Cell Press Reviews: Stem Cells to Model and Treat Disease

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Preface

We are very pleased to present Cell Press Reviews: Stems Cells to Model and Treat Disease, which brings together review articles from Cell Press journals in order to offer readers a comprehensive and accessible entry point into the rapidly advancing stem cell field. Articles were selected by the editorial staff at Cell Press with an eye toward providing readers an introduction to timely and cutting edge research written by leaders in the field. While Cell Press Reviews: Stems Cells to Model and Treat Disease is not an exhaustive overview of current stem cell technologies, our aim is to give readers insight into some of the most exciting recent developments and the challenges that remain. A wide range of topics are covered within this publication, including the safety and efficacy of stem cell treatments, the use of stem cells to regenerate organs, the use of reprogrammed stem cells to model and treat disease, and the use of stem cells in clinical applications such as HIV disease and stroke.

We are pleased to be able to include contributions from Shinya Yamanaka, recipient of the 2012 Nobel Prize in Physiology or Medicine, Professor at the Center for iPS Cell Research and Application at Kyoto University and at the Gladstone Institute of Cardiovascular Disease in San Francisco, CA, and President of the International Society for Stem Cell Research; George Q. Daley, Director of the Stem Cell Transplantation Program at Children’s Hospital Boston and Professor at Children’s Hospital Boston and Harvard Medical School; Irving Weissman, Director of the Institute of Stem Cell Biology and Regenerative Medicine at Stanford University School of Medicine; and many other prominent researchers in the field. Their insights will offer readers, both experts and those new to the field, a fascinating perspective into this critically important and evolving area of research.

Cell Press Reviews: Stem Cells to Model and Treat Disease is one in a series of books being published as part of an exciting new collaboration between Cell Press and Elsevier Science and Technology Books. Each book in this series is focused on a highly timely topic in the biological sciences. Editors at Cell Press carefully select recently published review articles in order to provide a comprehensive overview of the topic. With the wide range of journals within the Cell Press family, including research journals such as Cell, Cell Stem Cell, and Neuron as well as review journals like Trends in Molecular Medicine and Trends in Biotechnology, these compilations provide a diverse and accessible assortment of articles appropriate for a wide variety of readers. You can find additional titles at http://www.store.elsevier.com/CellPressReviews. We are happy to be able to offer this series to such a wide audience via the collaboration with Elsevier Science and Technology Books, and we welcome all feedback from readers on how we might continue to improve.

Chapter 1

Stem Cell Therapies Could Change Medicine… If They Get the Chance

Irving Weissman¹,*,    ¹Institute of Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA 94305 USA,    *Correspondence: irv@stanford.edu

Stem cell therapies have the potential to revolutionize the way we practice medicine. However, in the current climate several barriers and false assumptions stand in the way of achieving that goal.

Keywords

stem cell therapy; medicine

Cell Stem Cell Vol. 10, No. 6, June 14, 2012 © 2012 Elsevier Inc.

http://dx.doi.org/10.1016/j.stem.2012.05.014

Summary

Stem cell therapies have the potential to revolutionize the way we practice medicine. However, in the current climate several barriers and false assumptions stand in the way of achieving that goal.

Introduction

The first two precepts of the modified Hippocratic Oath, which all M.D. graduates pledge are, in paraphrase: first, do no harm; and second, the primary obligation of a physician is to the health of the patient (to which I add and future patients), and a physician will not let issues of race, creed, religion, politics, or personal ethics to stand between the patient’s health and his/her actions. The stem cell field, probably more than any I know of in medical science, is plagued by failures to act responsibly on both precepts.

While I am usually an optimist, I must admit that there is a possibility that we will continue to be in the Dark Ages of medicine for quite some time. I fear that therapies using purified tissue and organ-specific stem cells—the only self-renewing cells in a tissue or that can regenerate that tissue or organ for life—will remain elusive. Before I go further, just think about that statement: regenerate that tissue or organ for life. No pharmaceutical, no biotech-developed protein, and no other transplanted cells can do that. If we can deliver purified stem cells safely and effectively as a one-time therapy, we can change medicine, especially for diseases that drugs and proteins can’t touch. Moreover, if we manage the costs and charges carefully, this form of therapy could lower overall health care costs dramatically. This vision is based on solid scientific evidence that stem cells regularly maintain, and, if necessary, regenerate tissues in a homeostatically controlled process. So it’s worth the extra effort to find a way to make it happen.

Doing Harm

One of the barriers to practicing stem-cell-based regenerative medicine is the existence of fraudulent clinics and individuals who claim unproven therapies without underlying scientific backing. In many cases, they use cells that have never been tested experimentally for their stemness, have not been through IRB-approved protocols that demand experimental evidence to justify the human experiment, and lack both independent medical monitoring of patient safety and oversight by a state or country regulatory system such as the FDA. It is critical that, as the community that speaks for stem cell biology and stem cell medicine, we find ways to warn patients and caregivers effectively about these concerns (Taylor et al., 2010).

There is also a fine line between these clearly fraudulent practices and questionable ones that use the stem cell label, but are not in fact stem cell therapies. For example, cultures of adherent cells from bone marrow, cord blood, or adipose tissue are regularly claimed to be mesenchymal stem cells (MSCs), but in such cultures true stem cells that both self-renew and differentiate to mesenchymal fates such as bone, cartilage, fibroblasts, and adipocytes are rare. Mesenchymal stromal cells, as a population, may contain cells that produce immunomodulatory and/or angiogenic factors, but are not sufficiently purified or defined to be a characterized entity for research or clinical transplantation. Finding markers that help define these populations was an important step (Dominici et al., 2006), but until there is a better understanding of how many of these cells can self-renew and give robust regeneration, I do not think they should be called stem cells.

There are also many claims that mesenchymal and/or hematopoietic cells can transdifferentiate without gene modification to make brain, liver, heart, skeletal muscle, or other tissues. However, these claims lack rigorous scientific support (Wagers and Weissman, 2004). Highly visible athletes and politicians are among the many patients who have received such treatments. Recently, the Texas Medical Board approved a policy that allows licensed physicians to transplant investigational agents, including MSCs, with IRB approval but without a requirement for FDA approval of safety and efficacy. In my view, this lack of a requirement for FDA oversight and approval for both safety and efficacy is a giant step backward.

Another example of questionable stem cell practices comes from some commercial private cord blood banks. Cord blood does contain both HSCs and mesenchymal progenitors. The number of HSCs in each cord is sufficient to give rapid generation of blood only in infants and very small children, and above the age of ∼7, several HLA-matched cords are needed. The development of public cord blood banks is an important, life-saving advance for patients needing hematopoietic cell transplants but lacking matched donors. However, this activity is very different from the private cord blood banks that charge significant amounts to initiate freezing of cord blood cells and then maintain them in case the child from whom the cord is obtained needs therapy. These companies often list a broad range of diseases that now or someday will be treated with stem cells without warning the patients or caregivers that the evidence that cord blood cells will be useful for treating such diseases is still very limited, and in any case the stored cord blood has the same genetic background as the child from whom the cord was obtained. The overall cause of legitimate stem cell therapy would be greatly advanced by greater control and oversight of these and other organizations making unsupported claims about the potential of stem-cell-based treatments.

The Therapeutic Entity is the Stem Cell Itself

Very few adult stem cells have been prospectively isolated, and only prospectively isolated blood-forming stem cells (HSCs) and brain-forming stem cells (NSCs) have been transplanted in clinical trials (Baum et al., 1992; Uchida et al., 2000). Grafts of other tissues, such as skin and bone marrow, depend on the stem cells in that tissue, but prospectively isolated stem cells are usually not used. Instead of using cells as the therapy, as a general rule, large drug companies are approaching the use of disease specific iPSCs or adult stem cells as tools for chemical or protein screens to find compounds that can be taken as conventional drugs to treat diseases. Some of these efforts are focused on differentiated cells derived from stem cells, but others aim to address diseases where altered or insufficient numbers of stem cells are central to the disease. The principal property of stem cells that makes them special is their ability to self-renew and reconstitute cell populations. Inducing self-renewal in vivo could be difficult to achieve because many factors affect stem cell regulation. It seems unlikely that single molecules will be able to activate all of the necessary pathway genes appropriately to expand a stem cell pool and allow robust and physiologically significant regeneration. Thus, I think this approach is likely to fall short as a method to replace tissue stem cells in vivo, and efforts will need to focus more on transplanting the cells themselves. However, stem-cell-regulating agents derived from screening could still be used as adjuvants for transplanted stem cells.

At a broader level, HSCs themselves form a foundation on which the rest of the regenerative medicine field could be built. When engrafted, purified HSCs can replace the hematopoietic system. By doing so, they also render the host permanently tolerant to other organs, tissues, or tissue stem cells from the same donor without further immune suppression (Weissman and Shizuru, 2008). In the future, the isolation of HSCs and other tissue stem cells (e.g., NSCs) from the same donor could come from pluripotent stem cell lines, and not living or recently deceased donors. Pluripotent ESC or iPSC line production of HSCs is still not practical, and working out the pathways to achieve that objective remains a critical roadblock to expanding the field of regenerative medicine.

In Vivo Veritas

The experiments that validated human, purified HSCs for hematopoietic transplants and human brain-stem-cell-derived neurospheres for neural disease transplants used immune-deficient mice that were crucial in testing the potential therapeutic effectiveness of these cells in vivo (Weissman, 2002). Although the derivation of patient- and disease-specific iPSCs can allow experiments in a petri dish, the disease pathogenesis caused by inherited mutations would be more completely understood if the cells could mature in a more physiological setting. One way to study them would be to develop blastocyst chimeras that are implanted and allowed to develop. Mouse ESCs and iPSCs can already be studied using this type of approach. Currently, human ESCs/iPSCs do not form chimeras if placed in mouse blastocysts and implanted. However, human pluripotent stem cell lines are mainly at the epiblast stage, and not the preimplantation blastocyst, and even mouse epiblast cells cannot form long-term blastocyst chimeras. If the substantial practical and ethical issues could be overcome, blastocyst chimeras with human iPSCs might provide insights into the cellular and molecular mechanisms of human disease pathogenesis, and the gene expression programs that allow embryonic tissue stem cells to mature.

An Unexpected but Potent Barrier: Business Development

Growing up in America, it is obvious to all of us that the transition from discovery to therapy almost always involves for-profit entities. Ingenuity and innovation are hallmarks of our society, and so it is natural that the prospective identification and isolation of adult or tissue stem cells leads to business enterprises. I myself have cofounded several companies that have done discovery, preclinical proof of principle, and even phase I/II clinical trials in the stem cell field. Each has succeeded in the discovery and preclinical phases, but found that the results of the clinical trials can take a back seat to business decisions. For example, SyStemix Inc. was a 1988 Palo Alto startup that identified a method to prospectively isolate and transplant clinically relevant numbers of human HSCs. The company entered a relationship with Sandoz, Inc. to explore autologous and allogeneic HSC therapies. Purification of mobilized peripheral blood HSCs resulted in depletion of various metastatic cancer cells by 115,000– to 245,000-fold (Prohaska and Weissman, 2009), and thus could be used to reconstitute the hematopoietic system after therapy with a reduced risk of reintroducing tumor cells. This finding led to clinical trials.

Twenty-two patients with metastatic breast cancer underwent transplantation of previously mobilized HSCs after very-high-dose chemotherapy. Although the trials were small, two hypotheses were tested: (1) can one improve the outcome of patients with chemoresistant metastases? And (2) can one improve the outcomes of relapse patients with both metastases and chemoresponsive cancers? The therapy did not help the patients with chemoresistant breast cancers. However, at 3 years the chemoresponsive cohort who received cancer-depleted HSCs appeared to be doing better than patients with standard mobilized peripheral blood transplants. At that point, Sandoz merged with CIBA to form Novartis, and within a few years the stem cell program was cancelled. Last year Antonia Müller and Judy Shizuru published the follow-up of the patients 13–15 years later (Müller et al., 2012). One-third of the patients who received purified HSCs were still alive, contrasting with the 7% overall survival of 78 contemporaneous Stanford patients with stage IV breast cancer who received standard, unpurified, mobilized peripheral blood transplant therapy. Of the five long-term surviving patients who had received purified HSCs, four had no recurrence of their breast cancers.

Attempts to reinitiate the program in another startup, helped by Novartis management, were halted when consultant oncologists advised investors that stem cell therapies in breast cancer had failed, citing a study indicating that stem cell rescue of high-dose chemotherapy patients with metastatic breast cancer was no better than chemotherapy alone, that is, only ∼6% disease-free survival at 2 years (Stadtmauer et al., 2000). However, Stadtmauer et al. transplanted unfractionated mobilized blood, not purified HSCs, and no amount of evidence about the difference could counter the words stem cell in the title of the NEJM article. This particular problem could have been avoided by more rigorous editorial standards regarding the use of the term stem cell, and I would argue that improved accuracy in this respect would benefit many areas of the field.

How can we resolve this conflict of goals, that of a company to make a profit, and that of the biomedical researcher to advance medical science for the benefit of patients? The largest and best funding experiment I have seen so far comes from the California Institute of Regenerative Medicine. CIRM’s charter allows it to fund promising stem-cell-based discoveries to and through phase I trials, taking out the risk that leaves our field bereft of suitable funds and in the valley of death. However, to overcome the types of problems that the SyStemix trial encountered, this funding would need to be taken beyond initial trials to a point at which the evidence for clinical efficacy was irrefutable.

In Closing....

So, whom have I failed to annoy here? In one way or another, I have called out almost all of the different stakeholder groups involved in developing stem cell therapies. I wish I had a better story to tell, but I am convinced that we need to identify and reveal those who directly or indirectly do harm with phony medicines, and those who generate barriers to finding and transplanting adult tissue/organ stem cells for financial, religious, political, or other reasons. Unless we do, it will be difficult to usher in the era of stem cell regenerative medicine. Remember, right now our patients, friends, and families are contracting diseases that have a very short window of opportunity in which regenerative therapies can save them, and each delay removes a cohort of them from possible cures. We should not fail them.

References

1. Baum CM, Weissman IL, Tsukamoto AS, Buckle AM, Peault B. Proc Natl Acad Sci USA. 1992;89:2804–2808.

2. Dominici M, Le Blanc K, Mueller I, et al. Cytotherapy. 2006;8:315–317.

3. Müller AM, Kohrt HE, Cha S, et al. Biol Blood Marrow Transplant. 2012;18:125–133.

4. Prohaska SS, Weissman IL. Biology of Hematopoietic Stem and Progenitor Cells: Thomas’ Hematopoietic Cell Transplantation. In: Applebaum F, Forman S, Negrin RS, Blume K, eds. Oxford: Wiley-Blackwell; 2009;:36–63.

5. Stadtmauer EA, O’Neill A, Goldstein LJ, et al. Philadelphia Bone Marrow Transplant Group. N Engl J Med. 2000;342:1069–1076.

6. Taylor PL, Barker RA, Blume KG, et al. Cell Stem Cell. 2010;7:43–49.

7. Uchida N, Buck DW, He D, et al. Proc Natl Acad Sci USA. 2000;97:14720–14725.

8. Wagers AJ, Weissman IL. Cell. 2004;116:639–648.

9. Weissman IL. N Engl J Med. 2002;346:1576–1579.

10. Weissman IL, Shizuru JA. Blood. 2008;112:3543–3553.

Chapter 2

Why Is It Taking So Long to Develop Clinically Competitive Stem Cell Therapies for CNS Disorders?

Olle Lindvall¹,*,    ¹Lund Stem Cell Center, University Hospital, SE-221 84 Lund, Sweden,    *Correspondence: olle.lindvall@med.lu.se

The remarkable advancements in basic stem cell research with implications for several central nervous system disorders have so far not been translated into clinically effective therapies. Here I discuss some of the underlying problems and how they could be overcome.

Keywords

stem cell; therapy; cost; animal model; CNS; patient; pathology

Cell Stem Cell, Vol. 10, No. 6, June 14, 2012 © 2012 Elsevier Inc.

http://dx.doi.org/10.1016/j.stem.2012.04.004

Summary

The remarkable advancements in basic stem cell research with implications for several central nervous system disorders have so far not been translated into clinically effective therapies. Here I discuss some of the underlying problems and how they could be overcome.

Introduction

The first attempt to treat a central nervous system (CNS) disorder with cell transplantation took place three decades ago (Backlund et al., 1985). In this study, autologous adrenal medulla cells were implanted into the striatum of Parkinson’s disease (PD) patients to provide a local catecholamine source, but the beneficial effects were minimal. A few years later, human fetal mesencephalic tissue rich in dopaminergic neuroblasts was transplanted to the striatum in PD patients. These clinical trials established some important basic principles of cell therapy for CNS disorders: grafted neurons can replace dead host neurons in the diseased, 50- to 60-year-old human brain, reinnervate denervated areas, release transmitter, and, in some patients, give rise to therapeutically valuable effects (Lindvall and Kokaia, 2010). Based on these findings, stem-cell-based therapy for PD has been regarded as a low-hanging fruit, with the requirement for successful treatment being seemingly simple, namely to generate large numbers of standardized dopaminergic neurons for transplantation from stem cells. However, despite major efforts in basic and clinical research, there is still no clinically competitive cell therapy for PD or any other CNS disorder. Clinical trials with stem cells, often of bone marrow origin, are ongoing in, e.g., stroke, amyotrophic lateral sclerosis (ALS), and spinal cord injury (http://www.clinicaltrials.gov), but whether they will show efficacy is unclear. From my perspective, there are several major problems that explain why the clinical translation of stem cells for neurological disease is so difficult, as outlined below.

The Problem of Generating the Right Cells and Understanding Their Mechanisms of Action

Stem cells can act in brain diseases by replacing those cells that have died, but they can also restore function through other mechanisms (Lindvall and Kokaia, 2010). In the case of cell replacement, disease pathology determines which cells have to be generated from the stem cells. Different cells will be needed for different diseases. Substantial improvement in PD and ALS will require cells with the properties of dopaminergic and motor neurons, respectively. The situation for cell replacement in Alzheimer’s disease (AD) is much more complex because the stem cells would have to be predifferentiated in vitro into many different types of neuroblasts for subsequent implantation into a large number of brain areas. Similarly, in stroke there is a loss of several different types of neuron, glial cells, endothelial cells, and parenchyma. These broad defects raise the question of whether it is realistic to expect that clinically valuable improvement in disorders like AD or stroke could be achieved through cell replacement.

Importantly, efficacious cell replacement will require the generation of the correct neuronal phenotype. For example, in PD it is not sufficient to generate just any type of dopaminergic neuron. Rather, to induce substantial clinical benefit, the human stem-cell-derived dopaminergic neurons must exhibit the specific properties of the neurons that have died, i.e., the substantia nigra neurons (Lindvall et al., 2012). A recent study did succeed in showing efficient conversion of human embryonic stem cells into bona fide substantia nigra dopaminergic neurons using a differentiation protocol guided by developmental principles (Kriks et al., 2011). These cells ameliorated PD symptoms after transplantation in animal models without forming tumors.

For optimum recovery in many CNS diseases, neuronal replacement and at least partial reconstruction of circuitry should probably be the long-term goal. However, a large number of experimental studies in animal models of these disorders have demonstrated that stem cell delivery gives rise to functional improvements that cannot be explained by neuronal replacement. These beneficial effects may also be relevant in clinical settings. For example, systemic or intracerebral delivery of neural and other stem cells in stroke models has been reported to lead to improvements by trophic actions, modulation of inflammation, promotion of angiogenesis, remyelination and axonal plasticity, and neuroprotection (Lindvall and Kokaia, 2010). The functional effects can be enhanced if the stem cells have been genetically modified to secrete various factors such as trophic molecules. For clinical competitiveness, it is necessary, though, that the efficacy and safety of the stem-cell-based approach is superior to that of available treatments (e.g., drugs) acting on the same targets. Clinical trials are ongoing in stroke and ALS with delivery of stem cells, which are intended to act not by neuronal replacement but instead through one or more of the other presumed mechanisms. However, it is conceivable that effective therapies will not be developed until the mechanisms of action of the stem cells are much better understood and can therefore be optimized.

The Problem of Using the Right Animal Model and Behavioral Tests

Available animal models of CNS diseases do not mimic all aspects of the pathology of the human condition, which may explain lack of efficacy of cell therapy when it is translated to the clinical setting (Lindvall et al., 2012). For example, animal models of PD are mostly based on lesions of the nigrostriatal dopaminergic pathway, induced by toxins, and studies of sensorimotor functions. These models do not imitate the clinical disorder, which has many nonmotor and motor features with nondopaminergic pathology outside the substantia nigra. Attempts to develop transgenic models of PD have been pursued in recent years, but these represent only partial models of the core pathologies. For efficient clinical translation, better animal models that reflect the complex pathology and pathogenesis of CNS disorders accurately have to be developed through collaboration between basic scientists and clinicians. Many current models use otherwise healthy, young animals, which again is distinct from the clinical situation in many neurodegenerative diseases, where patients are often older, with concurrent diseases and chronic medication. For example, stroke patients frequently also suffer from hypertension and diabetes.

The animal models may not be able to fully predict the adverse events, toxicity of the cell product, immune and other biological responses, and risk for tumor formation that would occur after implantation of cells into patients. A lesson can be learned from the clinical trials with fetal dopaminergic cell therapy in PD. When troublesome graft-induced involuntary movements (so-called dyskinesias) were observed in patients (Freed et al., 2001), this side effect came as a surprise because none of the preclinical studies in rodent and primate models of PD had observed any adverse responses of this type. The risk of tumor formation from cells derived from pluripotent cells also makes clinical translation difficult. For example, life expectancy is virtually normal in PD patients, and therefore even a minor risk of tumor formation associated with stem cell therapy would be unacceptable. It is difficult to assess the clinical tumor risk with human embryonic stem cell derivates using preclinical xenograft studies (Erdö et al., 2003). Thus, for clinical translation, there will need to be rigorous mechanisms for determining the tumorigenicity of stem cells and their derivatives.

A prerequisite for application in patients must be a demonstration in an animal model that a given cell-based approach induces substantial improvement of clinically relevant functional deficits (Lindvall et al., 2012). For example, in rodent models of PD, behavioral improvement after stem cell therapy is often reported as a reversal of rotational asymmetry in animals with unilateral lesions of the nigrostriatal dopaminergic system. While this test gives a good measure of the dopamine-releasing capacity of the grafts, the deficit does not reflect any symptom seen in PD patients. Other behavioral tests are available but have only been used in few studies. Basic scientists and clinicians together have to develop functional and behavioral tests that assess deficits in animals resembling the impairments in patients with CNS disorders.

The Problem of Distribution and Progression of Pathology

Even if stem cells improve function in a specific area by neuronal replacement or other mechanisms, effective therapy is hindered if there is concurrent degeneration in other brain regions or if such changes develop after transplantation. For example, dopaminergic denervation in areas not reached by the intraputaminal grafts, such as the ventral striatum, in PD patients with fetal dopaminergic grafts counteracts the symptomatic relief following transplantation (Piccini et al., 2005). Similarly, even if replacement of motor neurons in the spinal cord of ALS patients did work, central motor neurons such as corticospinal neurons, which also degenerate in ALS, would most likely have to be replaced for effective, life-saving restoration of function. For successful, long-term clinical efficacy of stem cells in chronic neurodegenerative disorders, patient selection will be crucial, and neuronal replacement probably has to be combined with a neuroprotective therapy to hinder disease