; Tao et al., 2009). Despite these data, the term MSCs has been controversial, as a denitive demonstration of their stemness by singlecell isolation and in vivo serial transplantation experiments has been lacking (Bianco et al., 2013). These multipotent cells are found in various fetal and adult human tissues, including bone marrow (BM), umbilical cord blood (UCB), liver, and term placenta (Battula et al., 2007; Erices et al., 2000; Yen et al., 2005; Zvaier et al., 2000). MSCs are multipotent and have low immunogenicity, and therefore are considered as potential candidates for a variety of clinical applications (Jung et al., 2012; Stappenbeck and Miyoshi, 2009), including cartilage reconstitution and the treatment of rheumatoid arthritis, acute osteochondral fractures, spinal disk injuries, and inherited diseases such as osteogenesis
imperfecta (Guillot et al., 2008). However, to date, these cells have been poorly characterized, which raises signicant concerns because human trials using MSCs are currently under way. MSCs can be retrospectively identied based on their ability to form colony-forming unit broblasts (CFU-Fs) in vitro (Friedenstein et al., 1974). Traditionally, the isolation of MSCs from unfractionated whole BM (WBM) has relied on their adherence to plastic dishes. This technique gives rise to heterogeneous cell populations that frequently are contaminated with osteoblasts and/or osteoprogenitor cells, fat cells, reticular cells, macrophages, endothelial cells, and hematopoietic cells (Pittenger et al., 1999). Prolonged culture is often required to remove these contaminants and obtain a reasonably pure population of MSCs. However, during this process, the differentiation, proliferation, and migration potency of the MSCs gradually diminishes as the cells acquire a more mature phenotype (Kim et al., 2009; Rombouts and Ploemacher, 2003). In an effort to overcome these problems, investigators have made an intense effort to identify reliable MSC surface markers that could facilitate the prospective isolation of colony-initiating cells. Various surface markers, including CD49a, CD73, CD105, CD106 (VCAM-1), CD140b, CD146, CD271
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Direct Isolation Method for Functional Human MSCs
Figure 1. Screening of Putative Surface Markers for Prospective Identication of hMSCs (A) Human bone fragments were washed with PBS (BM cells) or treated with collagenase (CR cells). (B) Expression ratio of the indicated surface markers in the CD45 and GPA cell populations. The data are shown as the ratio of the expression of a given marker on CR cells versus BM cells (mean SEM, n = 5; *p < 0.1). (C) Colony-forming assay of a single marker population (2,000 cells) in CR cells at 14 days (mean SEM, n = 6 per group; *p < 0.05). (D) Assay measuring the negative linear relationship between the numbers of seeded cells (BM, CR, and CD45GPA CR cells) and sorted cells, using a surface marker in CR cells. (E) Clonogenic assay of single cells seeded into 96-well plates and cultured for 14 days (BM-MNC POIETICS were used in this assay; mean SEM, n = 3; *p < 0.05). See also Figure S1.
(LNGFR), MSCA-1, and STRO-1, have been used alone or in combination to isolate human MSCs (hMSCs) (Aslan et al., hring et al., 2006; Battula et al., 2009; Boiret et al., 2005; Bu 2007; Gronthos et al., 2003; Quirici et al., 2002; Sacchetti et al., 2007). CD49a, CD73, CD140b, and CD146 are widely expressed in stromal cells (e.g., pericytes and reticular cells) and thus are not unique to MSCs. STRO-1 is a popular MSC marker and is often used in combination with VCAM-1 for MSC isolation. However, these markers are also found on some hematopoietic cells, and additional markers, including CD45 and Glycophorin A (GPA), are required to exclude contaminating cells (Gronthos et al., 2003; Simmons and Torok-Storb, 1991). Therefore, the identication of a combination of cell surface markers specic to hMSCs has remained an important prerequisite for the repeated isolation of puried multipotent MSC fractions.
In the present study, we performed a comprehensive screening of putative surface markers to select the most useful ones for prospectively identifying a pure MSC population in human BM. We describe a signicantly improved method that enables the simple and reliable prospective isolation of MSCs based on their expression of LNGFR, THY-1, and VCAM-1.
RESULTS
Identication of MSC Markers We isolated fresh human BM cells using either the traditional method of ushing the BM or collagenase digestion of crushed bone (collagenase-released [CR] cells), as previously described for a murine MSC isolation procedure (Houlihan et al., 2012; Morikawa et al., 2009; Figure 1A).
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Figure 2. The LNGFR+THY-1+ Population Is Signicantly Enriched for CFU-Fs with Potent Differentiation Potential (A) Representative ow-cytometric proles of human BM stained for LNGFR and THY-1. (B) Numbers of CFU-Fs detected 14 days after plating 5,000 cells from each of the following groups: LNGFR+THY-1+, +/, /+, and / cells (mean SEM, n = 12 per group; **p < 0.01). (C) Assay of the negative linear relationship between the numbers of seeded LNGFR+THY-1+ and LNGFR+THY-1 cells. (D) Phase-contrast micrographs of a colony of LNGFR+THY-1+ cells (phase), showing the potential of a LNGFR+THY-1+ colony to differentiate into osteoblasts, chondrocytes, and adipocytes. Scale bar = 100 mm. (E) Phase-contrast micrographs of LNGFR+THY-1+ and LNGFR+THY-1 colonies (passage 1). Arrows point to cells with larger amounts of cytoplasm. Scale bar = 100 mm. (F) Secondary CFU-Fs assays were performed with single cells sorted from LNGFR+THY-1+ and LNGFR+THY-1 colonies after one passage (mean SEM, n = 3; **p < 0.01). (legend continued on next page)
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We initially examined the CFU-F potential of these two cell types by plating 103, 104, or 105 cells, and counting the wells with formed colonies. After a 2-week culture period, we found that the CFU-F frequency was far greater with CR cells than with BM cells using the standard isolation method (BM: 3.3% 3.3%, 7.4% 7.4%, 0% 0%; CR: 24.8% 5.2%, 35.6% 15.6%, 77.8% 22.2%; Figure S1A available online). Based on these ndings, we concluded that collagenase treatment and depletion of hematopoietic cells increases the frequency of CFU-F formation. This prompted us to search for putative cellsurface markers specic to this potent subpopulation. To that end, we used ow cytometry to screen >100 potential markers on both BM and CR cells. We specically looked for rare (<0.1%) surface markers whose frequency in the nonhematopoietic fraction increased following collagenase treatment. The frequencies of cells expressing some known MSC markers increased among CR cells, and in particular, cells expressing LNGFR and THY-1 increased signicantly in the CR fraction compared with ushed BM (CR/BM ratio: LNGFR: 8.75 2.3; THY-1: 6.70 1.3; Figure 1B). To assess the purication efciency of these potential markers (THY-1 and LNGFR) and compare them with previously validated MSC markers (MSCA-1, STRO-1, VCAM-1, CD73, CD105, and CD146), we performed CFU-F assays using CR cells based on their expression of each marker. The THY-1+ cells showed the greatest CFU-F frequency among the fractions tested (Figure 1C). A limiting dilution assay measuring the relationship between seeding density and colony-forming efciency also indicated that THY-1+ cells had the highest clonogenic potential (1 CFU-F per 150 cells seeded; Figure 1D). To determine whether a particular combination of surface markers could select for a more potent population of hMSCs, we examined cells that were positive for each selected marker (LNGFR, MSCA-1, STRO-1, VCAM-1, CD73, or CD105), combined with THY-1 expression (Figure 1E). Additionally, previously reported MSC marker combinations, including LNGFR+CD140a+ and LNGFR+CD146+, were tested (Figure S1B). Single-cell assays demonstrated that LNGFR+THY-1+ cells had the highest CFU-F potential (Figures 1E and S1B). Collectively, the LNGFR+THY-1+ cells demonstrated a CFU-F frequency that was 200,000 times higher than observed for unfractionated BM cells (LNGFR+THY-1+ cells versus BM cells: 1 per 56 cells seeded versus 1 per 1.2 3 106 cells seeded; Fig-
ures 1D and 1E). In addition, ow-cytometric analysis showed that the LNGFR+THY-1+ population contained the smallest percentage of hematopoietic cells (CD45+: 1.01%) and red blood cells (GPA+: 0.85%) among the combinations tested (Figure S1C). We conrmed by immunocytochemistry that the LNGFR+THY-1+ population was not contaminated by endothelial cells (CD31-staining negative) or osteogenic cells (osteocalcin-staining negative; Figure S1D). Therefore, two-color immunostaining with THY-1 and LNGFR is a reliable method for isolating MSCs from BM with minimal contamination from hematopoietic cells. hMSCs Exist Only in the LNGFR+THY-1+ Population Two-color staining for LNGFR and THY-1 of WBM revealed the presence of four distinct subpopulations (LNGFR+THY-1+, +/, /+, and /; Figure 2A). We isolated cells from each subpopulation and performed several in vitro assays to determine their characteristics. The traditional CFU-F and limiting dilution assays indicated that the number of colony-forming cells arising from each subpopulation was highest in the LNGFR+THY-1+ subpopulation, with a frequency of one in six (Figures 2B and 2C). When the in vitro mesenchymal lineage differentiation potential was investigated, CFU-Fs from LNGFR+THY-1+ cells robustly differentiated into adipocytes, chondrocytes, and osteoblasts (Figure 2D). In contrast, cells in the other subpopulations had extremely low CFU-Fs (1/6,300 in LNGFR+THY-1) or no CFU-Fs (LNGFRTHY-1 and LNGFRTHY-1+; Figures 2B and 2C). The few colonies that formed from LNGFR+THY-1 cells appeared to have nonbroblastic shapes (Figure 2E, arrows) and rarely formed secondary CFU-Fs when reseeded, although 60% of CFU-Fs from LNGFR+THY-1+ cells produced secondary colonies under the same conditions (Figure 2F). These results suggested that CFU-Fs in the LNGFR+THY-1 population were not hMSCs. Multipotent and self-renewing MSCs were only present in the LNGFR+THY-1+ fraction. Multicolor ow-cytometric analysis indicated that naive LNGFR+THY-1+ cells uniformly expressed CD49a, CD49d, CD73, CD140b, CD146, STRO-1 VCAM-1, and MSCA-1 (Figure 2G). In contrast, almost all of the cells were negative for hematopoietic lineage markers (CD3, CD14, CD16, CD19, CD20, CD45, CD56, and GPA), an embryonic stem cell marker (SSEA-1), and an endothelial/hematopoietic
(G) Flow-cytometric analysis of surface markers on LNGFR+THY-1+ cells, showing the percentage of cells that express the antigen (red line) versus a matched isotype control (gray). Lineage cocktail: CD3, CD14, CD16, CD19, CD20, and CD56. (H) Phase-contrast image of cultures of LNGFR+THY-1+ and LNGFRTHY-1+ cells after 14 days of culture in MethoCult medium (CFU-C assay). Scale bar = 100 mm. (I) Total numbers of CFU-Cs counted on day 14 (mean SEM, n = 3; **p < 0.01). BM-MNC POIETICS were used for all experiments in this gure. See also Figures S2 and S3.
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stem cell (HSC) marker (CD34; Figure 2G). To conrm that the LNGFR+THY-1+ population was devoid of hematopoietic progenitor cells, we performed a colonyforming units in culture (CFU-C) assay to detect hematopoietic progenitor cells. The results showed that LNGFR+THY-1+ cells produced no CFU-Cs (Figure 2H). However, LNGFRTHY-1+ cells, which exhibited no CFU-F potential, formed >40 CFU-Cs per 5,000 cells seeded under the same culture conditions (Figure 2I). These data strongly suggested that LNGFR and THY-1 could be used for the prospective identication of a rare and potent population of hMSCs. We looked for the presence of LNGFR+THY-1+ cells in a variety of other human tissues that contain MSC-like cells, including placenta (amnion, chorion, and decidua), fat, peripheral blood (PB, with or without G-CSF mobilization), and UCB (Lee et al., 2004; Nagase et al., 2009; Zimmerlin et al., 2010; Zvaier et al., 2000). LNGFR+THY-1+ cells were detected in the decidua but not the amnion or chorion of the placenta (Figure S2A). Decidual LNGFR+THY-1+ cells exhibited a high CFU-F potential equivalent to that of BM-derived LNGFR+THY-1+ cells (Figure S2B). Interestingly, both the LNGFR+THY-1 and LNGFRTHY-1 fractions, derived from adipose tissue, generated CFU-Fs, albeit less frequently than LNGFR+ THY-1+ cells (Figures S2C and S2D). We were unable to detect LNGFR+THY-1+ cells among 106 PB or UCB cells; however, a small number of LNGFR+THY-1+ cells that had clonogenic potential were present in G-CSF-mobilized PB (G-CSF mPB; Figures S2E and S2F). In a previous study (Morikawa et al., 2009), we reported that, after undergoing ex vivo expansion, intravenously injected MSCs were trapped largely in the lung and did not migrate to the BM compartment. To evaluate homing of cultured and naive LNGFR+THY-1+ cells to bone in vivo, we transplanted the cells into immunodecient mice (Figure S3A). Mice injected with 2 3 105 to 1 nbsp;3 106 LNGFR+THY-1+ cells expanded in culture, died from pulmonary embolism within a week, and no human cells could be identied in the recipients BM. In contrast, 3 months after the transplantation of 1 3 104 freshly isolated LNGFR+THY-1+ cells, we were able to collect human-derived cells from the recipients BM using speciesspecic antibodies. We performed immunohistochemistry after LNGFR+THY-1+ cell transplantation and observed the transplant human cells in mouse BM. Human THY-1positive cells were detected in lung, skin, and subcutaneous tissue; however, expression of a-SMA and alkaline phosphatase (ALP) was not detected (Figure S3B). To test whether these surviving human cells retained their stem cell properties, we sorted human THY-1+ cells and performed several in vitro assays (Figure S3C). Following expansion in vitro, the sorted human THY-1+ cells were
capable of colony formation and multilineage differentiation into bone, cartilage, and fat cells (Figure S3D). These data demonstrated that freshly isolated human LNGFR+ THY-1+ cells could home to the BM and retain their proliferative and multilineage differentiation potential in murine recipients, although the engraftment efciency was low. Dening Subpopulations of CFU-Fs Using Single-Cell Assays To further dene the differentiation and proliferation potential of LNGFR+THY-1+ cells, we monitored colony formation after single-cell seeding into a 96-well culture dish (Figure 3A). Interestingly, analysis of their growth kinetics revealed that single LNGFR+THY-1+-derived CFUFs could be divided into three distinct subgroups: rapidly expanding MSC clones (RECs: 16.4% 2.0%) that yielded >105 cells by day 14, moderately expanding MSC clones (MECs: 47.2% 2.9%) that took up to 21 days to yield 105 cells, and slowly expanding MSC clones that failed to generate 105 cells after 1 month of culture (SECs: 36.3% 2.9%; Figure 3B). The subgroups had different cellular morphologies. RECs were uniformly small and spindle-shaped, whereas MECs and SECs were polymorphic and contained cells with larger nuclei and enlarged cytoplasm (Figure 3C, arrows in phase images). The heterogeneity among these groups is also reected in the different cell size of RECs compared with MECs and SECs (Figure 3C). When the adipogenic and osteogenic differentiation efciencies of each subgroup were compared, there were clearly more adipogenic cells in RECs than in MECs or SECs (REC: 99.3% 0.33%; MEC: 58.1% 6.26%; SEC: 22.2% 1.87%; Figures 3D and 3E). In addition, RECs exhibited the greatest secondary colony-forming efciency (RECs: 33.3% 9.6%; MECs: 8.9% 3.5%; SECs: 1.0% 0.58%) after the single-cell sorting of each subgroup (Figure 3F). We established individual MSC clones derived from single LNGFR+THY-1+ cells (n = 145; Table S1). All RECs were multipotent and capable of differentiating into either all three lineages (adipogenic, chondrogenic, and osteogenic [37/46: 80%]) or bilineage cells (9/46: 20%). Although some MECs/SECs were unable to differentiate (6/48: 13% and 7/51: 14%, respectively), >60% of MECs/SECs showed either tri- or bilineage differentiation potential (38/48: 79% of MECs and 32/51: 62% of SECs), indicating that most of the CFU-Fs derived from LNGFR+THY-1+ cells were multipotent. These data suggested that there is a functional hierarchy among CFU-Fs derived from clonal LNGFR+THY-1+ cells, with RECs exhibiting the most potent stem-like characteristics. Cultured MSCs Undergo Cellular Senescence via P16INK4a We observed a greater increase in senescence-associated beta-galactosidase (SA-b-gal)-positive cells in MECs and
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Figure 3. Clonal Assay of LNGFR+THY-1+ Cells (A) Single-cell assay in a LNGFR+THY-1+ population. Representative image of a typical 96-well culture plate (day 21 after initial seeding) shows colony-forming wells. (B) Single-cell sorting and in vitro expansion reveals three distinct MSC subpopulations of rapidly, moderately, or slowly expanding MSC clones (RECs, MECs, and SECs, respectively). Growth curves of representative RECs, MECs, and SECs, and the percentage of each colony type present are shown. (C) Phase-contrast micrograph showing the cellular morphologies of RECs, MECs, and SECs. The arrows point to larger MECs and SECs with larger nuclei. Scale bar = 100 mm. (D) Differentiation of each colony type into adipocytes and osteoblasts. Scale bar = 100 mm. (E) Quantitative analysis of the differentiation capacity of each colony type (differentiation cells / nuclei; mean SEM, n = 6; *p < 0.05). (F) Average number of secondary CFU-Fs in wells for each cell type at 6 weeks (mean SEM, n = 3; *p < 0.05). BM-MNC POIETICS were used for all experiments in this gure. See also Table S1.
SECs compared with RECs, demonstrating that MEC/SEC cells undergo cellular senescence (Figures 4A and 4B). To assess the genomic stability of RECs, MECs, and SECs, we performed array-based comparative genomic hybridization (aCGH) to investigate aneuploidy and genomic abnormalities (Ben-David et al., 2011; Spits et al., 2008). We found that MECs and SECs accumulated nonoverlapping copynumber variations (CNVs), indicating de novo genomic DNA abnormalities, although no errors were identied in any of the RECs studied (Table S2). Since DNA damage induces premature senescence via cell-cycle regulatory proteins in many cell types (Galderisi et al., 2009), we
measured the gene expression of the tumor suppressor P14ARF and the cdk inhibitors P16INK4a and P21 in each group. A signicant increase in P16INK4a expression was observed in MECs and SECs compared with RECs, in contrast to P21 and P14ARF expression (Figure 4C). Therefore, cellular senescence in MECs and SECs might be triggered by de novo genomic DNA abnormalities and increased P16INK4a expression. Notably, a dose-response curve plotting the CFU-F number against the number of LNGFR+THY-1+ cells plated per well did not show a linear relationship. Instead, we obtained fewer than expected CFU-Fs from the higher cell
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Figure 4. Heterogeneity in the MSC Compartment (A) SA-b-gal assay of RECs, MECs, and SECs cultured in a glass chamber (at 6 weeks). Scale bar = 50 mm. (B) Quantitative analysis of SA-b-gal+ cells in RECs, MECs, and SECs (mean SEM, n = 3 per group; **p < 0.01). (C) Relative expression of P14ARF, P16INK4a, and P21 in 6-week cultured cells by real-time RT-PCR (mean SEM, n = 3; *p < 0.1, **p < 0.01; n.s., not signicant). See also Figure S4 and Table S2. concentrations (Figure S4A). In addition, we observed different growth kinetics with single REC, LNGFR+THY-1+ cells (1,000 cells), and WBM cells (1 3 106). Specically, single RECs continued to grow without senescence, generating >1012 cells after 10 weeks, whereas LNGFR+THY-1+ cells stopped proliferating after 6 weeks and yielded significantly fewer cells (1010 cells; Figure S4B). Logically, 1,000 LNGFR+THY-1+ cells should contain 170 CFU-Fs, with 30 comprised of RECs, 90 comprised of MECs, and 50 comprised of SECs. However, the total number of LNGFR+THY-1+ growth cells was much smaller than we anticipated. To analyze this phenomenon in detail and exclude the possibility of cellular overcrowding, we performed a coculture assay using RECs and SECs at low cell numbers (Figure S4C). Following 4 weeks in coculture, REC proliferation was markedly reduced in the presence of SECs (Figures S4D and S4E). These results suggest that the SEC-mediated inhibition of REC proliferation is independent of cell density. Therefore, the direct isolation of RECs should be the most effective way to obtain highly pure MSCs from mixed CFU-Fs. VCAM-1 and CD49d Are Specically Expressed in RECs Isolation of RECs is difcult because it requires single-cell sorting of LNGFR+THY-1+ cells followed by a minimum of 2 weeks in culture. Given the characteristics of RECs, which make them attractive for therapeutic applications, we attempted to identify REC-specic markers. Index sorting enabled us to identify cells in the LNGFR+THY-1+ fraction that gave rise to RECs. There was a higher concentration of RECs in the LNGFR and THY-1 bright fraction (66.6% in RECs); however, MECs and SECs were also present in the same region (51.4% in MECs, 45.0% in SECs; Figures S5AS5C). Similarly, measurements of cell size (forward scatter) and complexity (side scatter) failed to distinguish among the groups (Figure S5D). The differential expression of various cell-surface molecules was explored further in each of the three cell types (Figures 5A and S5E). The observation of moderate VCAM-1 and CD49d (Integrin a4) expression on RECs, in contrast to their absence on MECs and SECs, was particularly noteworthy (Figure 5A). VCAM-1 and Ki67 immunohistological staining indicated a higher proliferation in RECs (19%) compared with MECs or SECs (<5%; Figures 5B and 5C). Quantitative PCR analysis conrmed a signicantly higher expression of VCAM-1 in RECs compared with MECs and SECs (Figure 5D). Cell Motility Is Mediated via VCAM-1 in RECs VCAM-1 is known to bind to integrin a4b1 (VLA-4: CD49d/CD29), and this interaction is crucial for both rolling and adhesion of several cell types on the vascular endothelium prior to transendothelial trafcking
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Figure 5. VCAM-1 Identies Rapidly Dividing MSCs (A) Expression of the indicated surface markers in RECs, MECs, and SECs (6 weeks in culture). Shown is the percentage of cells that express the antigen (line) versus a matched isotype control (gray). (B) Immunocytochemistry of MSC subpopulation with VCAM-1 (green), Ki67 (red), and nuclei (blue). Scale bar = 100 mm. (C) Quantitative analysis of Ki67+ cells in RECs, MECs, and SECs (mean SEM, n = 3 per group; **p < 0.01). (D) Relative expression of VCAM-1 marker in cultured cells (6 weeks) by real-time RT-PCR (mean SEM, n = 3; *p < 0.05, **p < 0.01). See also Figure S5. (Papayannopoulou et al., 1998). A transwell migration assay revealed that RECs exhibited signicantly greater migration ability than MECs or SECs; however, the motility of the RECs was abrogated in the presence of function-blocking antibodies directed against VCAM-1 or CD49d (Figures 6A and 6B). Microlaments anchor to the cell membrane and are involved in cytoskeleton organization to produce effective cell motility (Gumbiner, 1996). F-actin is a major component of the microlaments that are formed by polymerization of monomeric actin (G-actin). The formation of F-actin is balanced by an equilibrium reaction of G-actin incorporation and dissociation; typically, however, nonmotile cells accumulate large bundles of microlaments known as stress bers (Schratt et al., 2002). We therefore analyzed stress-ber formation among the hMSC subgroups. Large tubular components (F-actin+) were detected in MECs and SECs, but not in RECs (Figure 6C). After cell culture with blocking antibody (anti-VCAM-1 or anti-CD49d), REC morphology was changed and stress-ber components were increased (Figures 6C and 6D). Therefore, the enhanced migration capabilities of RECs may be associated with expression of VCAM-1 and CD49d. The formation of stress bers is often accompanied by morphological changes of cells, typically characterized by enlargement of the cytoplasm (Wada et al., 2011). A comparison of cell widths (minor axis) clearly revealed that one REC cell was smaller than the others (Figure 6E). Notably, the cell width of RECs increased in response to VCAM-1 or CD49 blockade (Figure 6E). A recent report suggested that articial overexpression of LLP2A (ligand of CD49d) on hMSCs increases their migration to murine BM (Guan et al., 2012). Interestingly, VCAM-1 is also an equally effective ligand for CD49d. Following systemic infusion of RECs into murine recipients, there was no evidence of focal lung trapping,
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Figure 6. Expression of VCAM-1 and CD49d Correlated with the Migration Ability of MSCs (A) Migration assay in vitro. A total of 1.5 3 104 cells (RECs, MECs, and SECs) were resuspended in DMEM containing 1% FBS and 10 mg/ml aphidicolin, and allowed to migrate toward the culture medium supplemented with 20% FBS in the lower chamber. (B) Migration assay of RECs, MECs, and SECs with or without blocking antibody (Normal indicates no antibody treatment). The cells were incubated with each antibody (isotype control [mouse IgG1], anti-VCAM-1 [51-10C9], anti-CD49d [9F10], each at 10 mg/ml) for 30 min prior to assay (mean SEM, n = 3 per group; *p < 0.05, **p < 0.01). (C) The distribution of the indicated F-actin (green) and a-tubulin (red) was monitored by immunouorescence and microscopy (after 4 days). The small box represents a high-resolution image. Scale bar = 100 mm. (D) Quantitative analysis of F-actin+ cells (mean SEM, n = 3 per group; **p < 0.01). (E) Quantitative analysis of cell width (n = 30) in RECs, MECs, and SECs (**p < 0.01). (F) Imaging of transplanted cultured MSCs in vivo. A Luciferase image of cell migration in the recipient animals at 1 day postinjection of 1 3 105 cultured cells (infected with Venus-ffLuc lentivirus) is shown. (G) Quantitation of luciferase activity in recipient animals. Luminescent intensity was signicantly increased after injection of the cultured MSCs and MECs/SECs in lung regions (mean SEM, n = 3 per group; **p < 0.01).
conrming enhanced motility. In contrast, MEC/SECs were largely sequestered in the pulmonary vasculature (Figures 6F and 6G). These observations led us to speculate that VCAM-1 expression may be essential to maintain the RECs morphology and thus their in vitro and in vivo motility.
Further Purication of Functional MSCs Using VCAM-1 as a specic marker, we attempted to isolate RECs from cultured MSCs or directly from fresh BM cells. Based on ow cytometry, freshly isolated LNGFR+THY-1+ cells uniformly expressed VCAM-1 (Figure 2G), but this gradually decreased with time during ex vivo expansion
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Figure 7. Isolation of Functional MSCs in Culture and Fresh BM Cells (A) Culture-dependent reduction in the frequency of VCAM-1+ cells in the LNGFR+THY-1+ cell population. (B) Colony-forming potential of cultured VCAM-1+ or VCAM-1 cells derived from the LNGFR+THY-1+ cell population after 4 weeks and 8 weeks (mean SEM, n = 5; **p < 0.01). (C) Direct isolation of RECs from fresh BM cells. (D) Colony-forming potential of lowpositive or high-positive VCAM-1 cells in the LNGFR+THY-1+ cell population (mean SEM, n = 3; *p < 0.05).
(Figure 7A). We then resorted VCAM-1+ and VCAM-1 cells and compared the CFU-Fs of each cultured fraction. We found that VCAM-1+ cells had a far greater clonogenicity compared with VCAM-1 cells (Figure 7B). We then examined the utility of VCAM-1 for isolating RECs directly from fresh WBM (Figure 7C). After single-cell sorting into 96well plates, LNGFR+THY-1+VCAM-1hi+ (LTV) cells showed 2-fold higher CFU-Fs compared with LNGFR+THY-1+ (Figure 7D). Surprisingly, 87% of LTV CFU-Fs were RECs, whereas LNGFR+THY-1+- and LNGFR+THY-1+VCAM-1lo+derived CFU-Fs contained far less RECs (16.4% and 10.2%, respectively; Figure 7D). Therefore, VCAM-1 can be used as a marker for enriching multipotent/proliferative cells (RECs) from both culture-expanded MSCs and fresh BM cells.
DISCUSSION
Despite the widespread clinical use of hMSCs, there are fundamental gaps in our knowledge of basic MSC biology. In contrast to HSCs, the molecular mechanisms that maintain MSCs in their undifferentiated state or determine their differentiation pathways are still poorly understood. Due to the limitations of the methods used to isolate MSCs, it has not been possible to obtain a sufciently pure MSC population to address these questions. In the current study, we have demonstrated that LNGFR+THY-1+ cells in human BM constitute an extremely pure MSC population. The clonogenic potential of single-seeded LNGFR+THY-1+ MSCs, cultured in standard medium (Dulbeccos modied Eagles medium
[DMEM] + 20% fetal bovine serum [FBS]) exceeds that of other reported isolation methods (STRO-1+VCAM-1+ [Gronthos et al., 2003], MSCA-1+CD56 [Battula et al., hring et al., 2007], and 2009], LNGFR+CD140b+ [Bu LNGFR+CD146+ [Tormin et al., 2011]). Murine HSCs can be isolated based on CD34, c-Kit+, Sca-1+, and Lineage expression, which yields one HSC per ve cells (Matsuzaki et al., 2004). Our isolation method for hMSCs achieves a level of purity similar to that of mouse HSCs. Moreover, because LNGFR+THY-1+ cells contain the least number of hematopoietic cells, this combination enables the convenient isolation of MSCs based on the surface expression of only two antigens. This simple and easy isolation method should facilitate further studies and improve our basic understanding of the primary characteristics of MSCs. Using highly enriched primary MSCs, one can perform clonal experiments easily and efciently. Several groups have reported that MSCs are heterogeneous with respect to their morphological appearance (Kucia et al., 2007a, 2007b; Pittenger et al., 1999; Prockop et al., 2001). Our ndings conrm that CFU-Fs derived from a single LNGFR+THY-1+ cell can be divided into three functionally distinct subpopulations termed RECs, MECs, and SECs. RECs exhibited robust multilineage differentiation and self-renewal potency. We therefore consider RECs to be the most primitive and undifferentiated population in CFU-Fs. In a previous report, upregulation of p16Ink4a and p19Arf by loss of the transcriptional repressor Bmi-1 led to defective self-renewal of adult HSCs and neural stem cells, but was less critical for generating the differentiated progeny of these cell types (Park et al., 2003; Molofsky
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et al., 2003). In our results, MECs and SECs had increased expression of the two commonly known biomarkers for cellular senescence, SA-b-gal and P16INK4a. In contrast, P14ARF, which is generated by alternative splicing from the INK4a locus and P21, was not affected. We therefore speculate that the self-renewal capacity of cultured MSCs is mediated by an unknown mechanism rather than by BMI-1 regulation. VCAM-1 encodes a leukocyte adhesion molecule whose expression is restricted to endothelial cells and subpopulations of BM cells (Osborn et al., 1989). VCAM-1 binds to the integrin a4b1 and integrin a4b7 on circulating monocytes, granulocytes, and lymphocytes (Elices et al., 1990; Osborn et al., 1989). Increased expression of VCAM-1 has been reported in various cancer cell types, including breast, gastric, renal carcinoma, and melanomas (Ding et al., 2003; Minn et al., 2005). It has been suggested that the VCAM-1-CD49d interaction promotes cancer cell survival and migration (Klemke et al., 2007). In this study, we demonstrated that RECs have higher expression of VCAM-1 compared with MECs or SECs, and blockade of the VCAM-1-CD49d interaction signicantly diminished transwell migration and increased stress-ber formation. The functional difference between the groups relates to the organization of the F-actin cytoskeleton, since the rigid cytoskeletal structure inhibits their ability to transmigrate across the vascular endothelium (Morales-Ruiz et al., 2000). Therefore, we speculate that RECs interact with each other through VCAM-1-CD49d, thereby maintaining their primitive state, including self-renewal, multilineage potential, and migratory capacity. In this study we have described methods for the selective isolation of primitive hMSCs that may prove advantageous both experimentally and therapeutically. The identication of the combination marker LTV makes it possible to prospectively isolate functional hMSCs directly from BM. Using these cells, we may be able to tease out the complex molecular mechanisms that govern the undifferentiated state of hMSCs. Additionally, these markers may allow visualization of hMSCs in vivo, thereby helping to uncover their physiological and pathophysiological roles. The use of VCAM-1 as an REC-selective marker facilitates the isolation of enriched MSCs from a heterogeneous population of cultured MSCs that represents mixed CFU-Fs. Since we demonstrated that the coexistence of SECs alters the selfrenewal and differentiation potency of RECs in culture, the isolation of VCAM-1+ cells from cultured hMSCs may prove useful therapeutically. In conclusion, our data should help to unravel the fundamental cellular and molecular biology of MSCs. This information is critical if MSCs are to be used effectively and safely in regenerative therapy.
Tissue Collection
Heads of femurs were collected from patients (2280 years old) who had undergone hip replacement arthroplasty at Keio University Hospital, Tokyo. In total, 23 biological samples were obtained and used for the experiments described herein. Placenta, fat, PB, and UCB samples were also collected and stored in accordance with Keio University protocols and guidelines. Approval for the study was obtained from the Research Ethics Committee of the Keio University School of Medicine (No. 18-26), and patient recruitment was undertaken with written informed consent.
Cell Preparation
The heads of femurs were dissected and crushed with a pestle, after which the crushed bones were washed gently once in PBS to collect BM cells. The bone fragments were incubated for 2 hr at 37 C in DMEM (Invitrogen) in the presence of 0.2% collagenase (cell dissociation grade; Wako Chemicals) and 25 mg/ml deoxyribonuclease I (Sigma-Aldrich) to yield a suspension of CR cells. The CR and BM cells were then used in the initial experiments to dene prospective markers for the identication of MSCs. Placenta and fat-tissue experiments were also used for BM isolation protocols as described in the Supplemental Experimental Procedures.
Antibody Staining and Flow Cytometry

Cells were suspended in ice-cold Hanks balanced salt solution at 15 3 107 cells/ml and then stained for 30 min on ice with a monoclonal antibody as described in the Supplemental Experimental Procedures. The antibodies used were LNGFR-PE (Miltenyi Biotec), THY-1-FITC, and VCAM-1-APC (BD PharMingen, Biolegend). Flow-cytometric analysis and sorting were performed on a triplelaser MoFlo (Beckman Coulter), FACSVantage SE, or FACSCalibur ow cytometer (Becton Dickinson), and the data were analyzed using Flowjo software (Tree Star). Analysis of cell populations expressing varying combinations of LNGFR and THY-1 on their surface from the different tissues routinely demonstrated 99% purity by ow cytometry.
Limiting Dilution Assays

The sorted cells were seeded at different concentrations into 96well plates. After 14 days, wells with no colonies were included in the count for each population. The concentration of plated cells that resulted in 37% of the wells being negative for colony growth was taken as the cell concentration that yielded one CFU-F/well (Bacon and Sytkowski, 1987). Populations of sorted cells that were >99% pure were routinely prepared.
Single-Cell Sorting and Secondary CFU-F Assays

Flow-cytometric analyses and the collection of single cells for CFU-F assays were carried out using a FACSVantage SE cytometer. The cells were sorted directly into separate wells of a 96well plate containing 200 ml of medium using a CloneCyt automated cell deposition unit. Calibration of the clone sorting using uorescent beads showed that <1% of the wells received
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more or less than one bead. Secondary CFU-F assays were performed by ow cytometry, seeding single propidium iodide (PI)-negative cells (living cells) from a starting population of cultured cells.
Supplemental Information includes Supplemental Experimental Procedures, ve gures, and two tables and can be found with this article online at http://dx.doi.org/10.1016/j.stemcr. 2013.06.001.
DNA Preparation and Quality Assessments

Genomic DNA was isolated using a QIAGEN DNeasy Blood & Tissue Kit and Agilents recommended procedure from 5 3 106 cells (RECs, MECs, and SECs at 6 weeks) as described in the Supplemental Experimental Procedures.
ACKNOWLEDGMENTS
We thank T. Sunabori, H. Kanki, L. Lein, and Y. Aizawa for advice; K. Minoura (Agilent Technologies) for performing the aCGH analysis; and A. Miyawaki and C. Hara for the Venus-ffLuc construct. D.D.H. is funded by the Medical Research Council, UK. This work was supported by the Project for Realization of Regenerative Medicine and Support for the Core Institutes for iPS Cell Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT, to H.O. and Y.M.). This study was also supported in part by a Grant-in-Aid for Encouragement of Young Medical Scientists from Keio University, a Grant-inAid for Scientic Research (to Y.M.), and a grant-in-aid from the Global Century COE program of MEXT to Keio University. H.O. is a scientic consultant for SanBio Inc., Eisai Co. Ltd., and Daiichi Sankyo Co. Ltd. Received: February 3, 2013 Revised: May 31, 2013 Accepted: June 3, 2013 Published: July 11, 2013
Oligonucleotide aCGH Analysis

aCGH experiments were performed according to the manufacturers protocol (Agilent oligonucleotide array-based CGH for genomic DNA analysis, version 6.0, direct method) as described in the Supplemental Experimental Procedures. All aCGH data are available for viewing at the Gene Expression Omnibus (GEO) under accession number GSE34484.
Index Sorting Assay

Index sorting was carried out according to the instrument users guide (FACSVantage SE option and assembly; Becton Dickinson Biosciences). Index sorting facilitates the linkage of marker expression information from an individual cell with its ow-cytometric prole.
Migration Assays
Migration assays were performed using polyethylene terephthalate lters with 8 mm pores (BD Biocoat) separating the upper and lower chambers as described in the Supplemental Experimental Procedures.
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Imaging of Transplanted Cultured MSCs In Vivo

RECs, MECs/SECs, and cultured MSCs were expanded and then infected with Venus-ffLuc lentivirus (the Venus uorescent and luminescent fusion protein; Hara-Miyauchi et al., 2012). Venus+infected cells (1 3 105 cells) were then sorted and transplanted intravenously. One day postinjection, the treated mice were anesthetized and given D-luciferin (150 mg/kg body weight) i.p. To quantify the measured light, regions of interest (ROIs) were dened as lungs, and all values were examined from an equal ROI by Xenogen-IVIS 100-cooled CCD optical macroscopic imaging system (SC BioScience) as described in the Supplemental Experimental Procedures.
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ACCESSION NUMBERS
The Gene Expression Omnibus accession number for CGH array reported in this paper is GSE34484.
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Ar ticle Enhanced Hemangioblast Generation and Improved Vascular Repair and Regeneration from Embryonic Stem Cells by Dened Transcription Factors
Fang Liu,1,6 Suk Ho Bhang,2,6,9 Elizabeth Arentson,1 Atsushi Sawada,3 Chan Kyu Kim,1,7 Inyoung Kang,1,4 Jinsheng Yu,5 Nagisa Sakurai,1 Suk Hyung Kim,1,8 Judy Ji Woon Yoo,2 Paul Kim,2 Seong Ho Pahng,2 Younan Xia,2,9 Lilianna Solnica-Krezel,3,4 and Kyunghee Choi1,4,*
of Pathology and Immunology of Biomedical Engineering 3Department of Developmental Biology 4Developmental, Regenerative, and Stem Cell Biology Program 5Department of Genetics Washington University School of Medicine, St. Louis, MO 63110, USA 6These authors contributed equally to this work 7Present address: Soonchunhyang University School of Medicine, Seoul, Korea 8Present address: Samsung Research Institute, Seoul, Korea 9Present address: Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA *Correspondence: kchoi@wustl.edu http://dx.doi.org/10.1016/j.stemcr.2013.06.005 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
2Department 1Department
SUMMARY
The fetal liver kinase 1 (FLK-1)+ hemangioblast can generate hematopoietic, endothelial, and smooth muscle cells (SMCs). ER71/ETV2, GATA2, and SCL form a core transcriptional network in hemangioblast development. Transient coexpression of these three factors during mesoderm formation stage in mouse embryonic stem cells (ESCs) robustly enhanced hemangioblast generation by activating bone morphogenetic protein (BMP) and FLK-1 signaling while inhibiting phosphatidylinositol 3-kinase, WNT signaling, and cardiac output. Moreover, etsrp, gata2, and scl inhibition converted hematopoietic eld of the zebrash anterior lateral plate mesoderm to cardiac. FLK-1+ hemangioblasts generated by transient coexpression of the three factors (ER71-GATA2-SCL [EGS]-induced FLK-1+) effectively produced hematopoietic, endothelial, and SMCs in culture and in vivo. Importantly, EGS-induced FLK-1+ hemangioblasts, when codelivered with mesenchymal stem cells as spheroids, were protected from apoptosis and generated functional endothelial cells and SMCs in ischemic mouse hindlimbs, resulting in improved blood perfusion and limb salvage. ESC-derived, EGS-induced FLK-1+ hemangioblasts could provide an attractive cell source for future hematopoietic and vascular repair and regeneration.
INTRODUCTION
Pluripotent stem cells provide an exciting opportunity in the eld of basic as well as regenerative biology because of their unique capacity to differentiate in culture into all somatic cells that form an individual. Current efforts are aimed at generating a preferred somatic cell type by manipulating growth factors added to culture media. While these efforts have advanced the eld, derivation of a homogenous specic cell population from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) still remains as a major challenge in the eld. Successful derivation of a desired somatic cell lineage from ESCs or iPSCs would likely to be advanced by comprehensive understanding of how specication of that particular cell lineage is accomplished in the developing embryo. As for blood and vessel development, tracking a receptor tyrosine kinase fetal liver kinase 1 (FLK-1) expression has been instrumental. Specically, cell lineage tracing studies have demonstrated that FLK-1+ mesoderm contributes to primitive and denitive blood, endothelial cells, and cardiac and skeletal muscles (Lugus et al., 2009; Motoike et al., 2003).
FLK-1+ (or KDR+ in human) mesoderm isolated from ESCs or embryos can generate hematopoietic, endothelial, and smooth muscle cells as well as cardiac cells (Choi et al., 1998; Faloon et al., 2000; Yamashita et al., 2000; Ema et al., 2003; Huber et al., 2004; Kennedy et al., 2007; Kattman et al., 2006; Moretti et al., 2006; Yang et al., 2008). Importantly, hemangiogenic or cardiogenic potential of the FLK-1+ mesoderm can be segregated by the plateletderived growth factor receptor a (PDGFRa) expression in both mouse and human, such that, while the FLK1+PDGFRa cell population is enriched for the hemangiogenic potential, FLK-1+PDGFRa+ cell population is enriched for the cardiogenic potential (Kattman et al., 2011; Liu et al., 2012). Molecularly, there is an antagonistic relationship between the hemangiogenic and cardiogenic mesodermal outcome. For example, enforced Er71 expression leads to an increase in hematopoietic and endothelial cell output but a decrease in cardiac output. Conversely, Er71 deciency results in defective hematopoietic and endothelial cell output but enhanced cardiac outcome (Lee et al., 2008; Liu et al., 2012). Similarly, the hematopoietic program is inhibited
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Enhanced Hemangioblast Generation from ESCs
by enforced Mesp1 or Nkx2-5 expression, which promotes cardiac differentiation (Caprioli et al., 2011; Lindsley et al., 2008). Herein, we reasoned that hemangioblast generation from ESCs could be enhanced by inhibiting cardiac output with dened hemangiogenic factors. We presumed that the candidate factors should be preferentially expressed within the hemangioblast population, that they could individually skew toward the hemangiogenic output, and that the skewing effect could be most dramatic when the candidate factors were coexpressed. We identify ER71, GATA2, and SCL as core factors in hemangioblast development. Transient coexpression of these three factors during mesoderm formation stage robustly enhanced FLK-1+ hemangioblast (FLK-1+PDGFRa) production while concomitantly inhibiting cardiac outcome from differentiating ESCs. Such FLK-1+ hemangioblasts generated functional endothelial and smooth muscle cells in culture as well as in ischemic mouse hindlimbs, resulting in improved blood perfusion and limb salvage.
day 4. Enforced Gata2 expression appeared to mainly suppress FLK-1+PDGFRa+ cells without signicant increase in FLK-1+PDGFRa cells. As the analyses of the enforced Er71, Gata2, or Scl expression suggested that these three factors might function synergistically, we next determined if temporal coexpression of these three factors were even more powerful in the skewing effect. Thus, we generated A2 ES cells coexpressing Er71, Gata2, and Scl in a DOXinducible manner. Notably, only the FLK-1+PDGFRa cells, not FLK-1+PDGFRa+ nor PDGFRa+, in both serum (Figure 1A) and serum-free conditions (Figure 4E) were generated when we coexpressed the three factors. FLK1+PDGFRa+ cells were still suppressed at day 4. Hematopoietic and endothelial cell transcription factor and surface receptor genes were strongly upregulated in these FLK1+PDGFRa cells (Figure 1B). On the other hand, cardiac transcription factors, including Mesp1 and Islet1 as well as Pdgfra genes, were greatly suppressed in these FLK1+PDGFRa cells. Collectively, temporal coexpression of Gata2 and Scl together with Er71 results in the most powerful effect generating FLK-1+PDGFRa cells. ER71, GATA2, and Scl-Induced Hemangioblast Can Further Differentiate into Hematopoietic and Endothelial Cells In Vitro and In Vivo To determine if FLK-1+PDGFRa cells generated by the transient Er71, Gata2, and Scl coexpression, designated as ER71GATA2-SCL (EGS)-induced FLK-1+, were enriched for bona de hemangioblasts, we subjected day 3 embryoid body (EB) cells, control and DOX-treated, to blast colony assay, an in vitro measure of hemangioblast (Choi et al., 1998). Remarkably, DOX-treated EB cells, compared to controls, generated greatly enhanced number of blast colonies (Figure 1C). More importantly, blast colonies were readily generated with DOX from serum-free conditions plus bone morphogenetic protein 4 (BMP-4), which normally do not support blast colony formation (Figures 1D and 1E). To determine if indeed all blast colonies developed from FLK-1+ cells, we sorted FLK-1+ cells from day 3 EBs, control and DOX-treated, and subjected to blast colony assay. Again, FLK-1+PDGFRa cells generated from DOXtreated EBs produced an increased blast colony number compared to control FLK-1+ cells (Figure 1F). Blast cells are capable of generating blood, endothelial, and smooth muscle cells (Choi et al., 1998; Ema et al., 2003; Yamashita et al., 2000). To further validate that the differentiation potential of blast colonies generated from EGS-induced FLK-1+ cells were the same as the controls, we subjected individual blast colonies to further differentiation into hematopoietic, endothelial, and smooth muscle cells. Similar percentage of blast colonies from control and DOX-treated EBs generated both hematopoietic and adherent cells (Figure S1C). Adherent cells contained both CD31+ (or VE-CADHERIN+)
RESULTS
FLK-1+PDGFRa Cells from ESCs Can Be Potently Generated by Temporal ER71, GATA2, and SCL Coexpression We previously reported gene expression proling of the presumptive hemangioblasts, FLK-1+SCL+ (Chung et al., 2002; Lugus et al., 2007). Focusing on the genes that were preferentially expressed within FLK-1+SCL+ cells or FLK-1+ cells (Figure S1A available online), we determined if any of these could skew mesoderm toward the hemangiogenic FLK-1+PDGFRa outcome from ESCs. To this end, we generated A2 ESCs expressing single candidate genes in a doxycycline (DOX)-inducible manner (Ismailoglu et al., 2008). We induced candidate genes from day 2 and analyzed FLK-1 and PDGFRa expression between days 3 and 4, mesoderm formation, and patterning stage. While the majority of the candidate genes did not show skewing potential, we could detect skewing outcome toward FLK1+PDGFRa mesoderm with Er71-, Gata2-, or Scl-enforced expression (Figures 1A, S2A, and S2B). Parent A2 or other ESC line R1 showed similar differentiation potential generating FLK-1+ and/or PDGFRa+ cells (Figure S1B), indicating that genetic manipulation did not alter the differentiation potential of the A2 ESCs. Of the three factors, enforced Er71 expression readily generated FLK-1+PDGFRa cells as early as day 3. However, the FLK-1+ PDGFRa skewing effect was not effectively maintained at later time points, as we could detect PDGFRa expression on day 4 (Figure 1A). Enforced Scl expression resulted in slightly different kinetics, such that FLK1+PDGFRa cells were most abundant at a later time point,
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Stem Cell Reports

and SMA-a+ cells, indicating that DOX treatment resulted in enhanced hemangioblast generation without altering their differentiation potential (Figures 1G, 1H, and S1D). As expected, FLK-1+ cells, control and DOX-treated, robustly generated hematopoietic (CD45+ and CD41+) and endothelial (VE-CADHERIN+ and CD31+) cells when cultured on OP9 cells (Figure 1J and S1E). Gene expression analyses also corroborated with cell surface marker studies (Figure 1K). Lymphatic endothelial cell marker genes, including Prox1, Vegfr3, and Lyve1, were also highly expressed in EGS-induced FLK-1+ and differentiated progeny cells (Figures 1B, 1J, and 1K). Functional hematopoietic and endothelial cell outcome from EGS-induced FLK-1+ cells was further determined in vivo by Matrigel plug assay. Control FLK-1+ or EGSinduced, FLK-1+-derived GFP+CD31+ as well as GFP+SMAa+ cells were detected (Figures 1L, S1F, and S1G). The occurrence of GFP+CD45+ cells was more frequent from Matrigel plugs obtained from EGS-induced FLK-1+ cells (Figures 1L, S1G, and S1H). In some cases, such vessels were lled with round GFP+ but DAPI negative cells, suggesting these cells could be mature erythrocytes derived from FLK-1+ hemangioblasts (Figure S1G). Collectively, FLK-1+ cells, control or EGS-induced, contain functional progenitors for hematopoietic, endothelial, and smooth muscle cells. EGS-induced FLK-1+ cells are highly effective in generating blood, endothelial cells, and smooth muscle cells in vivo.
etsrp, gata2, and scl Inhibition Can Successfully Convert the Hematopoietic Field of the Zebrash Anterior Lateral Plate Mesoderm to a Cardiac Field To further ascertain that cardiogenic versus hemangiogenic outcome can be manipulated by the three hemangiogenic transcription factors, we determined if the cardiac eld can be extended by suppressing er71, gata2, and scl expression in zebrash. Fate-mapping studies in zebrash have established that, while blood and endothelial cells originate from the rostral part of the anterior lateral plate mesoderm (ALPM), cardiac cell lineages originate from the medial and lateral parts (Figures 2A and 2B; Schoenebeck et al., 2007). Moreover, limiting blood and vessel specication resulted in expansion of cardiac progenitors (Schoenebeck et al., 2007). We observed that etsrp morpholino (MO) injection alone was sufcient to convert the hemangiogenic eld to cardiac, as evidenced by hand2 expression in the rostral part (Figure 2C), with concomitant loss of i1a expression, a gene critical for the hemangioblast specication in zebrash (Liu et al., 2008). This effect was specic to the ALPM, as the lateral i1 expression was not affected (Figure 2K). More importantly, etsrp-decient embryos readily expressed hand2 in the rostral region of the ALPM (Figure 2D). scl MO injection also resulted in a similar extension of the cardiac eld (Figure 2E). Although gata2 MO did not extend hand2 expression rostrally, the hand2 expression domain was expanded within the medial/lateral part (Figure 2F). Whereas 75% of wild-type embryos coinjected with
Figure 1. Enhanced FLK-1+PDGFRa Hemangioblast Generation by Temporal Er71, Gata2, and Scl Coexpression (A) iEr71, iGata2, iScl, or iEGS (= iEr71-2A-Gata2-2A-Scl) EBs were treated with DOX from day 2 and analyzed for FLK-1 and PDGFRa at indicated times. Numbers indicate the percentage of live cells gated within each quadrant. Representative results from four independent experiments are shown. (B) FLK-1+ cells were sorted from day 3 iEGS EBs (DOX from day 2) and analyzed for gene expression. Expression levels relative to Gapdh are shown as mean SD of three independent experiments. (C and F) Day 3 iEGS EBs (DOX from day 2) were subjected to blast colony assay. Colony numbers are shown as mean SD of four independent experiments **p < 0.01, compared to DOX. (D) iEGS ESCs were differentiated in serum-free conditions with BMP-4 (10 ng/ml) and DOX added from day 0 and day 2, respectively, and replated on day 3, 3.25, or 3.5. Blast colonies and secondary EBs were counted on day 4. Colony numbers are shown as mean SD of four independent experiments, **p < 0.01, compared to DOX. (E) iEGS ESCs were differentiated as in (D) and analyzed for FLK-1 and PDGFRa on day 3.5. (F) FLK-1+ cells sorted from day 3 iEGS EBs (DOX from day 2) were subjected to blast colony assay. Colony numbers are shown as mean SD of four independent experiments **p < 0.01, compared to DOX. (G and H) Individual blast colonies were picked and cultured on type IV collagen-coated microtiter well in the presence of cytokines. Bright-eld images of hematopoietic and adherent cells are shown (G), scale bar: 50 mm. After removing hematopoietic cells, adherent cells were immunostained for CD31 (red), VE-CADHERIN (red), SMAa (green), and DAPI nuclear staining (blue) (H). Scale bar: 20 mm for CD31 and VE-CADHERIN; 50 mm for SMAa. (IK) FLK-1+ cells were sorted from day 3 iEGS EBs (DOX from day 2), cultured on OP9 cells for 6 days, and uorescence-activated cell sorting-analyzed for CD41, CD45, CD31, LYVE-1, and VE-CADHERIN (J) or gene expression (K). Expression levels relative to Gapdh are shown as mean SD of three independent experiments. (L) FLK-1+ cells were sorted as in (I), infected with GFP retrovirus, mixed in with OP9 cells and Matrigel, and injected subcutaneously into nonobese diabetic/severe combined immunodeciency/interleukin 2 Rg/ mice. After 2 weeks, Matrigel plugs were excised and assessed for GFP+CD31+ or GFP+SMAa+ by immunostaining. Nuclei are shown in blue with DAPI (scale bar: 50 mm). See also Figures S1 and S2 and Table S2.
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Stem Cell Reports

gata2 and scl MOs displayed the rostral extension of hand2 expression, 100% of the etsrp heterozygous (etsrpy1 1/+) embryos (Pham et al., 2007), coinjected with these two MOs showed anterior enlargement of hand2 expression (Figures 2G and 2J). Importantly, while 80% of embryos coinjected with etsrp and scl MO resulted in rostral hand2 extension, 100% of the embryos showed conversion of the hemangiogenic eld to cardiac when coinjected with all three MOs (Figures 2H and 2I). These results suggest the cooperativity among Er71, Scl, and Gata2 in promoting hemangiogenic mesoderm development. Collectively, simultaneous modulation of Er71, Gata2, and Scl expression in ESC or zebrash has profound effect on the hemangiogenic outcome. ER71, GATA2, and SCL-Mediated Genetic Program in Hemangioblast Development The earlier and robust effect of Er71, compared to Gata2 or Scl, on the FLK-1+ PDGFRa, skewing both in ESC and zebrash models, suggested that ER71 functions upstream of GATA2 and SCL. Indeed, we previously reported that Er71 expression in embryos and EBs is transient (Lee et al., 2008). The onset of Er71 expression in wild-type EBs precedes that of Gata2 and Scl expression (Figure S3A). Moreover, Gata2 or Scl expression in EBs was not detected in the absence of ER71 (Figure S3A). We also detected ER71 occupancy on the Scl promoter region (Figure S4). Moreover, Er71-enforced expression resulted in robust Gata2 and Scl upregulation. On the other hand, Gata2 or Sclenforced expression failed to upregulate Er71 expression (Figure S3B). We conclude that ER71 establishes Gata2 and Scl expression during hemangioblast development. To better understand genetic program-regulating hemangioblast formation, we compared global temporal gene expression changes by enforced ER71, GATA2, and Scl coexpression. Specically, we saw an upregulation of Er71, Gata2, and Scl expression by 6 hr after DOX addition, indicating that 6 hr DOX treatment was sufcient to induce these exogenous genes. Thus, RNA from day 2.25 (6 hr after DOX addition), day 2.5 (12 hr after DOX addition), and day 3 (24 hr after DOX addition) EB cells was subjected to microarray analyses. Notably, Fli-1, Lmo2, Esam, Sox7, Sox18, Hey1, Flk-1, and Cdh5 expression was signi-
cantly upregulated within 6 hr after DOX addition, suggesting that these genes are presumably direct targets of ER71, GATA2, and Scl (Figures 3A and 3B). Indeed, we previously demonstrated that Flk-1 and Ve-cadherin/Cdh5 are ER71 direct targets (Lee et al., 2008; Liu et al., 2012). Recent studies have reported that Lmo2 is also an ER71 direct target (Koyano-Nakagawa et al., 2012). While hematopoietic and endothelial cell genes became progressively upregulated over the course of 24 hr DOX treatment, cardiac genes, including Hand1, Isl1, and Mesp1, were progressively downregulated within the same time frame (Figures 3A and 3B). At the same time, Flk-1 expression and downstream signaling pathway genes were greatly upregulated by 24 hr after DOX addition (Figures 3C3E). Cell adhesion molecules as well as Bmp-4 and Bmp-6 were also upregulated. On the other hand, BMP-4 inhibitors, including Follistatin and Cer-1 gene expression, were greatly inhibited. WNT signaling and epithelial-mesenchymal transition (EMT) pathway genes were also signicantly downregulated (Figures 3C3E; Table S1). Collectively, our data suggested that enforced Er71, Gata2, and Scl coexpression resulted in activation of BMP and FLK-1 signaling and inhibition of EMT and WNT signaling. We further performed biochemical analyses to conrm the signaling pathways identied by the global gene expression proling. DOX treatment resulted in an increase of the total SMAD1/SMAD5/SMAD8 protein levels as well as a signicant enhancement of phospho-SMAD1/ SMAD5/SMAD8, indicating that BMP signaling was indeed activated (Figure 4A). AKT/protein kinase B (AKT) phosphorylation was greatly reduced, but ERK phosphorylation was strongly enhanced by DOX treatment (Figure 4A). As total AKT or ERK protein levels remained the same, ER71, GATA2, and SCL induce hemangioblasts by selectively activating the map kinase pathway. Phopho-glycogen synthase kinase 3b (GSK3b) levels at serine 9 were greatly decreased upon DOX treatment (Figure 4A). The total amount of GSK3b remained the same. DOX treatment also resulted in lymphoid enhancer-binding factor-1 (LEF1) inhibition (Figure 4A), a key component in WNT target gene expression. Potentially, ER71-, GATA2-, and SCL-mediated WNT inhibition is achieved both by GSK3b-bCATENIN as well as LEF1 inhibition. As for EMT,
Figure 2. Combinatorial Suppression of er71/etsrp, gata2, and scl Promotes Cardiogenic Output in Zebrash Anterior Lateral Plate Mesoderm (AI) In situ hybridization depicts hand2 and i1a expression in uninjected (A), control (B), and etsrp MO embryos (C; 3 ng), scl MO (E; 12.5 ng), gata2 MO (F; 33.4 ng), gata2 (33.4 ng)+scl (12.5 ng) MOs (G), etsrp (3 ng)+scl (3 ng) MOs (H), etsrp (3 ng)+scl (3 ng)+gata2 (33.4 ng) MOs (I)-injected embryos, and etsrp/ embryos (D). (J) Expression of hand2 in gata2 (33.4 ng)+scl (12.5 ng) MOs-injected etsrp+/+ and etsrp+/ embryos. The numbers show the severe phenotype. (AJ) Seven-somite stage, dorsal views, anterior to the left. (K) Lateral view of ia expression in uninjected, etsrp MO, or etsrp+scl+gata2 MO-injected embryos. Scale bar: 500 mm. See also Table S4.
Stem Cell Reports

A
Hours 6 12 24 DOX - + - + - +
C
DOX
12 h
+ -
24 h
+
Bmp4 Bmp6 Smad5 Chrd Cer1 Fst Kdr Flt1 Flt4 Nrp1 Nrp2 Mapk14 Itpr1 Akt2 Prkd1 Shb Pik3r2 Jun Wnt5a Wnt5b Wnt8a Fzd1 Fzd2 Fzd7 Nrcam Tcf4 Lef1 Itga9 Vav3 Tek Esam Cdh5 Rab13 Cgnl1 F11r Magi3 Amot Gja4 Bmp2 Wnt5a Mmp25 Adamts9 Vcan Itga8 Twist1 Snail1 Gsc Cdh11 Cdh2
Day of Differentiation (D) -DOX +DOX
- 4.66
4.66
D
Bmp4 Bmp6 Cer1 Fst Kdr Flt1 Flt4 Wnt5a Wnt5b Fzd7 Tcf4 Lef1 Tek Esam Cdh5 Itga8 Twist1 Snail1 Snail3 Gsc Cdh11 Cdh2
-8 -6 -4 -2 0 2 4 6 8
E
CERBERUS
EMT
- 5.51
Etv2 Gata2 Tal1 Hhex Lmo2 Lyn Runx1 Fli1 Erg Tie1 Tek Elk3 Klf5 Flt1 Kdr Eng Edn1 Eg7 Esx1 Sox17 Sox18 Cdh5 Hand1 Msx2 Isl1 Tbx6 Gata4 Mesp1 Mixl1 Actc1 Apln Vcan Ptprj Pdlim3 Myl7
15 10 5 0
Hhex
15 10 5 0
Lmo2
15 10 5 0
Fli1
2.25
2.5
3.0
2.25
2.5
3.0
2.25
2.5
3.0
6 4
Elk3
4 3 2 1 0
Sox17
1.5 1 0.5 0
Sox18
Blood/ Vessel
Gene Expression / Gapdh x103
2 0
2.25 2.5 3.0
2.25
2.5
3.0
2.25
2.5
3.0
0
2.25 2.5 3.0 2.25 2.5 3.0
0
2.25 2.5 3.0
0.25 0
2.25 2.5 3.0
0.02 0
2.25 2.5 3.0
2.25
2.5
3.0
Cardiogenesis
5 4 3 2 1 0 12 10 8 6 4 2 0
Hand1
1.5 1 0.5 0
Isl1
1.2 1 0.8 0.6 0.4 0.2 0

2.5 3.0
Mesp1
2.25
2.5
3.0
2.25
2.25
2.5
3.0
Pdgfr
2 1.5 1 0.5 0
Pdgfr
0.5 0.25 0
Mesp2
2.25
2.5
3.0
2.25
2.5
3.0
2.25
2.5
3.0
Adherens/Tight
7 6 5 4
Cdh5
0.08 0.06 0.04
Runx1
5 4 3 2 1 0
Mixl1
Wnt
1 0.8 0.6 0.4 0.2 0
Erg
1.5 1 0.5
Tie1
0.4 0.3 0.2 0.1
Ets1
VEGF
BMP4
5.51
CHORDIN BMP4 SMAD1 VEGF-A BMPR1A SMAD4
FOLLISTATION BMP6 SMAD5
NEUROPILIN VEGFR1 VEGFR2 -1 ERK1/2 C-JUN MEK1
VEGFR3 GRB2
Overexpressed gene Underexpressed gene Positive effect Negative effect
C-RAF-1 WNT5A TGF-BETA TCF(LEF) SNAIL1 VE-CADHERIN CLAUDIN1 E-CADHERIN N-CADHERIN OCCLUDIN FRIZZLED GSK3BETA DSH WNT WNT5B WNT8A FZD1 FZD2 FZD7
TWIST1
Fold Change (+DOX/-DOX in log2)

Day 2.5 Day 3

Stem Cell Reports

DOX treatment (from day 2 or 3) greatly suppressed N-CADHERIN expression (Figure 4B). At the same time, genes involved in EMT processes, including Snail1 and Twist1, were also suppressed by DOX addition (Figure 4C). CHIR99021, a GSK3b inhibitor, could partially rescue Snail1 inhibition by DOX treatment (Figure 4D) but not Mesp1 suppression or FLK-1+PDGFRa+ cardiac mesoderm (not shown). Notably, activation of the map kinase pathway and inhibition of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway was readily observed by enforced Er71 expression alone. SMAD1/SMAD5 activation and WNT inhibition was readily observed by enforced Gata2 and Scl expression, respectively (Figure 4A). This suggested that ER71, GATA2, and SCL may distinctly contribute to hemangioblast development, and the combined sum of ER71, GATA2, and SCL activity is required for the most efcient hemangioblast generation. Since the PI3K pathway appeared to negatively regulate hemangioblast formation, we tested if we could produce FLK-1+PDGFRa cells even more robustly by adding PI3K inhibitor. The percentage of FLK-1+PDGFRa cells generated was signicantly higher when we added DOX and PI3K inhibitor together (Figure 4E). On the other hand, FLK-1+PDGFRa cells were generated less when a map kinase inhibitor was added together with DOX, indicating that the map kinase pathway positively regulates the FLK1+PDGFRa cell formation (Figure 4E). EGS-Induced FLK-1+ Cells Can Potently Enhance Angiogenic Repair and Regeneration We tested if ESC-derived, EGS-induced FLK-1+ (= FLK1+(+DOX)) cells could be used for angiogenic repair and regeneration. Remarkably, mesenchymal stem cells (MSCs) delivered as spheroids were superior to single MSC cells in supporting vascular therapeutic activity in a mouse model of hindlimb ischemia, as they were protected from apoptosis and secreted higher levels of angiogenic
factors (Bhang et al., 2012; Shim et al., 2012). Thus, we determined if MSCs could be used as vehicles for delivering EGS-induced FLK-1+ cells. To this end, we used human MSCs (hMSCs; from female) to distinguish the contribution of EGS-induced FLK-1+ cells and MSCs in angiogenic repair. We rst evaluated if EGS-induced FLK-1+ cells could be protected from apoptosis while preserving endothelial differentiation potential by coculturing with hMSCs as spheroids. Specically, spheroids generated between EGSinduced FLK-1+ and hMSCs in different ratios (1:1, 1:5, and 1:10) were analyzed for the best condition that gave maximal cell survival of EGS-induced FLK-1+ cells after switching to hypoxia (1% oxygen) to mimic an in vivo ischemic environment. EGS-induced FLK-1+ cells under hypoxia readily became apoptotic when cocultured with hMSCs as single cells (Figures 5A and 5B). Spheroids containing only EGS-induced FLK-1+ cells also displayed multiple apoptotic cells (Figures 5A and 5B). However, EGS-induced FLK-1+ cells displayed signicantly enhanced survival when cocultured with hMSCs as spheroids (Figures 5A and 5B). Consistently, while Bcl-2 expression was readily detectable in (FLK-1+(+DOX)+hMSC) spheroids, p53 expression levels were high in single-cell culture or spheroids containing only FLK-1+(+DOX) cells (Figures 5C, S5A, and S5B). Of the conditions, the 1:5 group showed the maximal cell viability. Mitochondrial metabolic activity was also signicantly higher in the (FLK1+(+DOX)+hMSC) spheroids (1:5 ratio) compared to others (Figure S5C). Previous studies have demonstrated that hMSCs readily upregulated hypoxia-inducible factor 1a (HIF-1a) in spheroids in hypoxia (Bhang et al., 2012; Shim et al., 2012). Consistently, HIF-1a expression was readily detected in (FLK-1+(+DOX)+hMSC) spheroids, with the expression level being the highest in spheroids containing EGSinduced FLK-1+ cells and hMSCs in a 1:5 ratio (Figures 5C, S5D, and S5E). HIF-1a upregulation resulted in
Figure 3. ER71, GATA2, and SCL-Mediated Genetic Program Regulating Hemangioblast Development (A) Gene expression was assessed by microarray analysis at the indicated times (hr) after DOX addition. The average intensity for a given gene from three independent experiments is shown as heat map. Intensity values used in the heat maps are in log2 and normalized to mean of the DOX samples. Red shading indicates increased expression, and blue shading indicates decreased expression. (B) Kinetic expression levels of the indicated cell lineage markers over the time course of iEGS ESCs differentiation with (lled line) or without (dashed line) DOX is shown by qRT-PCR analysis. Data are presented as the mean values SD from four independent experiments. (C) Heat map presents the relative expression of a subset of genes at the 12 and 24 hr after DOX addition implicated in the indicated pathways. Intensity values used in the heat maps are in log2 and normalized to mean of the DOX samples at each time point. Data from three independent experiments are shown. (D and E) Networks of the pathway regulation were created using MetaCore. (D) Fold-change of pathway genes in +DOX versus DOX samples is shown. Data are presented as the mean values SD from three independent experiments. (E) Representative pathways that are focused on signicantly affected interactions. Signaling pathways involved in BMP-4 is in blue; VEGF, green; WNT, brown; and EMT, yellow. Overlapping circles contain differentially expressed genes common to both pathways. Red solid circles indicate upregulated genes, and blue solid circles indicate downregulated genes. Green arrows show activation, and red arrows show suppression. See also Figures S3 and S4 and Tables S1 and S2.
Stem Cell Reports

A
pSMAD1,5 S463,465 SMAD1 GAPDH
iEGS
-D O X +D O X
iGata2
-D O X +D O X
iEGS
T202,Y204
iEr71
-D O X +D O X
pERK1,2
ERK1,2
iEGS
-D O X +D O X
iEr71
-D O X +D O X
S9
iEGS
-D O X +D O X
iScl
-D O X +D O X
iEGS
-D O X +D O X
pAKT
S473
pGSK3
LEF1 GAPDH
AKT
GSK3
iEGS
-D O X +D O X
E-CAD N-CAD
N-CAD GAPDH
-DOX
+DOX
101 10 0 10 -1 10 -2 10 -3 10-4 Sox10 10 -5 10 -5 Col3a 10-4 10 -3 10 -2 10 -1 10 0 101 Mmp2 Fzd7 Cdh2 Vcan Jag1 Pdgfrb Wnt5b Snail1 Timp1 Krt7 Gsc Twist1
D
Gene/Gapdh x10 4
0.4 0.3 0.2 0.1 0
Mesp1
25 20 15 10 5 0
Snail1
- DOX
-DOX +DOX +DOX + 5uM CHIR99021
+DOX LY Wortmannin U0126 PD184161 +DOX -DOX 0.2 0.4 0.6 0.7 0.1 0.7 0.3 1.8 1.1 0.2 0.5 0.2 2.2 38 64 60 17 22
Figure 4. ER71, GATA2, and Scl-Mediated Signaling Pathways in Hemangioblast Development (A) iEr71, iGata2, iScl, or iEGS ESCs were differentiated in serum-free conditions with DOX added from day 2. Day 4 EB-lysates were analyzed for signaling molecules by immunoblotting. (B) iEGS EB cells were plated onto collagen I-coated slides on day 2 and stained with antibodies against E-CADHERIN (green) or N-CADHERIN (red) on day 4. N-CADHERIN protein expression levels in day 4 iEGS EBs (DOX) are also shown by immunoblot analysis. (C) Mouse EMT signaling SA-Bioscience QRTPCR array on iEGS day 4 EBs (DOX from day 2) are shown as scattered plots of fold changes normalized to three housekeeping genes for +DOX versus DOX. Red dots are downregulated genes in +DOX. (D) iEGS ESCs were differentiated, CHIR99021 and DOX added from days 24, and analyzed for Snail1 and Mesp1 expression. Expression levels relative to Gapdh are shown as mean SD of three independent experiments. (The asterisk shows p < 0.01 compared to +DOX group). (E) iEGS ESCs were differentiated in serumfree conditions, inhibitors and DOX added from day 2, and analyzed for FLK-1 and PDGFRa expression on day 4. PI3K inhibitors include LY294002 (10 mM) and Wortmannin (200 nM), MEK1/2 inhibitors include U0126 (10 mM) and PD184161 (10 mM). See also Table S2.
+DOX
-D O X +D O X
enhanced human vascular endothelial growth factor (VEGF), basic broblast growth factor (bFGF), hepatocyte growth factor (HGF) gene expression, and protein production as measured by secreted protein levels. Angiogenic factor production was most robust in spheroids containing EGS-induced FLK-1+ cells and hMSCs in a 1:5 ratio (Figures 5D, 5E, and S5F). Consequently, endothelial cells were efciently generated in spheroids containing EGS-induced FLK-1+ cells and hMSCs in a 1:5 ratio (Figures 5F and 5G). Spheroids containing only hMSCs did not express any endothelial cell genes (not shown). HIF-1a, mouse VEGF, or FGF expression was modest in spheroids generated with only EGS-induced FLK-1+ cells (Figure 5C; data not
shown). Vitronectin and laminin gene expression was efciently induced in all spheroids, indicating that threedimensional culture conditions augmented extracellular matrix protein production and cell-cell interactions (Figures 5H, S5G, and S5H). We conclude that ESC-derived, EGS-induced FLK-1+ cells can be protected from hypoxiainduced apoptosis and further differentiate into endothelial cells when cocultured with MSCs as spheroids. We assessed therapeutic efcacy of the (FLK1+(+DOX)+hMSC) spheroids in a mouse model of hindlimb ischemia. Specically, we induced hind-limb ischemia in female nude mice, intramuscularly injected spheroids containing EGS-induced FLK-1+ cells and hMSCs in a 1:5
Stem Cell Reports

Figure 5. hMSCs Promote EGS-Induced FLK-1+ Cell Viability and Endothelial Cell Differentiation when Cocultured as Spheroids Cells were cultured under hypoxic condition (1% oxygen). (A and B) Cell viability was determined by uorescein diacetate-ethidium bromide staining (A) (green: live cells; red: dead cells) or by neutral red assay (B) (*p < 0.01 compared to the FLK-1+(+DOX) spheroid group; three independent experiments). (C) RT-PCR analysis of Bcl-2, p53, hypoxia-inducible factor-1a, and angiogenic factors. Representative results from three independent experiments are shown. (D and E) Angiogenic factors released from single cells and spheroids were determined by ELISA (*p < 0.01 compared to the FLK-1+(+DOX) spheroid group; three independent experiments). (F and G) Quantication of Cd31 and Cdh5 expression was determined by qRT-PCR. Expression levels relative to Gapdh are shown as mean SD of three independent experiments. The asterisk indicates p < 0.01 compared to the FLK-1+(+DOX) spheroid group. (H) Immunoblot analysis of extracellular matrices (LAMININ and VITRONECTIN). See also Figure S5 and Table S2.
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ratio (3 3 106 cells/limb, 5 3 105 EGS-induced FLK-1+ cells + 2.5 3 106 hMSCs), and assessed the EGS-induced FLK-1+ cell contribution to the recovery of the hind-limb ischemia by the presence of Y chromosome-positive cells (parent ESCs are male, XY). Three days after ischemia induction, the (FLK-1+(+DOX)+hMSC) spheroid-recipient group contained signicantly less caspase-3 and Y-positive cells (apoptotic cells, arrows) in the ischemic limb compared to the FLK-1+(+DOX) spheroid-recipient group (Figures 6A6C). Y-positive cells were more abundant in the (FLK-1+(+DOX)+hMSC) spheroid-recipient group compared to the FLK-1+(+DOX) spheroid-recipient group (Figure 6D). (FLK-1+(+DOX)+hMSC) spheroid transplantation group displayed enhanced antiapoptotic gene expression (Bcl-2) and reduced proapoptotic factor (p53) gene expression (Figures 6E and 6F). Inammation and muscle degeneration was attenuated in the (FLK1+(+DOX)+hMSC) spheroid-recipient group compared to that of FLK-1+(+DOX) spheroid-recipient group, as judged by hematoxylin and eosin (H&E) staining (Figure 6G). Additionally, signicant amount of human VEGF and FGF2 were detected in mouse ischemic tissues in the (FLK-1+(+DOX)+hMSC) spheroid-recipient group compared to the FLK-1+(+DOX) spheroid transplantation group (Figures 6E and 6H). Twenty-eight days after ischemia induction, mice that received no treatment showed extensive muscle degeneration and inammation in the ischemic region (Figures 6I and 6J), resulting in a complete limb loss (95.5%; n = 22) or severe limb necrosis (4.5%; Figure 6K). Mice that received FLK-1+(+DOX) spheroids displayed similar muscle degeneration and limb loss (n = 5; data not shown). Mice that received hMSC spheroids did signicantly better with decreased limb necrosis (Figure 6K). However, these mice still showed muscle degeneration and inammation in the ischemic region (Figures 6I and 6J) and 27.3% of them ultimately lost limb (Figure 6K). Similar to mice
that received hMSC spheroids, mice that received (controlFLK-1++hMSC) spheroids also displayed decreased limb necrosis compared to no treatment group (Figure 6K). However, these mice still showed muscle degeneration and inammation in the ischemic region (Figures 6I and 6J) and 18.2% of them eventually lost limb (Figure 6K). No statistical difference was observed in brotic area between the two groups that received hMSC spheroids or (controlFLK-1++hMSC) spheroids. In contrast, mice that received (FLK-1+(+DOX)+hMSC) spheroids were protected from limb muscle necrotic damage (Figures 6I and 6J). The majority of the mice that received (FLK1+(+DOX)+hMSC) spheroids exhibited limb salvage (86.4%) with only mild limb necrosis (9.1%). Only 1 of the 22 mice (4.5%) lost limb (Figure 6K). Laser Doppler perfusion imaging analysis (Figure 6L) revealed that blood perfusion in ischemic limbs was signicantly improved in the group that received (FLK1+(+DOX)+hMSC) spheroids compared to the other groups. Seven days after treatment, the relative ratios of blood ow (ischemic to normal limb) were 35.6% 6.0% in the (FLK-1+(+DOX)+hMSC) spheroid-recipient group compared to the other groups (no treatment, 5.3% 2.2%; hMSC spheroid recipients, 13.2% 1.8%; controlFLK-1++hMSC spheroid recipients, 14.4% 2.8%; Figure 6L). By day 28, (FLK-1+(+DOX)+hMSC) spheroidtransplanted group signicantly improved the relative ratio of blood perfusion (72.6% 6.8%) compared to the other groups (no treatment, 3.1% 1.7%; hMSC spheroid recipients, 44.6% 7.7%; controlFLK-1++hMSC spheroid recipients, 47.4% 5.1%; Figure 6L). No statistical difference was observed between the hMSC spheroid and (controlFLK-1++hMSC) spheroid transplantation group. Mice that received (FLK-1+(+DOX)+hMSC) spheroids displayed signicantly enhanced CD31+ microvessel formation compared to the other groups (Figures 7A and 7C). SMA-a+ microvessel formation was also signicantly
Figure 6. Enhanced Ischemic Limb Salvage and Vascular Repair by EGS-Induced FLK-1+-hMSC Spheroid Transplantation (A) CASPASE-3 and Y-chromosome staining from day 3 samples. Apoptotic cells: CASPASE-3 (red), transplanted EGS-induced FLK-1+ cells: Y-chromosome (green), cell nuclei: DAPI (blue), respectively (scale bar = 50 mm). Arrows point to the apoptotic cells derived from EGSinduced FLK-1+ cells. (BD) The ratio of CASPASE-3 and Y-chromosome positive cells in the ischemic lesion (*p < 0.01, 24 mice/group). (E) Immunoblot analysis of anti- and proapoptotic factor and angiogenic paracrine factors from day 3 samples. (F) Quantication of Bcl-2 and p53 determined by qRT-PCR (*p < 0.01, 24 mice/group). (G) H&E staining of histological ischemic limb sections 3 days after transplantation (scale bar = 200 mm). (H) Amount of angiogenic factors produced in ischemic lesion as determined by ELISA (*p < 0.01 compared to the FLK-1+(+DOX)+hMSC spheroid group; 24 mice/group). (I and J) H&E staining (I) and Massons trichrome staining (J) of histological ischemic limb sections 28 days after treatments (blue indicates brosis in [J], scale bar = 200 mm). The asterisk indicates p < 0.01 compared to normal group; 22 mice/group. (K and L) Representative photographs of physiological status (K) and blood perfusion (L) of ischemic hindlimbs. Laser Doppler imaging 0, 7, 14, 21, and 28 days after treatments. All photographs were taken at the same magnication. In (L), p < 0.01 compared to no treatment group. See also Figures S6 and S7 and Table S2.
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Stem Cell Reports

enhanced by (FLK-1+(+DOX)+hMSC) spheroid transplantation (Figures 7B and 7C). Signicant number of EGSinduced FLK-1+-derived Y+CD31+ (Figures 7A, 7C, and S7A) or Y+SMA-a+ cells (Figures 7B, 7C, and S7A) were observed in the vasculature. Although a similar number of donor-derived (Y+) cells were detected 28 days after transplantation in mice that received control FLK-1+ or EGS-induced FLK-1+ cells, very few control FLK-1+-derived Y+CD31+ or Y+SMA-a+ cells were found in the vasculature (Figures 7A7C). Most of the control FLK-1+ or EGSinduced FLK-1+ progeny cells were found in close proximity to CD31+ or SMA-a+ microvessels. Many more bromodeoxyuridine (BrdU)-positive microvessels (newly formed microvessels) were detected in limb tissues transplanted with (FLK-1+(+DOX)+hMSC) spheroids compared to the other groups (Figure 7D). While human VEGF, bFGF, and HGF proteins were robustly detected (Figure 7D), human-specic SMA-a or CD31 expression was not detected (Figure 7C), suggesting that the contribution of hMSCs to the vascular repair in all conditions are restricted to paracrine effects. To determine if therapeutic effect was still observed with reduced cell number of EGS-induced FLK-1+ cells, mice (n = 8 each) were injected with 6 3 105 hMSC or (FLK-1+(+DOX)+hMSC) spheroids (1 3 105 EGS-induced FLK-1+ cells + 5 3 105 hMSCs) after ischemia induction and assessed for their recovery. Compared to the group that received no treatment or hMSC spheroids only, mice that received reduced number of (FLK-1+(+DOX)+hMSC) spheroids still dramatically enhanced angiogenesis, blood ow increase, and limb salvage with reduced muscle degeneration and tissue brosis (Figure S6). We also observed abundant Y+CD31+ or Y+SMA-a+ cells in nascent vessels (Figure S6). This indicated that vascular therapeutic effect could be achieved with as little as 1 3 105 EGS-induced FLK-1+ cells. Finally, we conrmed that EGS-induced FLK-1+ cell outcome was indeed restricted to vessel contribution in noninjury model. Specically, we injected ESCs or (FLK1+(+DOX)+hMSC) spheroids subcutaneously into mice. As expected, mice that received ESCs formed distinctive teratomas, which contained multiple tissue types, including cartilage, glands, neuroepithelium, bone, skin, and vessels
(Figures S7B and S7D). However, mice transplanted with (FLK-1+(+DOX)+hMSC) spheroids contained only endothelial and smooth muscle cells (Figures S7C and S7D).
DISCUSSION
We transiently coexpressed Er71, Gata2, and Scl during mesoderm formation stage in differentiating ESCs and greatly augmented hemangioblast generation, while nearcompletely blocking cardiac output. Similarly, we could convert hematopoietic eld of the anterior lateral plate mesoderm into cardiac by inhibiting all three hemangiogenic factors in zebrash. This implies that ER71, GATA2, and SCL form a central core network in hemangioblast development. Enforced ER71, GATA2, and SCL genetic program in ESCs resulted in activation of the BMP-4 and VEGF receptor 2 signaling pathways and inhibition of the PI3K and WNT signaling. Remarkably, each factor contributed uniquely to these signaling pathways, and the collective sum of these was essential for the nal hemangioblast outcome. From the characterizations of enforced Er71, Gata2, or Scl expression in ESCs and the three factor morphants in zebrash, we propose that ER71 function is upstream of GATA2 and SCL and is required at the commitment stage of hemangioblast generation. GATA2 and SCL maintain the hemangioblast phenotype by suppressing the cardiac program. Indeed, ER71 alone could signicantly induce hemangioblasts from ESCs. Moreover, etsrp inhibition alone was most powerful in blocking hemangiogenic outcome and converting it into cardiac eld in zebrash. In developing embryos and differentiating ESCs, Er71 expression is detected prior to Gata2 and Scl expression. Enforced Er71 expression strongly upregulated Gata2 and Scl expression within 12 hr. On the other hand, enforced Gata2 or Scl expression failed to upregulate Er71. Previous studies suggested that Gata2 and Scl are direct targets of ER71 (Kataoka et al., 2011). We also observed ER71 occupancy on the Scl promoter region. Recent studies that only the FLK-1+PDGFRa+ cardiac mesoderm is generated from Er71-decient ESCs (Liu et al., 2012), that vascular endothelial and endocardial progenitors differentiate as
Figure 7. EGS-Induced FLK-1+-Derived Endothelial and Smooth Muscle Cells Are Abundantly Found within Functional Vessels (A and B) Merged immunouorescent staining images of Y-chromosome and CD31 (A) or Y and SMAa (B) of ischemic hindlimb tissues 28 days after transplantation. Scale bar indicates 100 mm in low magnication and 70 mm high magnication. (C) Quantication of CD31 or SMAa-positive microvessel density in the ischemic region and microvessels with Y-chromosome signals (*p < 0.01 and #p < 0.05 compared to the normal group; 22 mice/group). Human- or mouse-specic gene expression for microvessels were also accessed with RT-PCR. (D) Newly formed microvessel density was analyzed by BrdU-positive microvessels and relative amount of microvessels in the ischemic lesion. (The asterisk indicates p < 0.01 compared to the normal group; 22 mice/group). See also Figures S6 and S7 and Table S2.
Stem Cell Reports

cardiomyocytes in the absence of etsrp (Palencia-Desai et al., 2011), and that cardiac program was readily derepressed in Scl decient endothelial cells (Van Handel et al., 2012) support this notion. We demonstrated that ESC-derived, EGS-induced FLK-1+ hemangioblasts generated functional endothelial and smooth muscle cells in vivo, as demonstrated by CD31+ as well as SMAa+ vessel structures produced from the EGS-induced FLK-1+ cells in Matrigel plugs. More importantly, we were successful in protecting EGS-induced FLK-1+ cells from apoptosis while preserving their differentiation potential by generating spheroids with hMSCs, which provided survival signal as well as paracrine angiogenic factors. Consequently, EGS-induced FLK-1+-derived endothelial and smooth muscle cells promoted vascular repair and regeneration in a mouse model of hind-limb ischemia. Control FLK-1+ cells were also able to differentiate into functional endothelial and smooth muscle cells. However, they were less efcient in their vascular repair and regeneration potential, probably due to their heterogeneous nature of both cardiac and hemangiogenic potential. Our studies are exciting, as there is great interest in applying stem cell-based therapies for peripheral arterial disease. Clinical trials, phase I and II, have shown that endothelial progenitor cells (EPCs), bone marrow-derived mononuclear cells, MSCs, or the combinations of these cell types are benecial for patients with limb ischemia or acute myocardial infarction (Katritsis et al., 2005; Lasala et al., 2010, 2012; reviewed in Critser and Yoder, 2010 and Minguell et al., 2013). Moreover, several studies have reported that pluripotent stem-derived mature endothelial cells could improve angiogenic repair and regeneration (Cho et al., 2007; Rufaihah et al., 2011). Currently, however, limitations still exist in applying either pluripotent stem-derived endothelial cells, EPCs, or MSCs clinically. For example, mature endothelial cells have a limited life span. Direct injection of ESC-derived endothelial cells into damaged ischemic area in animal models has met with limited success, due to apoptosis resulting in limited engraftment. We envision that ESC-derived FLK-1+ vascular progenitors would be ideal for future therapeutic applications in peripheral arterial disease. The current strategy of EGS-induced FLK-1+ cell delivery with MSCs as vascular tissue spheroids could also be extended to additional cell types, including EPCs and bone marrow mononuclear cells. In summary, we developed a powerful strategy generating hemangiogenic FLK-1+ progenitors from ESCs. The unlimited access to hemangioblasts, the ultimate hematopoietic and vascular progenitors, warrants further investigations on potential clinical applications requiring hematopoietic, vascular repair and regeneration. In the future, however, a strategy of deleting the exogenous genes
after hemangioblast induction should be considered. Derivation and future applications of hemangioblasts from human pluripotent stem cells in peripheral arterial diseases are warranted.
ESC Generation
Er71+/+ and Er71/ ESC lines and inducible ESC lines (iEr71 and iGata2) were generated as described previously (Lee et al., 2008; Liu et al., 2012; Lugus et al., 2007). Inducible Scl or Er71-2AGata2-2A-Scl (iEGS) ESCs were generated by targeting the tetresponsive locus of A2Lox cells (Ismailoglu et al., 2008), with the construct containing the coding sequence of Scl or Er71, Gata2, and Scl, which were linked by 2A peptides (de Felipe, 2002). Scl was fused to a Flag tag at the 30 end. After the correct targeting event was conrmed by a tet-responsive locus/complementary DNA vector-specic PCR, inducible Scl or Er71, Gata2, and Scl expression with 1 mg/ml DOX was veried by RNA and Immunoblot analyses.
Microarray and Pathway Enrichment Analysis

Global gene expression proles were analyzed using the GeneChip Mouse Gene 1.0 ST arrays (Affymetrix). Data were normalized, and expression values were modeled with Partek Genomics Suite (Partek). Differentially expressed genes were determined with signicant p value < 0.05 and false discovery rate < 0.25. Raw data are available at the Gene Expression Omnibus repository (accession number GSE45147). Based on the microarray data, pathway enrichment analysis and interaction networks building were performed using MetaCore Analytical Suite (GeneGo).
Mouse Hindlimb Ischemia

Hindlimb ischemia was induced in mice as previously described (Shim et al., 2012). Four-week-old female athymic mice (2025 g body weight; Jackson Laboratory) were anesthetized with xylazine (10 mg/kg) and ketamine (100 mg/kg). The femoral artery and its branches were ligated via skin incision using a 6-0 silk suture (Ethicon), along with the external iliac artery and all upstream arteries. The femoral artery was then excised from its proximal origin as a branch of the external iliac artery to the distal point, whereupon it bifurcates into the saphenous and popliteal arteries. All animals received humane care (Institutional Animal Care and Use Committee No. 20110260).
Transplantation of FLK-1+(+DOX)+hMSC Spheroids into Ischemic Mouse Hindlimbs

Mice were randomly divided into ve groups (n = 22 from three independent experiments). Normal mice that did not undergo any surgery or mice with surgery but without treatment were used as controls. hMSC, controlFLK-1++hMSC or FLK-1+(+DOX)+hMSC spheroids (3 3 104 cells/spheroid; 100 spheroids/limb; total 3 3 106 cells/limb; 0.5 3 106 EGS-induced FLK-1+ cells + 2.5 3 106 hMSC cells/limb) were intramuscularly injected into the gracilis muscle of the medial thigh.
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Laser Doppler Imaging Analysis

Laser Doppler imaging analysis was performed as described previously (Shim et al., 2012). A laser Doppler perfusion imager (Moor Instruments) was used for serial noninvasive physiological evaluation of neovascularization. Mice were monitored by serial scanning of surface blood ow in hindlimbs at days 0, 7, 14, 21, and 28. Digital color-coded images were scanned and analyzed to quantify a blood ow in ischemic regions from the knee joint to the toe. Mean values of perfusion were subsequently calculated. The morphological status of the ischemic limb was monitored for 4 weeks.
Chung, Y.S., Zhang, W.J., Arentson, E., Kingsley, P.D., Palis, J., and Choi, K. (2002). Lineage analysis of the hemangioblast as dened by FLK1 and SCL expression. Development 129, 55115520. Critser, P.J., and Yoder, M.C. (2010). Endothelial colony-forming cell role in neoangiogenesis and tissue repair. Curr. Opin. Organ Transplant. 15, 6872. de Felipe, P. (2002). Polycistronic viral vectors. Curr. Gene Ther. 2, 355378. Ema, M., Faloon, P., Zhang, W.J., Hirashima, M., Reid, T., Stanford, W.L., Orkin, S., Choi, K., and Rossant, J. (2003). Combinatorial effects of Flk1 and Tal1 on vascular and hematopoietic development in the mouse. Genes Dev. 17, 380393. Faloon, P., Arentson, E., Kazarov, A., Deng, C.X., Porcher, C., Orkin, S., and Choi, K. (2000). Basic broblast growth factor positively regulates hematopoietic development. Development 127, 19311941. Huber, T.L., Kouskoff, V., Fehling, H.J., Palis, J., and Keller, G. (2004). Haemangioblast commitment is initiated in the primitive streak of the mouse embryo. Nature 432, 625630. Ismailoglu, I., Yeamans, G., Daley, G.Q., Perlingeiro, R.C., and Kyba, M. (2008). Mesodermal patterning activity of SCL. Exp. Hematol. 36, 15931603. Kataoka, H., Hayashi, M., Nakagawa, R., Tanaka, Y., Izumi, N., Nishikawa, S., Jakt, M.L., Tarui, H., and Nishikawa, S. (2011). Etv2/ER71 induces vascular mesoderm from Flk1+PDGFRa+ primitive mesoderm. Blood 118, 69756986. Katritsis, D.G., Sotiropoulou, P.A., Karvouni, E., Karabinos, I., Korovesis, S., Perez, S.A., Voridis, E.M., and Papamichail, M. (2005). Transcoronary transplantation of autologous mesenchymal stem cells and endothelial progenitors into infarcted human myocardium. Catheter. Cardiovasc. Interv. 65, 321329. Kattman, S.J., Huber, T.L., and Keller, G.M. (2006). Multipotent k-1+ cardiovascular progenitor cells give rise to the cardiomyocyte, endothelial, and vascular smooth muscle lineages. Dev. Cell 11, 723732. Kattman, S.J., Witty, A.D., Gagliardi, M., Dubois, N.C., Niapour, M., Hotta, A., Ellis, J., and Keller, G. (2011). Stage-specic optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 8, 228240. Kennedy, M., DSouza, S.L., Lynch-Kattman, M., Schwantz, S., and Keller, G. (2007). Development of the hemangioblast denes the onset of hematopoiesis in human ES cell differentiation cultures. Blood 109, 26792687. Koyano-Nakagawa, N., Kweon, J., Iacovino, M., Shi, X., Rasmussen, T.L., Borges, L., Zirbes, K.M., Li, T., Perlingeiro, R.C., Kyba, M., and Garry, D.J. (2012). Etv2 is expressed in the yolk sac hematopoietic and endothelial progenitors and regulates Lmo2 gene expression. Stem Cells 30, 16111623. Lasala, G.P., Silva, J.A., Gardner, P.A., and Minguell, J.J. (2010). Combination stem cell therapy for the treatment of severe limb ischemia: safety and efcacy analysis. Angiology 61, 551556. Lasala, G.P., Silva, J.A., and Minguell, J.J. (2012). Therapeutic angiogenesis in patients with severe limb ischemia by transplantation of a combination stem cell product. J. Thorac. Cardiovasc. Surg. 144, 377382.
The results of qRT-PCR and ow cytometry analysis were analyzed by Students t test or one-way ANOVA (four group comparisons) with a Bonferroni multiple comparison posttest (GraphPad software). A p value <0.05 was considered signicant.
Supplemental Information includes Supplemental Experimental Procedures, seven gures, and four tables and can be found with this article online at http://dx.doi.org/10.1016/j.stemcr. 2013.06.005.
ACKNOWLEDGMENTS
We thank the Cell Sorter Cores of the Alvin J. Siteman Cancer Center (supported in part by an NCI Cancer Centre Support Grant No. P30 CA91842) and the Department of Pathology and Immunology at the Washington University School of Medicine. This work was supported by grants from American Heart Association Postdoctoral Fellowship 10POST4570022 (to F.L.) and National Institutes of Health grants HL63736 and HL55337 (to K.C.). Received: January 16, 2013 Revised: June 13, 2013 Accepted: June 17, 2013 Published: July 25, 2013
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Lee, D., Park, C., Lee, H., Lugus, J.J., Kim, S.H., Arentson, E., Chung, Y.S., Gomez, G., Kyba, M., Lin, S., et al. (2008). ER71 acts downstream of BMP, Notch, and Wnt signaling in blood and vessel progenitor specication. Cell Stem Cell 2, 497507. Lindsley, R.C., Gill, J.G., Murphy, T.L., Langer, E.M., Cai, M., Mashayekhi, M., Wang, W., Niwa, N., Nerbonne, J.M., Kyba, M., and Murphy, K.M. (2008). Mesp1 coordinately regulates cardiovascular fate restriction and epithelial-mesenchymal transition in differentiating ESCs. Cell Stem Cell 3, 5568. Liu, F., Walmsley, M., Rodaway, A., and Patient, R. (2008). Fli1 acts at the top of the transcriptional network driving blood and endothelial development. Curr. Biol. 18, 12341240. Liu, F., Kang, I., Park, C., Chang, L.W., Wang, W., Lee, D., Lim, D.S., Vittet, D., Nerbonne, J.M., and Choi, K. (2012). ER71 species Flk-1+ hemangiogenic mesoderm by inhibiting cardiac mesoderm and Wnt signaling. Blood 119, 32953305. Lugus, J.J., Chung, Y.S., Mills, J.C., Kim, S.I., Grass, J., Kyba, M., Doherty, J.M., Bresnick, E.H., and Choi, K. (2007). GATA2 functions at multiple steps in hemangioblast development and differentiation. Development 134, 393405. Lugus, J.J., Park, C., Ma, Y.D., and Choi, K. (2009). Both primitive and denitive blood cells are derived from Flk-1+ mesoderm. Blood 113, 563566. Minguell, J.J., Allers, C., and Lasala, G.P. (2013). Mesenchymal stem cells and the treatment of conditions and diseases: the less glittering side of a conspicuous stem cell for basic research. Stem Cells Dev. 22, 193203. Moretti, A., Caron, L., Nakano, A., Lam, J.T., Bernshausen, A., Chen, Y., Qyang, Y., Bu, L., Sasaki, M., Martin-Puig, S., et al. (2006). Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversication. Cell 127, 11511165. Motoike, T., Markham, D.W., Rossant, J., and Sato, T.N. (2003). Evidence for novel fate of Flk1+ progenitor: contribution to muscle lineage. Genesis 35, 153159.
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Ar ticle Reprogramming to Pluripotency Using Designer TALE Transcription Factors Targeting Enhancers
Xuefei Gao,1 Jian Yang,1 Jason C.H. Tsang,1 Jolene Ooi,1 Donghai Wu,2 and Pentao Liu1,*
Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China *Correspondence: pl2@sanger.ac.uk http://dx.doi.org/10.1016/j.stemcr.2013.06.002 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
2Key 1Wellcome
SUMMARY
The modular DNA recognition code of the transcription-activator-like effectors (TALEs) from plant pathogenic bacterial genus Xanthomonas provides a powerful genetic tool to create designer transcription factors (dTFs) targeting specic DNA sequences for manipulating gene expression. Previous studies have suggested critical roles of enhancers in gene regulation and reprogramming. Here, we report dTF activator targeting the distal enhancer of the Pou5f1 (Oct4) locus induces epigenetic changes, reactivates its expression, and substitutes exogenous OCT4 in reprogramming mouse embryonic broblast cells (MEFs) to induced pluripotent stem cells (iPSCs). Similarly, dTF activator targeting a Nanog enhancer activates Nanog expression and reprograms epiblast stem cells (EpiSCs) to iPSCs. Conversely, dTF repressors targeting the same genetic elements inhibit expression of these loci, and effectively block reprogramming. This study indicates that dTFs targeting specic enhancers can be used to study other biological processes such as transdifferentiation or directed differentiation of stem cells.
INTRODUCTION
Proper gene expression is a central part of development and a key to cellular homeostasis. Transcription factors (TFs) control gene expression, and a subset of them are regarded as master regulators for lineage development and/or identity maintenance (Spitz and Furlong, 2012). Master regulators often modulate gene expression through enhancers, which are important genetic elements that control the spatial and temporal expression of specic sets of genes (Levine, 2010). Epigenetic patterning of enhancers by the intricate interplay between DNA methylation, specic TFs binding, and histone modications has been demonstrated to occur before cell-fate decisions (Spitz and Furlong, 2012). Therefore, we hypothesized that a more effective and physiologically relevant regulation of gene expression can be achieved by direct manipulation of specic enhancers. Transcriptional-activator-like effectors (TALEs) are natural effector proteins secreted by plant pathogenic bacteria to modulate gene expression in host plants and to facilitate bacterial infection. TALEs contain a modular DNA binding domain consisting of highly similar tandem repeats of 3335 amino acids. The specicity of nucleotide recognition of each repeat is determined by two hypervariable amino acids at positions 12 and 13 (Boch et al., 2009; Cong et al., 2012; Moscou and Bogdanove, 2009; Streubel et al., 2012). The simple coding rule makes TALEs a unique tool to generate programmable effectors targeting a genomic region (Bogdanove and Voytas, 2011). TALE-
based designer transcription activators (A-dTF) or repressors (R-dTF) have been constructed by linking TALEs to activation or repression domains, respectively. These dTFs target specic promoters based on the assumption that the close proximity of the dTFs to the transcription start site (TSS) would modulate transcription (Bultmann et al., 2012; Geissler et al., 2011; Morbitzer et al., 2010; Zhang et al., 2011). Attempts were made to use A-dTFs to activate endogenous pluripotency loci such as Sox2, Klf4, Oct4, and c-Myc (Bartsevich et al., 2003; Bultmann et al., 2012; rez-Moreno et al., 2013; Zhang et al., 2011). For the Jua Oct4 locus, these experiments achieved modest activation but failed to demonstrate any physiological impact in reprogramming or other cellular processes. In this study, we chose the Oct4 and Nanog loci to investigate whether dTFs could regulate gene expression via their specic enhancers and whether the activation or repression could impact reprogramming to induced pluripotent stem cells (iPSCs) or affect embryonic stem (ES) cell differentiation. We report here that direct regulation of the endogenous pluripotency loci by dTFs targeting enhancers enables reprogramming of mouse embryonic broblast cells (MEFs)or epiblast stem cells (EpiSCs) to iPSCs in the absence of exogenous reprogramming factors OCT4 or NANOG. Therefore, dTFs targeting enhancers of genomic loci encoding key regulators can provide an effective approach for reprogramming to pluripotency and potentially for other applications such as transdifferentiation and directed differentiation of stem cells.
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Designer Transcription Factors Reprograms Cells
Figure 1. Reactivation of the Oct4 Locus by dTFs (A) The schematic diagram of TALE proteins and their binding sites at the Oct4 distal enhancer (DE). Color code for the amino acid positions 12 and 13 in a TALE repeat and the corresponding nucleotide in DNA: black NI for A, blue NG for T, red HD for C, and green NN for G. TALE proteins OD2, OD3, and OD4 bind inside the DE region, whereas OD1 and OD5 bind outside the DE. (B) Cloning of TALE protein coding DNA or dTFs into the PB vector. For ChIP analysis testing binding of TALEs to their target sequences, 3 3 HA tag was added at the C terminus of TALE proteins (upper panel). For activator dTFs (A-dTFs), the VP64 was added (lower panel). In all cases, mCherry was coexpressed with TALE proteins or dTFs via the T2A. N and C are the N and C termini of the TALE protein. CAG: the CAG promoter. PB-5TR and PB-3TR are the two ends of the PB transposon. NLS, nuclear localization signal. (C) Validation of TALE binding to the Oct4 locus in ChIP assay using an antibody to HA tag followed by qPCR amplifying the corresponding genomic DNAs. IgG was used as the control. (D) Luciferase assays to measure dTF activities. The 2.4 kb-Luc reporter has the DE, PE, and PP of the Oct4 locus, whereas the DDE construct lacks the DE. (E) qRT-PCR analysis of Oct4 mRNA levels in MEFs expressing the activator dTFs (A-dTFs) alone or plus CKS. All gene expression values are normalized to the expression of Gapdh. (F) Comparison of three dTFs, OD3, OD3-25, and OD3-37, on DNA binding, luciferase activities, and Oct4 mRNA levels induced by them in MEFs. Results are representative of three independent experiments and are mean SD, n = 3. *p < 0.01. See also Figure S1 and Table S1.
RESULTS
A-dTFs Targeting the Oct4 Distal Enhancer Activate the Locus We chose the mouse Oct4 locus to test functionality of dTFs because it is an essential pluripotency factor (Nichols et al., 1998), and reactivation of the locus is a critical step in reprogramming of somatic cells to iPSCs (Polo et al., 2012; Takahashi and Yamanaka, 2006). Specic genetic elements are shown to regulate proper Oct4 expression in stem cells of distinct pluripotent states (Bao et al., 2009; Minucci et al., 1996; Okazawa et al., 1991; Yeom et al.,
1996), namely, the distal enhancer (DE) in murine ES cells and germ cells, the proximal enhancer (PE) in EpiSCs, and the proximal promoter (PP). Because the DE is active specifically in ES cells, we constructed ve dTFs to target ve 19 bp sequences (OD1OD5) inside or outside the DE (Figure 1A; Table S1 available online). OD3 is on the 50 side of the multiple transcription factor binding sites of STAT3, TCF3, OCT4, SOX2, and NANOG in the DE (Chen et al., 2008; Young, 2011) (Figure 1A). We also targeted four 19 bp sequences (PP1PP4) in the Oct4 promoter as controls (Figure S1A). The TALE DNA binding domains were constructed using a modied Golden Gate cloning system
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(Sanjana et al., 2012) (Figure S1B). We next evaluated the DNA-binding property of these TALE proteins. To provide quantitation of binding, we fused 3 3 hemagglutinin (HA) tags with the C terminus of each TALEs (Figure 1B, upper panel). Chromatin immunoprecipitation (ChIP) quantitative real-time PCR analysis of ES cells after expression of the HA-tagged TALEs for 2 days showed all nine TALE proteins bound their intended sequences as indicated by the 7- to 12-fold enrichment compared to the immunoglobulin (Ig) G control (Figure 1C). We next investigated the ability of A-dTFs to activate the Oct4 locus. To make A-dTFs, we fused VP64 (Beerli et al., 1998) to the C terminus of the TALE proteins in a piggyBac delivery vector (Wang et al., 2008). The fusion protein is linked to mCherry by T2A peptide for convenient tracking of TALE protein expression (Figure 1B, lower panel). We rst examined the ability of A-dTFs to activate Oct4 expression in luciferase assay. MEFs were cotransfected with vectors expressing dTFs and a luciferase construct containing the 2.4 kb region covering all three upstream regulatory elements of the Oct4 locus (Figure S1A). Two days after transfection, three A-dTFs targeting the DE (A-OD2, A-OD3, and A-OD4) and three A-dTFs targeting the promoter (A-PP1A-PP3) substantially enhanced luciferase activities compared to the control construct (Figures 1D and S1C). Once the DE was deleted in the luciferase reporter (DDE in Figure S1A), none of the A-dTFs targeting the DE was able to activate the luciferase reporter indicating specicity of A-dTFs for the DE (Figure 1D). On the contrary, A-PP1, A-PP2, and A-PP3 still activated the reporter carrying only the Oct4 promoter region (Figure S1C). Consistent with the luciferase assay, expression of A-OD2, A-OD3, and A-OD4 in MEFs for 48 hr increased the Oct4 mRNA by 3- to 4-fold measured by quantitative RT-PCR (qRT-PCR) (Figure 1E), whereas all four dTFs targeting the promoter achieved lower mRNA levels, in contrast to the luciferase assay (Figure S1D). The Oct4 locus is silenced in MEFs through repressor complexes, chromatin modications, and DNA methylation. Three of the four transcription factors for reprogramming somatic cells to iPSCs, C-MYC, KLF4, and SOX2, are suggested to have roles in chromatin remodeling or bind the DE of the Oct4 locus (Yamanaka, 2008; Young, 2011). Coexpressing A-OD2/3/4 with C-MYC, KLF4, and SOX2 (CKS) in MEFs for 48 hr caused 10- to 20-fold increase of Oct4 mRNA with A-OD3 being the most potent (Figure 1E), indicating a synergistic interaction of dTFs with these transcription factors. In contrast, coexpressing A-PP1A-PP4 with CKS failed to substantially increase Oct4 mRNA levels (Figure S1D), highlighting the signicance of targeting the enhancer, rather than the promoter, in regulating gene expression by dTFs. In an attempt to further improve the potency of A-OD3, we made two new dTFs (A-OD3-25 and A-OD-37) recog-
nizing 25 and 37 bp sequences encompassing the sequence bound by A-OD3 (Figure 1A and Table S1). A-OD3-25 showed the similar DNA binding ability and promoted Oct4 mRNA expression and higher luciferase activities as compared to A-OD3, whereas A-OD3-37 was not competent in both assays (Figure 1F), suggesting that excessive peptide repeats may cause unnatural protein structure because naturally found TALEs have around 20 peptide repeats (Boch et al., 2009; Moscou and Bogdanove, 2009). To exclude the possibility that expression of the endogenous Oct4 in MEFs was due to general chromatin remodeling in the Oct4 genomic region by the VP64 domain, we examined expression of several neighboring genomic loci on the mouse chromosome 17, including Tcf19, Cchcr1, and H2Q-10 (Figure S1E), as well as Kcnk18, which has a stretch of DNA sequence (ACCCTGCCCCTCC) that is similar to the 19 bp region targeted by A-OD3 as shown in Figure 1A. The expression of these four loci were not substantially altered either by expression of A-OD3 alone or in combination with CKS (Figure S1F). Activation of the Oct4 Locus by dTFs Reprograms MEFs to iPSCs in Concert with C-MYC, KLF4, and SOX2 We next explored whether the endogenous Oct4 activation induced by dTFs has functional consequences and attempted reprogramming MEFs to iPSCs in the absence of exogenous OCT4. The PB (piggyBac) vectors containing doxcycline (Dox)-inducible dTFs and CKS were delivered to Oct4-GFP reporter MEFs via the piggyBac transposition (Figure S2A) (Wang et al., 2011). Expression of the reprogramming factors was induced by adding Dox in the medium (Figure 2A). PB transposition is efcient in mammalian cells (Wang et al., 2011); approximately 4% of MEFs survived electroporation and expressed the transgenes in the genome. As early as 5 days after transfection and Dox induction, microscopic GFP+ colonies (also mCherry+) were visually identiable in the combination of A-OD3 plus CKS or A-CKS (Figure 2B), whereas no GFP+ colonies were found in the control combination of exogenous OCT4 plus CKS (or OCKS) or A-PPs plus CKS, until day 11. We thus chose A-OD3 in the subsequent characterization of its function in reprogramming and in ES cells. Despite the rapid reactivation of endogenous Oct4 expression, A-OD3 was not sufcient to substantially enhance the reprogramming process compared to exogenous OCT4 because the combination OCKS caught up in terms of GFP+ colonies number at the late stage of reprogramming. On day 13, there were on average 68 GFP+ colonies in A-CKS dish compared to 141 colonies in the OCKS combination in three independent experiments (Figure 2C), consistent with the notion that high exogenous OCT4 levels facilitate late stages of reprogramming (Carey et al., 2011). On the other hand, although none of
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Figure 2. A-OD3 Replaces Exogenous OCT4 in Reprogramming Oct4-GFP MEFs to iPSCs (A) The time line for reprogramming MEFs to iPSCs using dTFs. MEFs under reprogramming were analyzed at several time points for various assay. The iPSC colonies were scored and picked on day 23 or 25. (B) Activation of the endogenous Oct4 locus detected by GFP expression. mCherry+ cells were imaged on day 5 after transfection. Scale bar: 200 mm. (legend continued on next page)
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A-PPs in combination with CKS caused rapid Oct4 locus reactivation, they eventually produced GFP+ colonies, with a reprogramming pattern similar to OCKS but with fewer colonies (Figure 2C; Table S2). The result suggested that A-PPs were also capable of inducing endogenous Oct4 reactivation in cooperation with CKS despite of a slower kinetics, potentially due to lower OCT4 expression. On day 5 and 11, 40% and 78% mCherry+ cells expressing A-CKS became GFP+. Expression of the endogenous OCT4 was conrmed in mCherry+ MEFs by immunostaining (Figure S2B). In contrast, no GFP+ cells were detected in cells expressing OCKS on day 5 and only 48% mCherry+ cells became GFP+ on day 11 (Figure 2D). To further investigate reactivation of the Oct4 locus by AOD3, the GFP+ cells from Oct4-GFP MEFs were harvested by uorescence-activated cell sorting (FACS) and analyzed as soon as they appeared. In the day 5 GFP+ cells reprogrammed by A-CKS, the Oct4 promoter started to be demethylated, but the locus was the only one activated among several pluripotency loci examined include Nanog, Zfp42 (Rex1), and Dppa3 (Stella) (Figures 2E and 2F). On the other hand, on day 11, GFP+ cells of both A-CKS and OCKS expressed low levels of key pluripotent genes besides the endogenous Oct4 (Figure 2F). Moreover, DNA demethylation was detected in the promoters of both the Oct4 and Nanog (Figure 2E). Therefore, rapid reactivation of the Oct4 locus facilitated by A-OD3 represents a necessary yet insufcient step in reprogramming. Additional epigenetic barriers at other key pluripotency loci still need to be overcome at the late stage of reprogramming (Plath and Lowry, 2011). Nevertheless, reactivation of the endogenous Oct4 locus by A-OD3 in MEFs under reprogramming marked the cells that were destined to become iPSCs. We ow-sorted cells expressing either A-CKS or OCKS (mCherry+) into three cell populations, GFPhigh, GFPlow, and GFP, on day 11. Cells were collected, counted, and replated (600 cells) on feeder cells to allow them to continue reprogramming (Figures 2A and 2G). qRT-PCR analysis conrmed the correlation between GFP expression and the endogenous Oct4 mRNA
level (Figure 2H). Interestingly, in cells expressing A-CKS, the GFPhigh cells formed 53 AP+ colonies (70% of the total colonies), and the rest (about 20 AP+ colonies) originated from GFPlow cells (Figure 2I). The GFP cells did not produce any colonies. On the other hand, AP+ colonies were formed from all the three cell populations expressing OCKS, with 48% (72) from GFPhigh, 45% (67) from GFPlow, and 7% (10) from GFP cells (Figure 2I). These results demonstrated that the levels of the endogenous Oct4 expression induced by the dTF were more predictive for successful reprogramming compared to expressing exogenous Oct4. Endogenous Oct4 activation is a critical and major limiting step in somatic cell reprogramming (Boiani et al., 2002; Hochedlinger and Plath, 2009). To investigate whether the reactivation of the endogenous Oct4 locus by A-OD3 could enhance reprogramming of somatic cells by the standard four Yamanaka factors OCKS, we cotransfected Oct4-GFP reporter MEFs with Dox-inducible expression vectors of OCKS and A-OD3 (A-OCKS). Coexpression of these factors produced GFP+ cells as early as 3 days after Dox induction (Figure S2C), indicating an even faster reactivation of the Oct4 locus comparing to A-CKS. Additionally, A-OCKS also produced more AP+ colonies (Figure S2D). Rex1 is expressed in mouse ES cells but not in EpiSCs and represents a better marker for ground-state pluripotency or for monitoring late stages of reprogramming (Brons et al., 2007; Tesar et al., 2007; Toyooka et al., 2008). To further demonstrate A-OD3s function in reprogramming, we repeated the experiments using the Rex1-GFP reporter MEFs where the GFP-IRES-Puro cassette was inserted into the Rex1 locus (Guo et al., 2011). iPSCs from these MEFs would be both GFP+ and Puror. In contrast to the rapid reactivation of the Oct4 locus in the aforementioned experiments, A-CKS only slightly accelerated reactivation of the Rex1 locus in the reporter MEFs, with GFP+ colonies appearing on day 20 compared to day 22 for the OCKS control (Figure 3A), again demonstrating that rapid reactivation of the Oct4 locus alone by A-OD3 is an early event in reprogramming. Dox was subsequently withdrawn after 14 days to select for Dox- or exogenous-factor-independent
(C) Quantitation of GFP+ colonies from MEFs expressing dTFs targeting the DE (A-OD3) or the promoter (A-PP1) at various time points of reprogramming. (D) mCherry+ cells were analyzed for GFP expression in ow cytometry on days 5 and 11. (E and F) The GFP+ cells were harvested by ow sorting and analyzed for DNA demethylation in the Oct4 and Nanog promoters and for gene expression. The percentages in (E) are the demethylated CpG in the promoters. (G) The reprogramming potential of MEFs with a reactivated Oct4 locus. Oct4-GFP MEFs under reprogramming were sorted into three populations based on GFP intensity on day 11. Six hundreds cells of each population were replated into a 6-well plate to allow colony formation. (H) Endogenous Oct4 expression in the three cell populations measured by qRT-PCR. (I) AP+ colony numbers from the replated cells scored on day 25. All gene expression levels are normalized to Gapdh. Results are representatives of three independent experiments and are mean SD. n = 3. *p < 0.01. yp < 0.05 A-CKS compared to OCKS. See also Figure S2 and Tables S2, S3, and S4.
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F G
Figure 3. Characterization of iPSCs Reprogrammed by A-CKS (A) GFP+ colonies from Rex1-GFP MEFs by A-CKS or OCKS at several time points during reprogramming. (B and C) Reprogramming of Rex1-GFP MEFs using various combinations of A-OD3 and the Yamanaka factors. Dox-independent Puro+ colonies were scored 25 days after transfection. (D) Detection of leaky expression in iPSC lines reprogrammed using Dox-inducible A-CKS. Primers specic for the exogenous CKS or for A-OD3 were used in RT-PCR. The three lines shown have no detectable exogenous factor expression in the absence of Dox. (E) Immunostaining of iPSC colonies for NANOG and SSEA1. DNA was stained with propidium iodide. Scale bars: 200.0 mm. (F) qRT-PCR analysis of expression of several pluripotency genes in iPSC line #3 and #5 reprogrammed by A-CKS. (G) iPSCs reprogrammed by A-CKS are able to differentiate to cells of all three germ layers in vitro. Antibodies used are as follows: neuronspecic class III b-tubulin; SMA (smooth muscle a-actin) and AFP (a-fetoprotein). Scale bars: 200.0 mm. (H) Chimera mouse generated using iPSC line #3 expression of Gapdh was used as the control in RT-PCR. Results are representatives of three independent experiments and are mean SD. n = 3. *p < 0.01. yp < 0.05 A-CKS compared to OCKS. See also Figure S3 and Tables S3 and S4.
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Figure 4. Changes of Histone H3 Modications at the Oct4 Locus Induced by A-OD3 (A) Differentiation of iPSCs produced by Dox-inducible OCKS or A-CKS and reexpression of the exogenous factors. (BE) Histone H3 modications H3K27me3 (B), H3K4me1 (C), H3K27ac (D), and H3K4me3 (E), were analyzed in the ChIP assay followed by qPCR. The relative enrichments were normalized to IgG, and a genomic region at the Tyr locus was used as the unrelated locus control. Values in x axis indicate the locations of PCR primers used qPCR in the ChIP assay. 0.3: 0.3 kb upstream of the TSS. Results are representative of three independent experiments in three cell lines and are mean SD. n = 3. yp < 0.05 A-CKS compared to OCKS. See also Figure S4 and Table S3.
colonies (Figure 2A). A-CKS produced around 60 Puror or GFP+ colonies per transfection of one million MEFs, whereas OCKS transfection produced about 120 such colonies (Figure 3A). On the other hand, A-OD3 was unable to effectively substitute either SOX2 or KLF4 in reprogramming (Figures 3B and 3C). We next examined Rex1 locus reactivation by A-OD3 plus OCKS or A-OCKS. Again Rex1-GFP+ colony appeared on day 20, slightly earlier than the OCKS control (Figures S3A and S3B). Reprogramming Rex1-GFP MEFs by A-OCKS also consistently led to roughly 1.5-fold more Puro+ colonies than expressing OCKS alone (Figure S3A). Therefore, even in the presence of exogenous Oct4, early reactivation of the endogenous Oct4 locus by the dTF still promoted reprogramming. iPSC Reprogrammed by A-CKS Are Pluripotent From the Puror iPSCs colonies produced by A-CKS, we picked 36 for characterization. From these 36 lines, seven were found not to express any of the exogenous reprogramming factors (Figure 3D), whereas the other lines still had expression due to leakiness of the Tet/On system. These nonleaky iPSCs were characterized in vitro and in vivo for their pluripotency. Both immunostaining and qRT-PCR analyses demonstrated that these
exogenous-factor- independent iPSC lines expressed key pluripotency genes at levels comparable to that in mouse ES cells (Figures 3E and 3F). The iPSCs retained the normal karyotype after 16 passages (Figure S3C). In vitro differentiation of the iPSCs produced somatic cell types representing all three germ layers (Figure 3G). Finally, chimeric mice were obtained using these iPSCs conrming their pluripotency in normal development (Figure 3H). The dTF Activator A-OD3 Causes Rapid Histone Modication Changes at the Oct4 Locus In ES cells, the Oct4 locus is marked by active histone modications such as H3K27 acetylation and H3K4 trimethylation, whereas, in MEFs, the Oct4 locus is transcriptionally repressed and is tightly packaged into nucleosomes marked by H3K9me3 and H3K27me3 (Mikkelsen et al., 2007). We next investigated the impacts of A-OD3 on histone modications at the Oct4 locus. To this end, we differentiated iPSC lines obtained by using Dox-inducible A-CKS or OCKS by all trans-retinoic acid for 14 days, and all the differentiated cells lost expression of pluripotency markers. Expression of the reprogramming factors was then induced, and cells were collected on days 0, 2, and 6 for ChIP analysis (Figure 4A).
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Many putative enhancer elements have been mapped in the genomes by their association with specic histone modications (Ong and Corces, 2011). We examined H3K4me1, H3K4me3, H3K27me3, and H3K27ac at eight specic sites in the 3.4 kb region upstream of the Oct4 locus TSS by ChIP assay. This genomic region encompasses the DE, PE, and PP. Compared to the OCKS, expression of A-CKS rapidly reduced H3K27me3 levels (Figure 4B) concomitant with increased levels of the active markers H3K4me1 (Figure 4C), H3K27ac (Figure 4D), and H3K4me3 (Figure 4E), as early as 2 days after Dox induction. In contrast, OCKS only induced similar changes six days after Dox induction (Figures S4AS4D). The dTF Repressor R-OD3 Targeting the Oct4 Distal Enhancer Induces ES Cell Differentiation The effectiveness of A-OD3 to reactivate the Oct4 locus prompted us to investigate whether a repressor targeting the same genetic element could negatively regulate the locus. We replaced the VP64 domain in A-OD3 and A-OD1 with the KRAB repressor domain of KOX1 (Margolin et al., 1994) to make mCherry-tagged Dox-inducible R-OD3 and R-OD1, which targets a region upstream of the distal enhancer as a control. We next tested the repressors in Oct4-GFP ES cells. In cells expressing R-OD3, the mCherry+ cells became GFPdim or GFP as soon as 3 days after Dox induction (Figure 5A). In contrast, R-OD1 had no obvious effect because the mCherry+ ES cells were still GFP+. We harvested mCherry+ cells by FACS at different time points of Dox induction and analyzed expression of Oct4 via either GFP expression or transcription level. After 3 days of R-OD3 expression, Oct4 mRNA levels were substantially decreased, and, on day 5, it was at about 10% of that in wild-type ES cells (Figure 5B). Flow cytometric analysis conrmed that on day 5, 86% of mCherry+ ES cells became GFP (Figure 5C). Concomitantly, Nanog, which is a target of OCT4, was also markedly downregulated in ES cells expressing R-OD3 (Figure 5B). By contrast, expression of R-OD1 did not noticeably decrease Oct4 mRNA or substantially increase GFP cells (Figures 5B and 5C).
Morphologically, the mCherry+GFP cells differentiated into trophectoderm-like cells and expressed high levels of Cdx2 and Eomes (Nichols et al., 1998; Niwa et al., 2005) (Figure 5D). ChIP analysis showed that ES cells stably expressing R-OD3 for 3 days (Figure 5E) had decreased levels of H3K27ac and increased H3K27me3 at the Oct4 locus, indicating silencing of the locus (Figures 5F and 5G). Expression of R-OD1, on the other hand, did not cause similar changes (Figures S5A and S5B). These results clearly demonstrated the effectiveness of the dTF repressor and also conrmed the essential role of the Oct4 distal enhancer in pluripotency. The dTF Repressor R-OD3 Targeting the Oct4 Distal Enhancer Blocks Reprogramming The effective repression of the Oct4 locus by R-OD3 provided an opportunity to examine the consequence of keeping the Oct4 locus inactive in reprogramming. Two experimental approaches were taken. In the rst case, we reprogrammed Rex1-GFP MEFs by expressing CKS and LRH1 (CKSL) under the constitutive active CAG promoter as LRH1 is reported to replace exogenous OCT4 in reprogramming by binding and activating the Oct4 locus (Heng et al., 2010). Expressing CKSL produced 44 GFP+ colonies scored 22 days after induction (Figure 6A), whereas coexpression of R-OD3 with CKSL produced no mCherry+GFP+ colonies (Figure 6B). Suppression of the Oct4 distal enhancer by R-OD3 therefore effectively blocked reprogramming. R-OD1, on the other hand, did not affect reprogramming. In the second approach, we reprogrammed Oct4-GFP MEFs by CAG-OCKS (constitutive expression) and Doxinducible R-OD3 (Figure 6C). In the presence of exogenous OCT4, reprogramming was not affected by R-OD3 (Figure S6). The iPSCs obtained expressed pluripotency genes at levels comparable to that in ES cells (Passage 0 in Figure 6D), except endogenous Oct4, which was suppressed by R-OD3. It further conrmed the effectiveness of repression of the Oct4 locus by R-OD3. We next examined the reversibility of R-OD3 repression on the Oct4 locus by withdrawing Dox and thus turning
Figure 5. Repressor dTF R-OD3 Blocks the Oct4 Locus Expression (A) Images of Oct4-GFP ES cells expressing two repressor dTFs: R-OD3 and R-OD1. Cells expressing dTFs are mCherry+. (B) Oct4 and Nanog expression in ES cells expressing R-OD3 or R-OD1 detected in qRT-PCR. (C) Flow cytometric analysis of Oct4-GFP ES cells on days 1 and 5 following expression of repressor dTFs (gated for mCherry+). (D) Differentiation of ES cells to trophoblast-like cells caused by R-OD3 and expression of Cdx2 and Eomes in these cells. (E) Diagram showing the PB vector expressing Dox-inducible R-OD3 for making a stable ES cell line. The repressor R-OD1 serves as the negative control. (F and G) Epigenetic changes at the Oct4 locus in ES cells expressing R-OD3 for 3 days measured in ChIP assay at the Oct4 locus. The relative enrichments were normalized to IgG, and a genomic region at the Tyr locus was used as the unrelated locus control. Values in x axis indicate the locations of PCR primers used in ChIP assay. 0.3: 0.3 kb upstream of the TSS. Scale bars: 200 mm. Results are representative of three independent lines and are mean SD. n = 3. *p < 0.01. yp < 0.05 day 5 compared to day 0. See also Figure S5 and Tables S3 and S4.
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A
GFP+ colony #
60
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Rex1 CKSL/ R-OD1 R-ODs Phase
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2A c-Myc 2A
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Electroporation +Dox
Picking mCherry+ colonies Passaging initially in Dox medium Day 23-25
Day 0 Oct4-GFP MEFs
D
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2.5 2 1.5 1 0.5 0
Figure 6. R-OD3 Suppresses the Oct4 Locus and Blocks Reprogramming (A) Reprogramming of Rex1-GFP MEFs to iPSCs by CKS plus LRH1 (CKSL) in the presence of repressor dTF R-OD3 or R-OD1. (B) The small number of colonies reprogrammed by CKSL in the presence of R-OD3 (mCherry+) were all GFP, indicating blocking of reprogramming. (C) Reprogramming of Oct4-GFP MEFs using CAG-OCKS and Dox-inducible R-ODs. mCherry+ iPSC colonies were picked and expanded in the presence of Dox. (D) Analysis of expression of endogenous Oct4, Nanog, and Zfp42 (Rex1) in iPSCs reprogrammed in (C) in either the presence (passage 0) or absence of Dox (passages 13). Expression in ES cells was used as the control. (E) Reactivation of the Oct4 locus monitored by GFP expression in iPSCs obtained in (C) once Dox was withdrawn. iPSCs became mCherry and GFP+ within three passages. All gene expression values are normalized to the expression of Gapdh. Scale bars: 200 mm. Results are representative of three independent experiments and are mean SD. n = 3. *p < 0.01. See also Figure S6 and Table S4.
Nanog
Oct4 (endo)
Zfp42
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Passage 0
off R-OD3 expression in iPSCs obtained above. The endogenous Oct4 mRNA gradually reached 30% of that in ES cells at passage 2 and reached similar levels at passage 3 (Figure 6D) as the cells switched from mCherry+/GFP to mCherry/GFP+ (Figure 6E). However, it should be noted that the continuous expression of exogenous factors in these iPSCs could inuence the repression reversibility in this experiment. Regulation of the Nanog Locus by dTFs Targeting the 5 kb Enhancer We next extended our ndings of enhancer regulation by dTFs to the Nanog locus. Studies of Nanog expression regulation have revealed an enhancer located at approximately 5.0 kb upstream of its TSS, which is a DNase I-hypersensi-
tive site and bound by OCT4, NANOG, SOX2, and ZFP281 (Levasseur et al., 2008; Loh et al., 2006). We rst made three A-dTFs (A-ND1, A-ND2, and AND3) targeting the respective 19 bp sequences inside or outside the 5 kb enhancer region (Figure S7A; Table S1). Luciferase assay in MEFs showed that A-ND2 could increase luciferase activities by more than 3-fold compared to the control (Figure S7B). EpiSCs are pluripotent cells established from developing epiblasts and express lower levels of NANOG (Guo et al., 2009; Silva et al., 2009). Exogenous Nanog transgene reprograms EpiSCs to naive iPSCs (Silva et al., 2009). To examine whether A-NDs were able to increase Nanog expression in EpiSCs and perhaps also to reprogram EpiSCs to iPSC, we transfected EpiSCs by lipofection with a PB construct
Stem Cell Reports

Figure 7. Regulation of the Nanog Locus by dTFs Targeting the 5 kb Enhancer (A) Expression of dTFs in reprogramming EpiSCs. The transfected EpiSCs were collected on day 2 for several assays including qPCR, qRT-PCR, and ChIP analysis, or allowed to be reprogrammed to iPSCs. (B) Nanog mRNA levels in EpiSCs expressing A-NDs. (C) H3K27ac levels at the Nanog locus in EpiSCs expressing A-NDs. (D) Reprogramming Oct4-GFP reporter EpiSCs to iPSCs by A-ND2. (E) GFP+ iPSC colonies from EpiSCs by A-ND2. (F) qRT-PCR analysis of several pluripotency genes in iPSCs reprogrammed by A-ND2. (G) Chimera derived from iPSCs from EpiSCs by A-ND2. (H) Decrease of Nanog mRNA levels in ES cells expressing R-ND2 in qRT-PCR analysis. (I) Efcient reprogramming of EpiSCs to iPSCs by Klf4 (K), which was suppressed by R-ND2 (R). Expressing a Nanog transgene rescues reprogramming (K+N+R-ND2). All gene expression values are normalized to the expression of the Gapdh gene. Scale bars: 200.0 mm. Results are representative of three independent experiments and are mean SD. n = 3. *p < 0.01. yp < 0.05 day 2 compared to day 0. See also Figure S7 and Tables S3 and S4.
expressing the Dox-inducible A-NDs (Figure 7A). The transfection and the PB transposition efciencies were estimated to be about 15% and 1%2%, respectively. Among the three dTFs, A-ND2 increased Nanog expression by 3-fold and reached comparable Nanog levels found in mouse ES cells (Figure 7B). A-ND2 also caused rapid epigenetic changes at the Nanog locus with H3K27ac levels being substantially increased only 2 days induction (Figure 7C), whereas A-ND1 had no obvious effects (Figure S7C). We then investigated A-ND2 in reprogramming Oct4-GFP EpiSCs (Guo et al., 2009) to iPSCs (Figure 7A). Expression of A-ND2 produced 21 iPSC colonies compared to 30 colonies when exogenous Nanog was expressed (Figures 7D and 7E). A-ND1 or A-ND3, however, did not yield any colony. iPSC lines generated by A-ND2 expressed key pluripotency genes at levels comparable to that in mouse ES cells
(Figure 7F). Adult chimeras were also derived from these iPSCs (Figure 7G). Therefore, the dTF targeting to the Nanog 5 kb enhancer was able to activate the locus and reprogram EpiSCs to iPSCs. We also made R-ND2 from A-ND2 by replacing the VP64 domain with the KRAB domain and investigated Nanog expression in ES cells. We transfected Nanog-GFP reporter mouse ES cells cultured in serum containing medium with the mCherry-tagged R-ND2 PB transgene. In ow cytometric analysis, more than 80% of mCherry+ ES cells became GFP/dim in 5 days indicating loss of Nanog expression (Figure S7D). Suppression of Nanog expression was conrmed in qRT-PCR, which showed that only 25% of Nanog mRNA left in cells expressing A-ND2 for 5 days (Figure 7H). The essential functions of NANOG for acquisition of ground-state or naive pluripotency have been
Stem Cell Reports

demonstrated in Nanog-decient ES cells (Silva et al., 2006, 2009). EpiSCs express little KLF4. Exogenous KLF4 reprograms EpiSCs to naive iPSCs (Guo et al., 2009). We re-examined the requirement of NANOG in KLF4-mediated EpiSCs reprogramming to iPSCs using R-ND2. We introduced a PBCAG-Klf4 transgene to EpiSCs via the PB transposition, which produced around 170 iPSC colonies scored on day 14 (Figure 7I). By contrast, if R-ND2 was coexpressed with the Klf4 transgene (Klf4 plus R-ND2 or K+R), fewer than ten colonies were obtained (Figure 7I), and none of them were mCherry+GFP+. Repressing Nanog by R-ND2 in KLF4-mediated EpiSC reprogramming was partially rescued using a Nanog transgene (K+R+N) (Figure 7I), conrming the essential function of NANOG in reprogramming EpiSCs to iPSCs. In summary, targeting the Nanog 5 kb enhancer by dTFs also enabled manipulation of the endogenous locus for reprogramming to pluripotency.
DISCUSSION
Regulation of gene expression is central in development and in homeostasis and is achieved by both cis- and trans-elements. Enhancers dictate the spatial and temporal patterns of gene expression during development and can drive progenitor cells to distinct cell fates. Recent studies have shown that cell-fate decisions and lineage commitment are regulated by epigenetic patterning at enhancers (Ong and Corces, 2011). One of the best-characterized enhancers in ES cells is the distal enhancer of the Oct4 locus, which controls Oct4 expression in ES cells and PGCs (Bao et al., 2009; Minucci et al., 1996; Yeom et al., 1996) and is marked by active histone modications and bound by multiple key pluripotency transcription factors (Chen et al., 2008; Young, 2011). We decided to target the Oct4 distal enhancer as a proof of principle for dTFs to regulate a key pluripotency locus. Previous attempts to activate the Oct4 expression were focused on targeting dTFs to the promoter, which only activated its expression in reporter assays but not effectively in MEFs or other somatic cells (Bultmann et al., 2012; Zhang et al., 2011), an observation that we were able to reproduce in this study. In contrast, A-OD3, which targets the region close to the binding sites of OCT4, SOX2, and NANOG at the distal enhancer, induces rapid histone modication changes and efciently reactivates the locus in MEFs. These results are consistent with a recent study that, in reprogramming, OCT4, SOX2, and KLF4 act as pioneer factors at enhancers of genes that promote reprogramming (Sou et al., 2012). Indeed, A-OD3, working together with SOX2, KLF4, and C-MYC, reprograms MEFs to bona de iPSCs, bypassing the need of exogenous OCT4. Furthermore, the rapid reac-
tivation of endogenous OCT4 by A-OD3 enhances reprogramming in the context of exogenous OCT4, SOX2, KLF4, and C-MYC. Besides replacing exogenous OCT4, using A-OD3 has helped reveal new insight in reprogramming. Endogenous Oct4 reactivation is believed to be an essential landmark and a bottleneck step for reacquisition of pluripotency (Kim et al., 2009) and being the only reprogramming factor recalcitrant to substitution by a family member (Nakagawa et al., 2008). Yet, we show here that early reactivation of the Oct4 locus alone by dTFs is not sufcient to complete reprogramming. Additional epigenetic changes in other pluripotency loci are still required despite robust reactivation of endogenous Oct4 in MEF cells. We used the VP64 transactivation domain to generate dTF activators. VP64 at the Oct4 distal enhancer would presumably recruit and interact with histone acetyltransferase p300 and transcriptional activation complexes (Ito et al., 2000; Milbradt et al., 2011) and induce epigenetic changes that facilitate binding of additional factors such as OCT4 itself at the distal enhancer. Replacing VP64 with the KRAB domain in the dTFs produces repressor dTFs. R-OD3 suppresses the Oct4 locus and induces ES cell differentiation and blocks reprogramming. The repression by R-OD3 could be reversed by coexpressing exogenous OCT4, SOX2, KLF4, and C-MYC in iPSCs. In addition to the Oct4 locus, a dTF targeting to the 5 kb Nanog enhancer also allows efcient regulation of this locus. The activator alone reprograms EpiSCs to iPSCs, whereas the repressor suppresses Nanog expression and permits dissection of NANOG requirements in reprogramming. This proof-of-principle study demonstrates that targeting key regulatory elements such as enhancers of key genes is an effective way to regulate their expression. dTFs could mimic the complicated transcription regulation by recruiting physiologically relevant factors to a specic locus. Reprogramming somatic cells to iPSCs is an inefcient process. Using dTFs rather than native transcription factors could eventually prove to be an alternative or even more efcient reprogramming approach. It can be envisioned that in the future a combination of dTFs (activators and repressors) targeting loci encoding master regulators could enable cellular transdifferentiation or direct stem cells to a specic cell lineage as master regulators for a number of lineages have been extensively studied. Two recent studies reported that one could achieve tunable gene activation by combinations of dTFs targeting the promoters (Maeder et al., 2013; Perez-Pinera et al., 2013); a similar approach may also be feasible to regulate enhancers. With the advances of next-generation sequencing technologies, genome-wide mapping of regulatory elements have identied thousands of enhancers and other elements
Stem Cell Reports

(Shen et al., 2012). Functional validation of these enhancers to investigate their roles in specic cell types or developmental stages presents a challenge. Advances in TALE cloning technologies now enable high-throughput assembly of dTFs at low cost (Reyon et al., 2012). dTFs may therefore provide a solution to functionally dissect the newly identied enhancers, including the recently reported super-enhancers (Whyte et al., 2013), in vitro or in vivo.
Supplemental Information includes Supplemental Experimental Procedures, seven gures, and ve tables and can be found with this article online at http://dx.doi.org/10.1016/j.stemcr. 2013.06.002.
ACKNOWLEDGMENTS
We thank the Sanger Institute RSF (James Bussell, Andrea Kirton, Michael Robinson, Robert Ellis, Sophie Jolley, and Marie Hitcham), Cytogenetic core (Fengtang Yang and Beiyun Fu), ow cytometry core facility (Bee-Ling Ng and William Cheng), Rebecka Kiff for technical assistance, Dr. Feng Zhang (Broad Institute of MIT and Harvard) for providing TALE repeat plasmids, and Professor Austin Smith, Dr. Jennifer Nichols, and Dr. Ge Guo for Oct4-GFP EpiSCs and Nanog-GFP ES cells. Dr. David Ryan critically read the manuscript. This work is supported by Wellcome Trust (grant number 098051). X.G. designed and did most of the experiments, analyzed and interpreted data, made all gures, and contributed to the writing of the manuscript; J.Y., J.T., J.O., and D.W. (supported by National Basic Research Program of China 2010CB945500 and the Strategic Exploration Grant of Stem Cells XDA01020303) did experiments, provided reagents, or provided intellectual input; and P.L. designed the experiments, supervised the research, and wrote the manuscript. Received: March 23, 2013 Revised: June 4, 2013 Accepted: June 5, 2013 Published: July 11, 2013
Mice
Housing and breeding of mice and experimental procedures using mice were according to the UK 1986 Animals Scientic Procedure Act and local institute ethics committee regulations.
Mouse ES and iPSC Culture

Mouse ES cells and iPSCs were normally cultured in M15 medium: knockout DMEM, 15% FBS (HyClone), 1 3 glutamine-penicillinstreptomycin (Invitrogen), 1 3 Nonessential Amino Acids (NEAA; Invitrogen), 0.1 mM b-mercaptoethanol (b-ME; Sigma), and 106 U/ml LIF (Millipore). We also cultured iPSCs in the chemically dened medium N2B27/2i/LIF.
Transfection of MEFs and Reprogramming to iPSCs

MEFs were prepared from 13.5 day postcoitum mouse embryos and were cultured in M10 (knockout DMEM plus 10% of fetal calf serum). MEFs were transfected by Amaxa Nucleofector (Lonza) program A-023 and were seeded on STO feeder cells for reprogramming.
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Established Oct4-GFP reporter EpiSCs (Guo et al., 2009) were cultured in N2B27/Activin/bFGF at the density of 6 3 105 cells per well in a 6-well plate coated with human bronectin for Lipofectamine 2000 (Invitrogen) transfection. Twenty-four hours after transfection, EpiSCs were split at 1:6 in 6-well plate and cultured in EpiSC culture medium containing Dox (2 mg/ml) for 1 day before the culture medium was changed to N2B27/2i/LIF and Dox (2 mg/ml). The medium was changed every 2 days. The GFP+ iPSC colonies were counted on day 14 posttransfection. Transfection and PB transposition efciencies were calculated similar to in MEFs.
ChIP Analysis
IgG and antibodies for the HA tag, H3K4me3, H3K4me1, H3K27ac, and H3K27me3 were used for ChIP analysis.
Statistical signicance was determined using a Students t test with two-tailed distribution. p values < 0.05 were considered as signicant. Data are shown as mean SD. Supplemental Information and Tables S1S5 include further details of materials and methods.
Stem Cell Reports

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Stem Cell Reports

Ar ticle Do Pluripotent Stem Cells Exist in Adult Mice as Very Small Embryonic Stem Cells?
Masanori Miyanishi,1,3 Yasuo Mori,1,3 Jun Seita,1 James Y. Chen,1 Seth Karten,1 Charles K.F. Chan,1 Hiromitsu Nakauchi,2 and Irving L. Weissman1,*
1Institute 2Division
for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, Tokyo University, Tokyo 108-8639,
Japan 3These authors contributed equally to this work *Correspondence: irv@stanford.edu http://dx.doi.org/10.1016/j.stemcr.2013.07.001 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
SUMMARY
Very small embryonic-like stem cells (VSELs) isolated from bone marrow (BM) have been reported to be pluripotent. Given their nonembryonic source, they could replace blastocyst-derived embryonic stem cells in research and medicine. However, their multiple-germ-layer potential has been incompletely studied. Here, we show that we cannot nd VSELs in mouse BM with any of the reported stem cell potentials, specically for hematopoiesis. We found that: (1) most events within the VSEL ow-cytometry gate had little DNA and the cells corresponding to these events (2) could not form spheres, (3) did not express Oct4, and (4) could not differentiate into blood cells. These results provide a failure to conrm the existence of pluripotent VSELs.
INTRODUCTION
During normal development, pluripotent cells from the inner cell mass give rise to several types of tissue-committed stem cells (TSCs), restricted in their differentiation potential. TSCs can self-renew and produce downstream progenitors and mature cells throughout life. It remains controversial (Wagers et al., 2002; Wagers and Weissman, 2004) whether some pluripotent cells self-renew as pluripotent stem cells (PSCs) and persist after birth (Beltrami et al., gler et al., 2004; 2007; Check, 2007; Jiang et al., 2002; Ko Krause et al., 2001; Serani et al., 2007). One group has recently identied bone marrow (BM)residing very small embryonic-like stem cells (VSELs) in both human and mouse as a putative nonembryonic source for PSCs (Druka1a et al., 2012; Kucia et al., 2006b; Wojakowski et al., 2009; Zuba-Surma et al., 2011). Mouse VSELs have been characterized to: (1) be very small (35 mm), (2) have a CD45Lineage(Lin) SCA-1+ phenotype, (3) express pluripotent marker genes (e.g., Oct4, Nanog), and (4) when cultured on the myoblast C2C12 cell line, form embryoid body-like spheres and then differentiate into multi-germ-layer cells. Moreover, these cells were reported to be directly obtainable from BM or mobilized peripheral blood without culture. Thus, their pluripotency should be reproducible in independent laboratories, a requirement for a scientic nding to be generally accepted (Jasny et al., 2011). However, recent studies have shown the lack of stem cell characteristics in VSELs isolated from mouse (Szade et al., 2013) or human cord blood (CB) (Danova-Alt et al.,
2012), while another study conrmed the existence of mouse VSELs and their ability to give rise to lung cells (Kassmer et al., 2013). We sought to recapitulate the previous ndings on relating to mouse VSELs, focusing on the DNA content of the small-sized fraction and on its hematopoietic lineage potential. We discovered an anomaly in separating cells by forward scatter (FSC) using different types of cell sorters and, more signicantly, could not nd PSCs within the VSEL fraction of mouse BM.
RESULTS
Identifying and Characterizing Candidate VSEL Cells We sought to identify VSELs from mouse BM by using commonly reported VSEL characteristics (Kucia et al., 2006a; Ratajczak et al., 2011; Zuba-Surma et al., 2008). We fractionated BM samples using uorescence-activated cell sorting (FACS). Dead cells and debris were excluded as Ratajczak and colleagues previously reported (ZubaSurma et al., 2008). To minimize the risk of missing candidate cells, SCA-1+ cells (including SCA-1lo) beyond the threshold dened by the uorescence minus one (FMO) method (Herzenberg et al., 2006) were included, and only the obvious Lin+ cells were excluded from the Lin fraction (which included Linlo cells). Consequently, the LinSCA-1+ gate was more inclusive than that described elsewhere for VSELs and included all cells reported to be in the VSEL fraction (Kucia et al., 2006a; Ratajczak et al., 2008a, 2011). LinSCA-1+ events were subdivided into three fractions
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Failure to Detect VSEL in Mouse Bone Marrow
Figure 1. Adult Mouse Bone Marrow CD45LinSCA-1+ Cells Enriched in the FSClo Region Contain Little DNA (A) FACS plots of BM cells from wild-type mice. The initial gate (left middle) was based on dened-size microspheres (left upper) and expected to include both HSCs and VSELs. After excluding dead cells (left bottom), we focused on the LinSCA-1+ fraction (center upper), subdividing these into FMO-dened CD45 (dashed line in center middle), CD45int, and CD45hi fractions (center bottom). The frequency of FSClo (<10 mm microspheres) and FSChi (>10 mm microspheres) cells in each subfraction is indicated (right panels). (B) Frequency of FSClo (left) or FSChi (right) cells among the CD45, CD45int, and CD45hi subfractions of LinSCA-1+ events. The mean SD of data from 22 independent experiments are shown. (C) Analysis of DNA content by SYTO16 staining. The threshold of high SYTO16 positivity was determined to include 99% of unfractionated BM cells (upper right). The vertical line in the lower plots indicates the position of 10 mm microspheres. See also Tables S1 and S2. according to CD45 expression (Figure 1A; Table S1 available online). We used the FSC intensity of 10 mm microspheres to dene the cutoff point between FSClo and FSChi events. As previously reported (Zuba-Surma et al., 2008), the CD45 fraction contained many more FSClo cells than FSChi cells. Conversely, the CD45hi fraction contained many more FSChi cells than FSClo cells (Figure 1B; Tables S1 and S2). FSClo events could include erythrocytes, vesicles, or cell fragments and/or debris. Since the original group has stated several times that VSELs are diploid (Kucia et al., 2008; Ratajczak et al., 2007, 2008b), we analyzed DNA content of FSClo events using SYTO16, a cell-membrane permeable DNA dye. We found that SYTO16hi events, representative of diploid cells, were present in about 98% of the CD45hi LinSCA-1+ fraction but only 10% of the CD45/int LinSCA-1+ fraction (Figure 1C). The remaining events in the CD45/intLinSCA-1+ gate showed much lower intensity for SYTO16, indicating that these were likely to be cell fragments and not diploid cells. Furthermore, most of the CD45/intLinSCA-1+SYTO16hi cells were in the FSChi gate (Figure 1C). This indicated that our VSEL candidates were the relatively larger cells in the population analyzed, unlike the VSELs described in previous reports (Kucia et al., 2006a; Ratajczak et al., 2011; Wojakowski et al., 2009; Zuba-Surma et al., 2008, 2009). Flow Cytometry in Assessing Size of Candidate VSELs We hypothesized that this discrepancy was due to the type of ow cytometer used: Ratajczak et al. (2011) mainly used a
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Figure 2. Evaluation of Cell Size by Different Cell Sorters (A) Dened-size microspheres (upper) and whole BM cells (lower) were analyzed by FACSAria (left) and MoFlo (right). Dashed lines indicate the FSC of 10 mm microspheres. The positions of lymphocytes (Lym) and granulocytes (Gra) are indicated in the lower panels. (B) On a FACSAria, BM cells were sorted by expected size (26, 610, and >10 mm) based on their FSC relative to microspheres (upper) and then reanalyzed on the same FACSAria (middle) and on a MoFlo (lower). Vertical lines indicate the positions of 2, 6, and 10 mm microspheres on each machine. (C) FACS plots of diploid (SYTO16hi) CD45/intLinSCA-1+ cells (candidate VSELs; left) and CD45hiLinSCA-1+ cells (HSCs; right) analyzed by FACSAria (upper) or MoFlo (lower). Vertical lines indicate the position of 10 mm microspheres in each sorter, and horizontal lines indicate the threshold of SYTO16 positivity. (D) Comparison of the sizes of candidate VSELs (n = 34) and HSCs (n = 100) determined with an image ow cytometer. Each dot represents a cell, and each line with error bars represents the mean SD. See also Figure S1. MoFlo (Beckman Coulter) (Zuba-Surma et al., 2008, 2009), but we used a FACSAria (BD Biosciences). Thus, we analyzed the same BM sample on a FACSAria and MoFlo. Based on the FSC intensity of unfractionated BM, 10 mm microspheres were positioned to the left of lymphocytes on the FACSAria but in between lymphocytes and granulocytes on the MoFlo (Figure 2A). Using the FACSAria and the MoFlo, we sorted the BM sample into three subpopulations, based on the FSC intensity of dened-size microspheres. We then reanalyzed each of these subpopulations on both the FACSAria and MoFlo. Critically, a given population of cells seemed smaller (based on microsphere reference FSC intensities) on the MoFlo than on the FACSAria (Figures 2B and S1A). However, uorescence intensity of the cell-to-cell comparison did not differ between the sorters (Figure S1B). Finally, we evaluated the FSC of candidate VSELs and hematopoietic stem/progenitor cells on FACSAria and MoFlo. Notably, more than 70% of candidate VSELs were detected in the FSChi gate on FACSAria, whereas >80% of them were in the FSClo gate on MoFlo (Figure 2C). To address the concern that ow cytometer FSC based on microspheres is not a reliable indicator of absolute size, we
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Figure 3. No Evidence for the Pluripotency of CD45/intLinSCA-1+ Cells (A) Real-time RT-PCR analysis of Oct4 expression in subfractions of LinSCA-1+ cells and in ESCs (BM cells were prepared as a pool of four mice and then sorted in quadruplicate and subjected to RT-PCR; the data are shown as mean SD). ND, not detected. (B) Representative images of 8 day progeny of FACS-puried LinSCA-1+ populations from Actin-EGFP mice. Cells were cocultured with C2C12 cells in DMEM supplemented with 2% FCS. Scale bar, 50 mm. (C) The total number of GFP+ cells detected by FACS analysis at day 8 coculture of either 1,000 CD45/intLinSCA-1+ events or 1,000 CD45hiLinSCA-1+ events (n = 11 for each from three independent experiments); the red line indicates the mean. (D) FACS analysis of Oct4-derived EGFP expression on culture day 8. On day 0, the following were plated on C2C12 cells: no cells (far left column) or BM cells from Oct4-EGFP or Actin-EGFP mice, sorted by the indicated phenotypes. Data shown are representative of three independent experiments. See also Figure S2 and Table S3. measured the size of cells with an image ow cytometer (FlowSight, Amnis). We captured 2 3 106 BM cells and identied 34 events having the immunophenotype corresponding to the candidate VSELs (Figure S1C). Their mean diameter was nonsignicantly smaller than that of a randomly selected population of 100 CD45hiLinSCA1+SYTO16hi events (7.06 versus 7.51 mm; p = 0.82; Figure 2D). However, only the candidate VSEL fraction contained few very small-sized (<5 mm) particles, as previously shown in fetal liver-resident VSELs (Zuba-Surma et al., 2009). Assessment of VSEL Candidates for Indicators of Pluripotency To determine whether the CD45/intLinSCA-1+ fraction contained any pluripotent cells, we compared Oct4 expression levels among freshly puried CD45/intLinSCA-1+ and CD45hiLinSCA-1+ cells and a mouse embryonic stem cell (ESC) line using quantitative RT-PCR with four different primer pairs. We could not detect Oct4 expression in CD45/intLinSCA-1+ (both SYTO16hi and SYTO16lo) cells at all (Figure 3A). These results contrast with previous reports (Kassmer et al., 2013; Kucia et al., 2006a; Ratajczak et al., 2011; Shin et al., 2010). Next, we performed a sphere-forming assay using a mouse myoblastic C2C12 cell line (Kucia et al., 2008; Shin et al., 2010). The C2C12 cell line is known to initiate its own differentiation and form muscle tubules when cultured in low serum concentrations (Yaffe and Saxel, 1977). We observed that, when cultured alone, C2C12 cells aggregated spontaneously and sometimes formed spherelike structures (Figure S2A). Therefore, to distinguish
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between spheres originating from candidate VSELs and spontaneous aggregation of C2C12 cells, we isolated candidate VSELs from Actin-EGFP transgenic mice and cocultured them with C2C12 cells. Some GFP+ cells proliferated and formed small clusters (Figure 3B) but never the spheres described in previous reports (Kucia et al., 2008; Shin et al., 2010). Eight-day progeny was harvested and analyzed for the absolute number of GFP+ cells by ow cytometry. The mean number of GFP+ cells initiated from the CD45/int LinSCA-1+ fraction was signicantly lower than that from the CD45hiLinSCA-1+ fraction: 27.2 versus 1764; p = 0.0002, respectively (Figure 3C). We repeated this assay with BM samples harvested from Oct4-EGFP transgenic mice. However, we could not detect any GFP+ cells in day 8 cultures of the following originally plated cells: 1,000 CD45/intLinSCA-1+, 5,000 CD45hiLinSCA-1+, or 105 unfractionated BM cells (Figure 3D). The addition of leukemia inhibitory factor and/or usage of mouse embryonic broblasts instead of C2C12 cells did not affect the results (data not shown). Grown GFP+ cells from both CD45/intLinSCA-1+ and CD45hiLinSCA-1+ fractions had similar morphologies: an unlobulated nucleus and many vacuoles in voluminous cytoplasm, suggestive of macrophages (Figure S2B). Furthermore, ow cytometry analysis conrmed these GFP+ cells expressed high levels of CD45 (a hematopoietic lineage marker) and CD11b (a macrophage marker; Figure S2C). Since C2C12 cells are reported to secrete macrophage-colony stimulating factor (M-CSF) into the culture supernatant (Ghosh-Choudhury et al., 2006), it is possible that they may induce macrophage differentiation to either CD45/intLinSCA-1+ or CD45hiLinSCA-1+ cells via M-CSF signaling. To demonstrate the pluripotency of a particular cell, clonal expansion without differentiation from a single cell in vitro is an indispensable step. However, the C2C12 cells did not work to induce this expansion in candidate VSELs. Therefore, we sought another way to demonstrate that CD45/intLinSCA-1+ cells are pluripotent or at least could even generate any tissue-specic lineage. We focused on testing hematopoietic potential because (1) a previous report showed that transplantation of cultured VSELs could reconstitute the hematopoietic system (Ratajczak et al., 2011) and (2) methods of purication and functional analyses of hematopoietic stem cells (HSCs) have been established (Spangrude et al., 1988; Uchida and Weissman, 1992). If hematopoietic potential cannot be recapitulated, the cells in the VSEL fraction must not be pluripotent. Freshly isolated CD45LinSCA-1+ BM cells (including both FSClo and FSChi) did not proliferate under hematopoietic culture conditions (Figures S3A and S3B). A recent report indicated that 5-day coculture with the OP9 stromal
cell line (i.e., OP9 priming) is critical for VSELs to be designated for a hematopoietic lineage (Ratajczak et al., 2011). In accordance with previous reports (Nakano et al., 1994; Seiler et al., 2011), we found that, when cocultured with OP9 cells, an ESC line was capable of hematopoietic differentiation (data not shown); this served as a positive control. However, CD45LinSCA-1+ cells could not generate hematopoietic colonies in the methylcellulose assay, even after OP9 priming (Figures 4A and 4B). The addition of various hematopoietic cytokines during the OP9 priming did not affect the results (data not shown). Furthermore, CD45LinSCA-1+ cells failed to produce any hematopoietic cells under the following additional conditions: (1) serial plating (Ratajczak et al., 2011); (2) including SYTOX-Blue positive fraction; (3) using only the SYTO16hi cells obtained from ten mice; (4) using cells from H2KBCL-2 transgenic mice (Domen et al., 1998); or (5) using cells sorted on a MoFlo machine (data not shown). These observations indicate that the FMO-dened CD45LinSCA-1+ fraction, at least in vitro, lacks hematopoietic potential, as recently described both for mouse VSELs (Szade et al., 2013) and their human counterparts (Danova-Alt et al., 2012). CD45intLinSCA-1+FSChi cells with Limited Hematopoietic Potential Originated from HSCs Within the remaining CD45hiLinSCA-1+ fraction, 38.0% of CD45hiLinSCA-1+FSChi cells but no CD45hiLinSCA-1+ FSClo cells formed hematopoietic colonies. Also, 1.86% of CD45intLinSCA-1+FSChi cells formed hematopoietic colonies (Figure 4B). Many colonies from the CD45int LinSCA-1+FSChi fraction contained fewer cells than those from the CD45hiLinSCA-1+FSChi fraction, and their differentiation potential was restricted to nonerythroid cells and tended to skew to the monocyte/macrophage lineage (Figures 4C and S3C). In addition, when compared to CD45hi LinSCA-1+FSChi cells, CD45intLinSCA-1+FSChi cells showed a more indented nucleus, lower levels of SCA-1, and higher levels of Lin and side scatter (Figures S3D and S3E). These ndings suggest that the CD45intLinSCA-1+ FSChi cells are at a lineage stage downstream of the CD45hi LinSCA-1+FSChi cells. However, the exact lineage stage from which granulocyte-macrophage progenitors can develop may vary depending on conditions such as the type of cytokine cocktail (Rieger et al., 2009). Therefore, we cannot denitively determine the stage of the initially plated cells that gave rise mainly to macrophages in this assay. To directly evaluate whether the colony-forming cells in the CD45intLinSCA-1+FSChi fraction were progeny of HSCs or independent of hematopoietic lineage cells, we engrafted EGFP-HSCs into uncolored mice. The experimental design, summarized in Figure 4D, was similar to that described in a previous report (Hall et al., 2007). Three
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Figure 4. HSCs Are the Only Contributor to Postnatal Mouse Hematopoiesis (A) Representative images on culture days 5 and 10. Each FACS-puried BM LinSCA-1+ fraction from Actin-EGFP mice was cocultured with OP9 cells for the rst 5 days (i.e., OP9 priming) and transferred to methylcellulose for an additional 5 days (i.e., methylcellulose expansion). The threshold between FSClo and FSChi was dened by 10 mm microspheres. Scale bar, 100 mm. (B) The number of colony-forming units (CFUs) from 103 cells of each LinSCA-1+ fraction. Data shown are mean SD of four independent experiments. ND, not detected. (C) Proportion of colonies. From ve independent colony assays, 199 colonies derived from CD45intLinSCA-1+FSChi cells and 250 colonies derived from CD45hiLinSCA-1+ FSChi cells were picked up, cytospun, and stained with the May-Giemsa method to determine the cell types included. CFU-M, CFU-macrophage; CFU-G, CFU-granulocyte; CFU-GM, CFU-granulocyte/macrophage; CFU-Mix, CFU-erythroid and myeloid cells. (D) Schematic of in vivo experiments. (E) The number of CFUs from 103 cells of each LinSCA-1+ fraction. Data shown here are mean SD. (F) Ten day progeny of CD45intLinSCA-1+ FSChi or CD45hiLinSCA-1+FSChi cells were harvested with OP9 stromal cells and analyzed by FACS. Live cells were gated and tested for the expression of CD45 and GFP. A total of 5 3 104 events were recorded. Data were similar in three independent experiments. See also Figures S3 and S4.
months after the mice underwent transplantation of GFPexpressing HSCs, 98% of their peripheral blood cells (not shown) and 96.8% of their BM granulocytes were GFP+ (Figure S4A). The frequencies of CD45, CD45int, and CD45hi fractions within LinSCA-1+ gate in the chimeric mice were comparable to those in age-adjusted nonirradiated syngeneic mice (Figure S4B). We evaluated the frequency of colony-forming cells in each fraction. As in the experiments with mice that did not receive transplants, a limited number of colony-forming cells were detectable in the fraction of CD45intLinSCA-1+FSChi and CD45hiLinSCA-1+FSChi cells (Figure 4E). Analysis
by uorescent microscopy and ow cytometry conrmed that all of the colonies derived from CD45intLinSCA-1+ FSChi cells (81/81) and CD45hiLinSCA-1+FSChi cells (926/926) expressed GFP (Figure 4F). This indicates that the CD45intLinSCA-1+FSChi cells that demonstrated hematopoietic potential originated from HSCs. Furthermore, recipient CD45/int cells, including the previously reported highly radiation-resistant VSELs (Ratajczak et al., 2011), failed to respond to this critical myelosuppressive condition and generate HSCs and/or their downstream progenitors in vivo. In other words, HSCs, reported to have little developmental plasticity (Wagers et al.,
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2002), are the only contributors to postnatal mouse hematopoiesis.
DISCUSSION
The existence of pluripotent cells after the preimplantation blastocyst stage has not been demonstrated, except in the case of germline stem cells. Therefore, VSELs and other PSCs in postnatal mice must be independently veried to change how we view events in embryogenesis and fetal development. PSCs in postnatal humans, if they exist, would have an extremely signicant impact on society because of their potential applications in research and regenerative medicine. In fact, the discovery of VSELs has already led to a commercial venture, Neostem, to bring their potential to human therapies. One of its nancial backers, the Vatican, has announced in two symposia at the Vatican that these cells represent an ethical alternative to ESCs derived from humans (International Vatican Conference, November 911, 2011 and April 1113, 2013, http://www.stemforlife.org/vatican-initiative). In contrast to these optimistic ndings, some groups have recently reported on their failure to detect VSELs. To accept a discovery as a scientic fact in the stem cell eld, we previously proposed that all of the following three criteria are critical (Weissman, 2007): (1) the initial discovery must be published in fully peer-reviewed journals; (2) the experiment as published must be repeatable in many independent laboratories; and (3) the phenomenon described should be so robust that other experimental methods must reveal it. And in the case of stem cells, the regeneration derived from their transplantation should be rapid, robust, and lifelong. It is not infrequent that, although the initial ndings are peer-reviewed, subsequent attempts to independently replicate the ndings fail (Begley and Ellis, 2012). We therefore sought to employ a logical strategy to examine all the cells within the CD45/intLinSCA-1+ population in mouse BM, even going as far as to include dead cells and cell debris in the analysis. Besides surface markers, the main criteria for the VSELs as dened by the original group are the following two points: (1) very small in size (35 mm) and (2) pluripotency. First, we tried to clarify whether this fraction contains any viable cells of the reported small size. We found that about 90% of all the events in this gate are indeed relatively small and have much less DNA than that of diploid cells according to SYTO16 staining measured on FACSAria (Figure 1C). This suggests that these relatively very small events are simply cell debris or fragments; subcellular particles, such as exosomes and blebs from dying cells, could be included. As mentioned above, it is unreliable to determine absolute cell size by FSC intensity (Figure 2). Furthermore,
any modications such as xation or cytospin have the potential to affect the cell size. Therefore, we used an image ow cytometer and directly measured the size of candidate events without any modication. This experiment revealed that most of the SYTO16lo events lack the round shape of cells and are jagged, suggestive of dead cells and/or cell debris. SYTO16hi cells, both from CD45/intLinSCA-1+ and CD45hiLinSCA-1+ are not statistically different in size and the majority are not small (<5 mm). However, we were still able to detect an exceedingly rare population of CD45/intLinSCA-1+SYTO16hi events, which we sought to evaluate for pluripotency. Since the original group reported that VSELs can survive and differentiate to the hematopoietic lineage on OP9 cells, we sought to evaluate these candidate VSELs by the same assay, namely blood colony formation. When we cultured all the SYTOX-Blue-positive cells in the CD45/int LinSCA-1+ fraction (without size discrimination), no colony formation was detected. This indicates logically that SYTOX-Blue staining method can separate dead cells functionally; there are no viable VSELs in the SYTOX-Blue-positive fraction. We were unable to detect any hematopoietic cells among the SYTOX-Blue-negative CD45LinSCA-1+ cellsthe ideal candidates for VSEL cellsunder the following additional conditions: (1) serial plating (previous reports showed serial plating would recover cell growth; Ratajczak et al., 2011); (2) using only the SYTO16hi cells obtained from ten mice; (3) using cells from H2K-BCL-2 transgenic mice (to improve viability by inhibiting programmed cell death; Domen et al., 1998); or (4) using cells sorted on a MoFlo machine (data not shown). Since we have shown that size may not be a reliable parameter in isolating VSEL, we broadened our search by not using the size criteria while maintaining the immunophenotype (CD45/intLinSCA-1+FSChi). Compared to CD45hiLin SCA-1+FSChi cells, CD45intLinSCA-1+FSChi cells showed much less hematopoietic differentiation potential, and it never matched CD45hiLinSCA-1+FSChi cells, even after serial plating, in contrast to the original groups report (Figure 4B; data not shown; Ratajczak et al., 2011). However, given our observation of some hematopoietic output from the CD45intLinSCA-1+FSChi fraction, we sought to determine the developmental origin responsible: HSC or candidate VSEL. All of the hematopoietic differentiation potential in the CD45intLinSCA-1+FSChi fraction were derived from prospectively isolated and transplanted HSCs (Figure 4F), suggesting that the only cells with hematopoietic differentiation potential in the CD45intLinSCA-1+FSChi cells are hematopoietic stem/progenitor cells. Failure to detect hematopoietic lineage potential seriously calls into question the existence of pluripotent stem cells in CD45LinSCA-1+ cells. However, this nding does not deny the existence of other multi- or oligopotent
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stem cells with other lineage potentials. For instance, Morikawa et al. (2009) reported that CD45LinSCA-1+ platelet-derived growth factor receptor a+ cells in adult mouse BM can give rise to mesenchymal stromal cells both in vitro and in vivo. At the time of our writing this report, a few independent groups have replicated select aspects of VSEL: Bhartiya et al., 2012 reported VSEL-derived TSCs existed in mammalian ovary or in human BM and CB (Parte et al., 2011) and Sovalat et al. (2011) claimed VSELs could be isolated from human BM or granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood. However, all of these other groups evaluated only the existence of VSEL by the analysis of the cell size and immunophenotype without evaluating the pluripotency criteria, as criticized by the others in the eld (Ivanovic, 2012). In fact, other recent reports failed to detect the stem cell properties of mouse (Szade et al., 2013) or human VSELs (Danova-Alt et al., 2012). When only one or a few markers of cells rather than the function of cells themselves are assayed, artifactual or nonphysiological expression of single markers can lead to the interpretation that a cell type rather than a marker is being studied. The only assays acceptable for functionally identifying stem cells are those that assess (1) self-renewal; (2) differentiation to clonal progeny of the cell types inferred from the name of the cell; and (3), for adult tissue outcomes, robust and sustained regeneration. The transfer of donor markers to host cells could occur by cell fusion and, theoretically, by exosome transfer, to the extent that exosomes are shown to be physiological entities and not cell culture artifacts. We conclude that most CD45/intLinSCA-1+ cells in mouse BM were not as small as previously reported. Moreover, even despite broadening our search to include larger cells as candidates, we could not nd a single instance of CD45/intLinSCA-1+ cells that (1) expressed Oct4, (2) proliferated to form spheres in cultures, or (3) demonstrated the ability to generate cells of the hematopoietic lineagethree functional aspects of pluripotency that were previously reported. These results suggest the absence of embryonic-like pluripotent cells in postnatal mouse BM. Additional rigorous data would be needed to demonstrate their existence both in humans and mouse before clinical application, including the derivation of the same repertoire of normal tissue cells currently demonstrated with ESC and/or induced pluripotent stem cells.
tained in our laboratory. The Oct4-EGFP transgenic strain (Tg [Pou5f1-EGFP]2Mnn/J) was purchased from the Jackson Laboratory. The C57Bl/6J-W41/W41 strain was kindly provided by Dr. Susan L. Hall, Loma Linda University. We used 412-week-old female and male mice. All animal procedures were performed in accordance with the International Animal Care and Use Committee and the Stanford University Administrative Panel on Laboratory Animal Care.
Cell Preparation and Staining

BM cells harvested from bilateral femurs and tibias with a ushing method were treated with BD Pharm Lyse Buffer (BD PharMingen). These cells were stained for 30 min with phycoerythrin (PE)-conjugated lineage antibodies (Lin), which consisted of anti-CD45R/ B220 (clone; RA3-6B2, nal concentration; 4 mg/ml), anti-T cell receptor (TCR) b (H57-597, 4 mg/ml), anti-TCR gd (GL3, 4 mg/ml), anti-Ly-6G/C (RB6-8C5, 8 mg/ml), anti-CD11b (M1/70, 4 mg/ml), and anti-Ter119 (TER-119, 4 mg/ml); allophycocyanin-Cy7-conjugated anti-CD45 (30-F11, 8 mg/ml); and biotin-conjugated anti-Ly6A/E (SCA-1) (E13-161.7, 10 mg/ml) followed by streptavidin-PE-Cy5 (1 mg/ml). All the antibodies were purchased from BD. One micromolar SYTO16 (Life Technologies) was added to evaluate DNA quantity.
Flow Cytometry
Sorting and analyses were performed on multilaser-equipped FACSAria II and, where otherwise indicated, on a MoFlo cell sorter or FlowSight image ow cytometer. Dead cells were distinguished by adding 1 mM of SYTOX-Blue dead cell stain (Life Technologies). To minimize contamination, a second round of sorting was performed. The automatic cell deposition system was used for single-cell assays. Data were analyzed with FlowJo software (Tree Star) or IDEAS software (Amnis).
Isolation of HSCs and Candidate VSELs

By comparing the FSC of dened-size microspheres (Flow Cytometry Size Calibration Kit, Life Technologies) and that of BM cells, we evaluated relative cell size. The SCA-1 channel was separated between negative and positive by the FMO method. We further divided LinSCA-1+ cells into three subfractions according to the CD45 expression level: the upper limit of the CD45 subpopulation was dened by the FMO method; the CD45hi subpopulation by the obvious positive peak; and the CD45int area as that between CD45 and CD45hi. HSCs and candidate VSELs were expected to reside in the CD45hiLinSCA-1+FSChi and CD45LinSCA-1+ (and/or CD45intLinSCA-1+) FSClo populations, respectively.
Sphere Formation Culture

A C2C12 mouse myoblast-derived cell line was maintained in Dulbeccos modied Eagles medium (DMEM) containing 20% fetal calf serum (FCS). For the sphere formation assays, freshly isolated 1 3 103 BM LinSCA-1+ cells from Actin-EGFP mice were: (1) plated on the 1 3 105 C2C12 cells seeded the previous day on a 24 well plate and (2) cultured for 8 days in DMEM with 2% FCS. Half of the culture media was changed every day. The grown cells/clusters were trypsinized, harvested, passed through a 100 mm lter, and analyzed by FACSAria. The cells were also
Mice
C57Bl/Ka-Thy1.2 Ly5.1 (B/Ka), C57Bl/Ka-Thy1.1 Ly5.1 (BA), C57Bl/Ka-Thy1.1 Ly5.1-Tg (actin-EGFP), and C57Bl/Ka-Thy1.1 Ly5.1-Tg (pH2K-BCL-2) mouse strains were derived and main-
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observed by uorescence microscopy (DMI 6000B, Leica Microsystems).
2009), and (4) TaqMan probe set Mm.PT.51.7439100.g (Integrated DNA Technologies). Primer sequences and amplicon sizes are listed in Table S2.
Nonfeeder Culture for Hematopoietic Differentiation

For liquid cultures, puried populations were suspended in 12 well plates with the following medium: Iscoves modied Dulbeccos medium (IMDM; Life Technologies) supplemented with 20% FCS, antibiotics, 10 ng/ml of mouse (m) recombinant interleukin 3 (IL-3), 10 ng/ml mouse stem cell factor (SCF), and 10 ng/ml mouse Flt3 ligand (R&D Systems). For clonogenic analyses of CD45hiLinSCA-1+ (including HSCs) and CD45/intLinSCA-1+ (including VSELs) faction, cells were cultured 10 days in IMDMbased methylcellulose medium (Methocult GF M3434; StemCell Technologies), which contained FCS, bovine serum albumin, 2-mercaptoethanol, recombinant human (h) insulin and transferrin, as well as recombinant mSCF, mIL-3, hIL-6, and human erythropoietin. All cultures were incubated in a humidied chamber in 5% CO2. Colonies were scored and picked up for making cytospin preparations to dene cell components.
The nonparametric Mann-Whitney test was applied to all comparisons of mean values after F-test evaluation of variances. All statistical analyses were performed on Prism 5 software (GraphPad).
Supplemental Information includes four gures and three tables and can be found with this article online at http://dx.doi.org/10. 1016/j.stemcr.2013.07.001.
ACKNOWLEDGMENTS
We thank L. Jerabek and T. Storm for laboratory management; T.J. Naik for technical assistance; A. Mosley, C. Wang, and A. McCarty for animal care; Dr. S.L. Hall, Loma Linda University, for providing W41/W41 mice; and J. Ooehara, Y. Yamazaki, and M. Otsu for supporting experiments of side-by-side comparison between MoFlo and FACSAria. This study was supported by fellowships from the Toyobo Biotechnology Foundation, the Uehara Memorial Foundation, Human Frontier Science Program (to M.M.), and the Japan Society for the Promotion of Science (to Y.M.) and by grants from the US National Institutes of Health (R01 CA086065 and U01 HL099999 to I.L.W.), the California Institute for Regenerative Medicine (RT2-02060 to I.L.W.), and the Leukemia & Lymphoma Society (700709 to I.L.W.). H.N. is a shareholder and a member of the scientic advisory board of ReproCELL and Megakaryon and a member of the scientic advisory board of Shionogi. I.L.W. is a member of the scientic advisory board of StemCells. Received: June 2, 2013 Revised: July 2, 2013 Accepted: July 3, 2013 Published: July 24, 2013
OP9 Coculture Assay

OP9 coculture was performed as previously reported (Ratajczak et al., 2011). OP9 cells were maintained in a-MEM (MEMa Nucleosides Powder, Life Technologies) with 10% FCS. Freshly isolated LinSCA-1+ cells (CD45, CD45int, or CD45hi) were plated on OP9 cells in 12 well plates with a-MEM plus 20% FCS and cultured for 5 days. Cultured cells were then trypsinized, centrifuged, and replated in methylcellulose medium with OP9 cells. For serial passage, methylcellulose cultures were trypsinized, centrifuged, and replated in new methylcellulose medium. Colonies were scored and picked up to make cytospin preparations and stained by the May-Giemsa method.
Purication of Long-Term HSCs and Transplantation

BM mononuclear cells from actin-EGFP mice were enriched for c-kit+ cells by using anti-c-kit microbeads (Miltenyi Biotec) and sorted for propidium iodideLin(CD3/CD4/CD8/Mac-1/Gr-1/ B220/CD19/Ter119)CD34CD150+Flk2c-kit+SCA-1+ long-term HSCs. Eight-week-old W41/W41-recipient mice (Reith et al., 1990), hematopoietic-decient due to mutations in the W locus encoding the c-kit gene, received 400 cGy total body irradiation followed by intravenous injection of 100 sorted long-term HSCs via the retro-orbital vein. Peripheral blood and BM cells were analyzed by ow cytometry after transplantation to evaluate chimerism.
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RNA Extraction and Real-Time RT-PCR

Prior to quantitative RT-PCR, 1 3 10 of each of the following cell types were sorted by ow cytometry: CD45/intLinSCA-1+ SYTO16lo, CD45/intLinSCA-1+SYTO16hi, CD45hiLinSCA-1+ SYTO16hi, and SSEA-1hi ESCs. RNA extraction, reverse transcription, and PCR amplication were performed by using CellsDirect One-Step qRT-PCR Kit (Life Technologies) according to the manufacturers instructions. Gene-specic products were monitored by the 7900HT Fast Real-Time PCR System (Life Technologies). Four different design of primer pairs for Oct4 messenger RNA were used: (1) designed by Niwa group (Toyooka et al., 2008), (2 and 3) designed by Ratajczak group (Kucia et al., 2006a; Liu et al.,
3
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Differentiation and Isolation of iPSC-ECs

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Fractionation of Cytoplasmic and Nuclear Proteins

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Gene Expression Analysis

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MDSC Suppression Analysis

Quantication of HGF Secretion

REFERENCES RNAi Experiments

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