NRC 3184

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TIMELINE
Cancer stem cells: an evolving concept

Long V.Nguyen, Robert Vanner, Peter Dirks and Connie J.Eaves
Abstract | The cancer stem cell (CSC) concept derives from the fact that cancers are
dysregulated tissue clones whose continued propagation is vested in a biologically
distinct subset of cells that are typically rare. This idea is not new, but has recently
gained prominence because of advances in defining normal tissue hierarchies, a
greater appreciation of the multistep nature of oncogenesis and improved methods
to propagate primary human cancers in immunodeficient mice. As a result we have
obtained new insights into why the CSC concept is not universally applicable, as
well as a new basis for understanding the complex evolution, phenotypic
heterogeneity and therapeutic challenges of many human cancers.
Three centuries after Robert Hooke1 first
proposed that cells are the units of life, our
understanding of cancer had advanced to
the point that many scientists believed that
a broad range of cures were within imminent reach. This led to the signing of the US
National Cancer Act of 1971 and an infusion
of resources to conquer the war against cancer within a decade. Interestingly, this decision coincided with the early growth of the
stem cell field as a biological science. Forty
years later, the naivety of the expectations
of 1971 is apparent. But the ensuing marriage of stem cell biology and oncology has
inspired new ideas and approaches that are
now fuelling renewed hope for significant
progress in the nearfuture2.
The clonal nature of tumours is a
consequence of their development being
dependent on the sequential accumulation
of multiple rare genetic or epigenetic events,
although strong evidence indicates that the
interactions of even fully malignant cells
with their environment also help to determine the extent of their deviant behaviour
and continuing evolution. Such a process
predicts that the first altered cells will probably resemble the primitive normal elements
of the tissue in which they arise, including
their responses to many of the same molecular control mechanisms. This is perhaps
best exemplified in studies of leukaemia3
but is also underscored by a long history
of detecting, in tumours, features of both
primitive and mature normal cells from the

tissue of origin, particularly when tumours
are detected at an early stage. Conversely,
the deregulated expansion of premalignant
cells is likely to reflect a perturbation in
the molecular mechanisms that normally
control cell numbers in the tissue of origin.
At the same time, both the multistep nature
of oncogenesis and the consequences of the
progressively pronounced perturbations are
likely to produce much greater heterogeneity
in the genetic, epigenetic, morphological,
phenotypic and functional characteristics
of the cells that make up a given tumour,
regardless of itsorigin.
The concept of somatic stem cells comes
from early observations in the eighteenth
century that lower organisms can regenerate multiple tissues and organs4 (TIMELINE).
We now know that regenerative potential is
an essential and physiologically regulated
property of multiple, partially specialized
subsets of stem cells that are present in different mammalian tissues5. These stem cells
can generate either one or both daughter
cells with the same poised but uncommitted
differentiation options (a self-renewal division), or they may generate progeny that exit
the stem cell state to begin the stepwise, and
ultimately irreversible, process of differentiation into specialized end cells that usually
lack further proliferative ability (a differentiation division) and that often have limited
survival. Regeneration requires conditions
NATURE REVIEWS | CANCER
that favour self-renewal divisions, whereas

homeostasis requires that the outcomes of
self-renewal and differentiation divisions are
balanced.
The dependence of establishing a stable
malignant phenotype on the accumulation
of a series of rare events makes it unlikely
that this process will happen within the
number of cell divisions that are required to
produce fully differentiated, non-dividing
progeny from most normal tissue stem
cells. By contrast, normal tissue stem cells
constitute a life-long reservoir of cells with
active mechanisms for self-renewal. These
considerations make stem cells obvious
candidates for accruing the events that can
generate a fully malignant cell population,
and are the basis of the original concept
of cancer stem cells (CSCs). However, the
genetic and phenotypic heterogeneity of
malignant populations, including those with
tumour-initiating properties, now demand
more complex models to describe the cell
ular make-up of many cancers by the time
they have become symptomatic (BOX1).
This has been accompanied by increasing
diversity in the field about what is meant by
the term cancer stem cell. There is therefore
increasing confusion and controversy about
the degree to which the concept of CSCs can
engender new insights into the biology of
tumours, or how to develop more effective
anticancer treatment strategies.
In this Timeline article, we trace the historical milestones that have shaped the concept of what normal stem cells are and then
use this as a backdrop to discuss how these
principles and related methodologies have
been adapted over time to investigate CSCs.
We have chosen to define CSCs as the cells
within a malignant clonal population that
can propagate the cancer under the assumption that these cells must be eradicated to
achieve cures. Importantly, this definition
assumes that not all of the cells within a
population of malignant cells have this property otherwise it would not be necessary
to distinguish CSCs with a separate designation. This definition also implies that CSCs
are responsible for generating all of the cells
within the malignant population that lack
cancer-propagating ability (as well as those
cells that perpetuate it). It also implies that
VOLUME 12 | FEBRUARY 2012 | 133
2012 Macmillan Publishers Limited. All rights reserved
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the choice of these alternative fates by CSCs
is embedded in an intrinsically established
intracellular molecular response network
that is likely to be related to the tissue from
which the CSCs originate and that the loss
of cancer-propagating ability is not readily
reversible invivo. Notably, this definition
does not make any inference about either
the cell in which the first oncogenic event
occurred or the cell type in which a fully
malignant phenotype was first manifested.
Stem cells in normal tissues
Blood cell formation has served as a paradigm
for the later development and testing of
ideas about the properties and regulation
of normal stem cells in many tissues (for
example, in the skin68, corneal limbus9,10,
brain1113, breast 1416, skeletal muscle17,18,
intestine19,20, lung 21,22 and prostate2325), and
the coordinated processes that normal stem
cells undergo to generate mature progeny
(TIMELINE). In many cases, the presence of a
stem cell population was first suggested by
histological evidence of cells that seemed
to be undifferentiated among or adjacent to
other cells that seemed to be differentiated.
However, functional validation and the
purification and further characterization
of normal tissue stem cells had to await the
development of methods that allowed their
growth and differentiation to be assessed at
a single-cell level. This is why invivo clonal
tracking, limiting-dilution transplantation,
and clonal culture methodologies have been
important steps in the development of this
field. The most generally accepted end point
to establish the existence of a stem cell is the

demonstration of a sustained regeneration of
all elements of the tissue, with the sustained
aspect being assumed to reflect the operation of a durable self-renewal capability of
the initial cell. Durable self-renewal refers
to the ability of certain cells in a tissue to
divide many times perhaps indefinitely
without activating a state of readiness to
differentiate into the mature cells that are
characteristic of thattissue.
Equally important for stem cell characterization has been the development of
assays that allow stem cell quantification.
This requirement has most often relied on
the demonstration of a positive and linear
doseresponse relationship between the
number of cells that are introduced into
the assay and the number of stem cells that
are detected. Limiting-dilution strategies
that are based on the presence or absence
of a regeneration end point have also been
frequently used. The validity of these assays
also depends on the existence of a linear
cell doseresponse relationship. The utility
of assays for stem cells also relies on their
specificity. For example, it may be difficult
to devise conditions (either in culture or
invivo) that support the production of not
only all of the mature cell types of the tissue, but also the survival and self-renewal
of the stem cells from which the mature cell
types arise. The inevitable vagueness of the
term maintained (which is used to infer the
durable self-renewal of a stem cell population) also requires that the term be
operationally defined and rationalized.
In spite of these caveats, functional

assays for normal stem cells have been very
useful for identifying their phenotypes,
and hence for the development of powerful strategies for the prospective isolation
and more direct analysis of these typically
rare cells. Together, these approaches have
enabled the discovery of many genes and
components of transcriptional networks and
signalling pathways that are important for
the functionality of different stem cell populations. It is interesting to note that many of
these interactomes are shared by multiple
stem cell types26,27, although a complete and
specific molecular description of the stem
cell state has not yet been generated for any
particulartissue.
Assays that detect and quantify stem
cells retrospectively based on functional
end points that measure their expressed
potentialities have also been important for
the discovery that some of the key properties of stem cells may vary at different
stages in the development of an organism,
or even at the same stage. For example, in
many tissues such as the haematopoietic
system, the skin, the corneal limbus, the
brain and skeletal muscle the stem cells
in the fetus may be rapidly dividing but
those in the adult show a relatively slow
turnover rate8,17,2830. However, in other
adult tissues such as the small intestine some cells with bona fide stem
cell activity remain in an actively dividing
state31, although a more quiescent, reserve
population may also exist 32. The differentiation programmes, proliferative states
Timeline | Milestones contributing to the understanding of normal stem cells and cancer stem cells
The first
observations
of tissue
regeneration
in animals4
Myeloproliferative disorders
described as panmyelopathies
with a possible common
multipotent haematopoietic
cell origin50
The first limiting dilution transplants of murine

leukaemia-producing cells as a forerunner of
later normal stem cell and CSC assays79
Undifferentiated multipotent embryonal cancer
cells from a murine testicular teratocarcinoma
found to give rise to all three germ layers172
Development of the
CFUS assay and its
use to detect stem cell
properties43,147
Proof of the clonal

origin of mouse
teratocarcinomas44
The first prospective

differential isolation of
different populations of
intermediate progenitor cells
(from the haematopoietic
system), thus supporting the
idea of a hierarchical process
of differentiation174
18th century 1941 1950 1955 1959 1960 1961 1963 1964 1967 1969
Teratocarcinomas found to
contain both differentiated and
undifferentiated cells, leading to
the idea that the undifferentiated
cells represent multipotent
cancer cells41
The first experimental evidence

of transplantable HSCs51
Discovery of the Ph chromosome as a

consistent clonal marker of human CML53
The first demonstration of regenerated

clones of blood cells bearing unique
radiation-induced marker chromosomes52
The first use of limiting dilution

analysis to quantify a stem cell
population (of HSCs)173
The first invivo

clonal assay for
CSCs (in a
murine model of
lymphoma)80
Molecular evidence of
the clonal origin of CML
(from X-chromosomeinactivation studies)57
Boxes with a black keyline indicate events related to stem cells in normal tissues. Boxes with a red keyline indicate events related to cancer stem cells. ALL, acute lymphoblastic
leukaemia; AML, acute myeloid leukaemia; CFUS, colony-forming unit-spleen; CML, chronic myeloid leukaemia; CSC, cancer stem cell; HSC, haematopoietic stem cell;
Ph, Philadelphia chromosome; RFLPs, restriction fragment length polymorphisms
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and the underlying regulatory molecular
mechanisms of normal tissue stem cells are
complex, and they can vary even for stem
cells within the same tissue8,3339.
It is also important to note that many
phenotypic markers that are key to the differential isolation of stem cells may not
be essential for the functional integrity of
stem cells. These include some markers that
have proven the most useful for the purification and characterization of stem cells.
Changes in the expression of such markers
may therefore have no effect on the differentiation and self-renewal properties of the
cells that are isolated using these markers.
Conversely, the expression of such markers
may vary under different developmental or
physiological conditions (for example, when
a stem cell changes its proliferative status),
thus obviating the utility of marker expression as a direct indicator of a stem cell. For
these reasons, very few phenotypic markers
have proven to be reliable surrogates for
enumerating stem cells, particularly when
stem cells have been physiologically or
experimentally perturbed.
Initial clues to the concept of CSCs
The first connection between cancer and
stem cells dates back to the nineteenth
century when histological similarities were
noted between tumours and embryonic
tissue. This gave rise to the embryonal
rest theory, which postulates that cancers
are caused by cells with properties similar
to those of the early embryo40. By 1941,
teratocarcinomas were recognized as
Leukaemic cells found

to behave as a
self-maintaining
population with no
influx of stem cells from
an unrecognized
precursor
compartment175
(19791983) Evidence of a
common lymphoidmyeloid
pluripotent stem cell in
humans that is the target of
the Ph translocation and
leukaemia initiation58,59,120
malignant structures that contained many

types of differentiated cells, suggesting
their origin from tumorigenic stem cells41
(TIMELINE). Pierce et al.42 further noted the
higher mitotic activity of the morphologically undifferentiated cells in teratocarcinomas and implicated these cells as
candidate teratocarcinoma stem cells. This
view was additionally supported by the
finding that the yield of teratocarcinomas
correlated with the frequency of transplanted cysts that contained such undifferentiated embryonal carcinoma cells42.
Meticulous tracking also showed that
the appearance of differentiated cells in the
teratocarcinomas occurred after an initial
phase of embryonal carcinoma cell proliferation similar to that seen during normal
embryogenesis. Four years later in 1964
and threeyears after the publication of
the first clonal assay for a primitive normal
tissue cell (in the haematopoietic system)43
Pierces group provided unequivocal
proof of the clonal origin of mouse teratocarcinomas from single transplanted multipotent malignant cells44. These findings
led Pierce and colleagues45,46 to formulate a
more general view of cancer as a caricature
of normal development in which more
malignant cells not only propagate the
cancer but also give rise to a more benign
population ofcells.
Other early clues about CSCs took root
in clinical observations. For example, in
1930 medulloblastoma was suggested to
originate from embryonal rests, termed
medulloblasts, that failed to differentiate.
Xlinked RFLPs used to

establish the first
multistep genetic model
of tumorigenesis in a
non-haematopoietic
human cancer
(colorectal cancer)75
The first use of limiting

dilution principles to
quantify and characterize
HSC populations with
long-term repopulating
activity invivo159 and
invitro157,158
Early in life these medulloblasts form

tumours that resemble the germinal zones
in the embryonic cerebellum47,48. This
concept has recently gained support with
the suggestion that different subtypes of
medulloblastoma have unique developmental origins that reflect the different sensitivities of disparate precursor compartments
to specific oncogenic mutations49. Another
20years later, Dameshek50 used his pathological observations of the multi-lineage
involvement of all of the myeloproliferative
disorders to insightfully speculate that these
diseases share a common origin from a
multipotent haematopoietic stem cell. This
speculation was followed several years later
by the first experimental evidence of such a
cell51,52 (TIMELINE).
The discovery in 1960by Nowell and
Hungerford53 of the Philadelphia (Ph) chromosome and its unique association with
chronic myeloid leukaemia (CML) provided the first genetic evidence that human
malignancies are usually individual clones
of cells and may have specific mutational
causes. The subsequent demonstration
of the Ph chromosome in all of the major
non-lymphoid lineages of blood cells from
patients with chronic phase CML54,55 confirmed Damesheks prediction of a probable
common origin of the CML clone from a
transformed, but still multipotent, haematopoietic stem cell. This evidence was further reinforced by the results of unfortunate
concurrent attempts to use blood from
patients with CML as a transient source
of granulocytes for highly leukopaenic
Demonstration that
leukaemic stem cells from
patients with AML
reconstitute the full
spectrum of phenotypes in
the malignant populations
that they regenerate in
transplanted mice116
Demonstration that different

subtypes of human ALL can
originate at distinct stages of
haematopoietic
differentiation183
1970 1976 1979 1984 1987 1988 1989 1992 1997 2003 2005
The first clonal assay for non-haematopoietic

progenitor cells (skin epithelial progenitor
cells)6
(19761981) Establishment of a hierarchical
model of haematopoietic differentiation
from evidence of different types of
progenitors that undergo coupled
restrictions in their potentials for
differentiation and proliferation153155,176,177
The first demonstration

of a highly selective
positive marker (CD34)
for a rare subset of
primitive human
haematopoietic cells178
The first success in

prospectively isolating
stem-like cells to near
homogeneity (as
shown by the CFU-S
assay), achieved using
a combination of cell
surface markers179
Development of a
quantitative colony assay
for neural stem cells11
(20032004) The first demonstration

of CSCs in human solid tumours
(breast and brain cancers)85,111
(19922000) Demonstration that biologically

distinct stages of haematopoietic differentiation
can be prospectively isolated as separate viable
cell populations180182

PERSPECTIVES
BCRABL1targeted drugs. In patients
with chronic phase CML, these drugs usually eliminate most of the Ph-positive cells
and induce apparent complete remissions.
But, because these drugs are poorly active
against the stem cells of chronic phase
CML62,66,67, the CML clone usually regrows
rapidly after the treatment is stopped, even
after many years of treatment 68.
Clonal evolution of cancers
The first genetic evidence of the clonal
progression of cancer in patients was provided in 1963by studies of a case of CML
that was described by Levan, Nichols and

Norden69. CML is usually diagnosed in
a so-called chronic phase when mature
blood cell production by the clone is still
essentially normal. If adequately treated at
this stage, CML may not subsequently proceed to the otherwise inevitable blast phase
that resembles an acute leukaemia. What
Levan etal.69 observed was the coexistence
in the same patient of Ph-positive cells that
differed in their sex chromosome complement, one set being 46Ph;XY and the other
being 47Ph;XXY; other somatic cells were
normal (46;XY). Given the low probability
Box 1 | Hierarchical versus stochastic models of tumour cell heterogeneity

The hierarchical model (see the figure) assumes that cancer stem cells (CSCs) represent a
biologically distinct subset within the total malignant cell population. It is the only model that is
relevant to the designation of malignant tumour-propagating cells as CSCs, although it does not
require that CSCs are functionally or genetically homogeneous. According to this model, a pool of
CSCs can only be maintained by cells that have both CSC potential and, by definition, the ability to
give rise to progeny with self-limited proliferative capacity. This model precludes the concept of
reversibility between intrinsically determined states of unrestricted and limited proliferative
potential. However, it accommodates the possibility that CSCs, like their normal counterparts, may
retain responsiveness to (and even dependence on) external cues to elicit their intrinsically
determined potentialities for survival, growth and differentiation, irrespective of how perturbed the
process of differentiation may be. The clinical implication from this model is that the elimination of
all CSCs will inevitably terminate the growth of the tumour, and that failure to do so leaves open the
possibility of relapse.
By contrast, the stochastic model (see the figure) assumes that every cell within a tumour has the
same potential to act as a CSC, and that their variable activities are at least partially determined by
some stochastically varying intrinsic factor. This means that their activities are not totally determined
by the environment in which the cells are found. Experimentally, the stochastic model can mimic a
hierarchy in which the CSCs are rare and produce cells with diminished proliferative capacity,
because only a proportion of cells at any given moment exhibits CSC activity.
To distinguish these models from one another requires the use of clonal tracking methods to
demonstrate whether there is stochastic determination of CSC activity on serial propagation. It is
important that CSCs are functionally defined. Therefore they cannot be identified solely on the basis
of phenotypic or transcriptomic profiles, which may fluctuate differently from the stem cell
behaviour that is detected functionally.
Cell divisions
Normal
Hierarchical model
Normal stem cell

Normal progenitor cell
Normal terminally
dierentiated cell
Apoptotic or necrotic cell
Cell divisions
Cell divisions
patients. The transient engraftment of these

recipients with circulating Ph-positive cells
provided the first indication of transplantable, leukaemic stem cells in the circulation
of CML patients56. A few years later in the
late 1960s, Fialkow and colleagues57 generated formal proof of the clonal origin of all
of the major lineages of blood cells (with
the exception of T cells) in the neoplastic
clone that is present in patients with
CML. They studied a series of female
patients with CML who were heterozygous
for different isoforms of the Xlinked
glucose-6phosphate dehydrogenase (G6PD)
gene. Females who are heterozygotes for
G6PD alleles express only one G6PD isoform
in any given cell, but have approximately
equal distributions of cells expressing each
isoform throughout their somatic tissues. By
contrast, in female patients with CML who
were also G6PD heterozygotes, all of the red
blood cells, platelets and granulocytes were
found to contain only one isoform. This
finding showed definitively that all of these
mature blood cell types were derived from
a single deregulated multipotent cell that
must have also been able to self-renew, as is
expected of a haematopoietic stemcell.
Later studies showed that chronic phase
CML clones also frequently included some
B-cell-lineage progeny 58,59. In addition, progenitors of all of the lineages were present, as
is expected of a clone in which the process
of differentiation is largely preserved but in
which the control of proliferative activity is
significantly deregulated60,61. Interestingly,
subsequent studies showed that the stem
cells of chronic phase CML resemble their
normal counterparts in being a largely quiescent population, and they even display
a lower than normal self-renewal ability
when stimulated to divide62. These features
point out how a hierarchically structured
neoplasm may use different mechanisms
at sequential stages of differentiation to
expand the clone and endow its members
with a competitive advantage. They also
illustrate why targeting the point of greatest
expansion within the hierarchy of a neoplastic clone may provide only a temporary
remission.
Subsequent studies showed that mimicry of chronic phase CML using mouse
cells that were genetically engineered to
express BCRABL1 (the pathogenic fusion
oncogene that is created during the formation of the Ph chromosome63) depends on
an initial and appropriate targeting of a
haematopoietic stem cell64,65. The clinical
relevance of this finding is also well established from a decade of experience with
Stochastic model
C
A
B
ne lone lone
C
C
Cancer stem cell
Cancer cell with diminished
proliferative ability
Cancer cell with no proliferative ability
Clo
Nature Reviews | Cancer

PERSPECTIVES
of two primary CML clones arising independently in the same individual, their
observation suggested the formation of a
subclone that was marked by a duplication
of the Xchromosome. Subsequent studies documented the acquisition of other
cytogenetic alterations in emerging subclones that heralded the transition to blast
crisis and thus implicated a number of
genes in this transition70. More recent work
has shown that BCRABL1 induces a state
of genomic instability in primitive CML
cells, thus providing a potential mechanism
for the clonal evolution that is characteristic
of this disease71,72.
Concurrent evidence of a similar phenomenon emerged from studiesof the
DNA content of cells in solid tumours73,74.
Although difficulties in obtaining
metaphase preparations from these
cells inhibited the assessment of their
clonal origin and evolution, these issues
became addressable by the application of
the approaches that were introduced by
Fialkow 57 and subsequently adapted to a
molecular platform by Vogelstein and colleagues75. The Vogelstein group then used
this platform to show for the first time that
human colorectal tumours are clonally
derived and that 75% of these tumours are
characterized by a loss of chromosome 17p.
Their subsequent examination of colorectal
tumours at various stages of progression
allowed the construction of the first multi
step genetic model of tumorigenesis in a
solid tissue. Although the exact sequence
or pattern of genetic alterations is now
known to be variable, this model has
proven to be an important framework for
understanding many other examples of
carcinogenesis. Importantly, recent studies
have shown that the mutations that were
found in human colorectal cancer generate intestinal carcinomas in mice only
when forced to occur in the stem cells76.
This is consistent with a similar patho
genetic sequence in the genesis of human
gastrointestinalcancers.
CSC assays
To investigate the cellular mechanisms that
are responsible for tumour propagation,
initial efforts focused on the transfer from
one organism to another of tumour fragments or large numbers of tumour cells.
Then, in 1937, Furth and Kahn77 showed
that transplanting a single cell from a
murine leukaemia could successfully reinitiate the leukaemia in 5% of recipient
animals. Unfortunately, this result could
not distinguish between two alternative
possibilities: either that the assay was inefficient in detecting cells that all possessed the
same leukaemia-initiating potential, or that
there was an intrinsically heterogeneous
population of initial cells of which only a
small fraction possessed leukaemiainitiating ability. Later evidence from several anecdotal clinical reports suggested
that primary human leukaemia had a
similar efficiency of disease re-initiation56,
even when immunological barriers to
prevent engraftment were reduced78. Such
observations highlighted the need for new
models to investigate the biological properties of malignant cells with transplantable
tumorigenic ability. Attempts to quantify
these cells began in earnest in 1959 with
the introduction by Hewitt and Wilson79
of limiting-dilution transplants. Four years
later, Bruce and Van Der Gaag 80 reported
the first invivo colony assay for a mouse
lymphoma-initiating cell. This was adapted
from the colony-forming unit-spleen
assay that was described by Till and
McCulloch (BOX2).
The subsequent creation of a series of
genetically modified mice with increasing
severities of immunodeficiencies81 then
enabled analogous xenograft experiments
to detect and quantify cells that are present
in many primary human tumours and
have tumour-initiating activity8295 (FIG.1).
These xenograft models are considered the
gold standard in the human CSC assay
field, and they have been very powerful in

enabling studies of the biology and therapeutic responses of human CSCs. However,
these models also have caveats. First, most
primary tumour cells do not grow auton
omously and may depend on factors that
are not cross-species active in both mice
and humans9698. Second, the mutated gene
that causes severe combined immuno
deficiency in many of the mouse strains
used is a DNA repair gene99. This makes
the mice hypersensitive to DNA-damaging
treatments, thus potentially compromising the utility of these strains to assess the
responses of engrafted human CSCs to
DNA-damaging drugs. A third caveat is
the immunodeficiency in these mice. This
facilitates their engraftment with human
cells but also provides a system that lacks
many elements that are now thought to
substantially influence tumour growth100103.
Finally, the ~2year lifespan of mice may
compromise the detection of slow-growing
human tumours and could possibly impose
unknown, age-related differences in host
factors on the behaviour of tumours.
Conversely, tumour cells with transient,
but not permanent, self-sustaining ability
may be prematurely identified as CSCs in
immunodeficient mouse assays if the assays
are not of sufficient duration104. Such variables probably explain some of the reported
differences in the observed frequencies and
properties ofCSCs.
Box 2 | Quantitative stem cell assays for haematopoietic stem cells

The first experimental evidence for haematopoietic stem cells was the observation that lethally
irradiated mice that had been protected by an injection of bone marrow cells developed a
haematopoietic system that was stably chimeric51 and included clones of host cells bearing unique
radiation-induced marker chromosomes52. A few years later, Till and McCulloch43,147 devised a
method for quantifying transplantable bone marrow cells. They called these cells colony-forming
units-spleen (CFUS) because they formed macroscopically visible colonies in the spleen following
injection into irradiated mice. They further showed that CFUS were multipotent and had
self-renewal ability (as shown by their generation of daughter cells with the same CFUS properties).
Invitro colony assays for mouse and human haematopoietic cells were introduced a few years
later. Further characterization of cellular phenotypes and properties showed that many distinct
intermediate progenitor cell types with clonogenic activity invitro could be detected. However,
most of these cells were restricted to producing one or two lineages of mature blood cells, had
little self-renewal ability and, in the mouse, showed little overlap with CFUS148156. The
identification of these intermediate progenitor cells provided the first indication of the existence
of a hierarchy of sequential early stages of haematopoietic progenitor differentiation. Later, these
stages were described more explicitly by their different cell surface marker phenotypes.
Intermediate progenitor cells were visualized in other tissues also but were often referred to as
transit amplifying cells.
By the 1980s, CFUS were shown to be prospectively separable from cells with long-term
haematopoietic repopulating activity. New assays were then introduced that used limiting-dilution
principles to detect cells that could sustain progeny outputs for many weeks either invitro on
stromal feeder layers157,158 or invivo in irradiated hosts159. Additional experiments showed that
these cells could be isolated at very high purities35,160162. The generation of suitably
immunodeficient mice has allowed similar limiting-dilution transplant assays and purification
strategies to be developed for analogous types of human haematopoietic cells163165.

PERSPECTIVES
In vivo
Heterogeneous
human tumour
Tumour
growth
Human
CSC-containing
cell population
Primary immunodecient mouse

Dissociate
Cancer
stem cell
Other
cancer
(non-stem)
cells
In vivo
Tumour
growth
Regenerated human Secondary immunodecient mouse
CSC-containing cell
population
Figure 1 | Xenograft assays to measure human CSCs. Cancer stem cells (CSCs) are most rigorously
Nature Reviews
| Cancer
and specifically defined by their demonstrated ability to produce a progressively
growing
tumour
consisting of cells that resemble those in the original tumour. For this reason, CSCs are also frequently
referred to operationally as tumour-initiating cells. Limiting-dilution transplants or other clonal tracking strategies are typically used to determine the frequency of CSCs in the initial tumour-derived cell
suspension. Ideally, the tumours that form in primary hosts are again tested for their content of cells
with CSC activity (demonstrable in injected secondary hosts) to formally confirm that the initial CSCs
had self-replicating ability. These principles apply both to measurements of the CSC frequency and
the total CSC content, either in the bulk population or in isolated subpopulations of dissociated
tumour cells. The most sensitive assays are those in which there is no immunological difference
between the host and the tumour. When this is not possible (such as for human tumours), xenografts
into highly immunodeficient mice are used.
The important development of invitro

colony assays for fibroblasts105 and then for
normal haematopoietic cells (BOX2)
stimulated efforts to identify conditions
that would support the clonal growth of
malignant cells invitro. Successes with
different types of human leukaemic cells
were first reported in the early 1970s106,107
and stimulated attempts to achieve similar
results with cells from solid tumours108.
However, the studies of cells from solid
tumours were stymied for many years
by a lack of understanding of the growth
requirements of normal cells from these
tissues. Only after more than a decade were
several such factors identified; for example,
fibroblast growth factor (FGF) and epidermal growth factor (EGF). Then, in 1992,
Reynolds and Weiss11 showed that EGF
and FGF could stimulate primitive, multipotent neural cells to proliferate in liquid
suspension cultures and to generate
clonal clusters of cells that they called
neurospheres. The novel aspect of their
approach was the use of non-adherent
culture conditions for cells that had been
assumed to be anchorage-dependent (that
is, requiring attachment to a suitable surface to proliferate). Interestingly, it was
soon found that similar conditions would
support the growth of many other types

of primitive normal and malignant cells
invitro. This enabled the first evidence to
be obtained of a brain tumour stem cell
population109112. Subsequently, similar
conditions were reported to support the
growth of human breast cancer 113, colon
cancer 89, melanoma114 and prostate cancer
cells115. However, although such spheres
can be derived from single cells, most
studies of primary tumours using this
methodology have plated the initial cells
in liquid cultures at much higher densities.
The rapid cell aggregation that then occurs
confounds interpretation of the meaning of
spherecounts.
CSC purification
A classic concern with stem cell assays is
whether their selectivity reflects an important, intrinsic set of molecular properties
(that are assumed to be irreversible) in a
subset of cells or a generalized inefficiency
in the assay (for example, inefficient access
to a certain site or inadequate exposure to
required growth factors). One approach
to resolve these possibilities is to query
whether the cells that are identified by the
assay possess different features from the other
cells (FIG.2). In its most stringent application
to cancers, a CSC assay would require demonstrating that the malignant population
contains a phenotypically distinct subset
of cells that can produce the same range of
malignant progeny in recipient mice as was
present in the original tumour (FIG.1). This
paradigm was first introduced by Bonnet
and Dick116 in their studies of human acute
myeloid leukaemia (AML), and has subsequently been extended to many solid
tumours8587,89,9195, but with incomplete
reproducibility 90.
An important and interesting common
theme has been the finding that differentially expressed markers on the normal
stem cells of the tissue in which a cancer
has arisen are frequently useful for the
identification and isolation of the CSCs.
Examples include the CD34+CD38low or
CD34+CD38 phenotype of many human
AML stem cells, the CD133+ or CD15+
phenotype of human brain tumour CSCs
and the CD44+CD24low or CD44+CD24
phenotype of human breast tumour CSCs.
However, the reproducibility of these findings remains controversial90,117. The reasons
for many of the reported discrepancies are
not yet clear. Possible explanations may
include differences in whether the tumour
population being assessed is freshly isolated or has been passaged previously in
mice. Differences in the techniques that
are used to dissociate the tumour and to
isolate various cellular subsets from it, and
the strain of host mouse used may also be
contributing factors. Additional sources
of confusion include the lack of consistent
stringent end points of stem cell activity
and the intermingling of data from cell
lines or even briefly cultured malignant
cells with data from freshly isolated
primarycancers.
The measurement of CSCs is clearly a
rapidly evolving area in which more diverse
strategies may enable CSC populations to be
isolated at higher purities. This could eventually make single-cell transplants practical
and allow meaningful, direct analyses of the
cells responsible. No doubt progress along
these lines will continue to require the iterative testing of new phenotypic properties. In
particular, a goal is to identify properties that
are both essential for the invivo tumourpropagating activity of CSCs and detectable
by experimental procedures that do not
require the killing of thecells.
Genomic instability
Cells possess a battery of mechanisms
to preserve the integrity of their DNA.
Structural changes (such as deletions,
PERSPECTIVES
duplications and various rearrangements)
as well as point mutations can initiate
biological changes that promote tumour
formation. Therefore, it has long been
assumed that the perturbation of mechanisms that control genomic stability are
probably major contributors to the oncogenic process. Results from recent studies
have highlighted the complex numbers and
dynamics of subclones that characterize
many human cancers. These include the
remarkable finding that even rare mutations can be independently recurrent 118,119.
Thus, although a linear progression model
may best describe the earliest stages of
oncogenesis, a multitude of subclones
simultaneously appearing and disappearing each driven by a different subset of
CSCs (BOX1) is more relevant to depicting many clinical realities. In addition, the
low frequency of CSCs in many tumours
(<1 per 1,000 cells) makes these cells
largely inaccessible to current genomic or
transcriptomic analysis methods, unless
these analyses are coupled to powerful
purification strategies that are not yet widely
available. Thus, the interpretation of data
that are derived using many of the new and
powerful genomic tools may still be limited
in their ability to identify which genomic
changes represent driver events or important
moleculartargets.
CSCs may not be cancerous stem cells
The broad concept of CSCs is frequently
confused with the narrower concept of normal stem cells becoming cancerous (cancerous stem cells). The multistep nature
of oncogenesis does not require or even
assume that all changes leading up to the
establishment of a malignant stem cell will
occur within the original stem cell compartment of the affected tissue. (Nevertheless,
it does seem likely that the first oncogenic
event would often have occurred in such
cells, at least in tissues in which cell turn
over is rapid.) Historical evidence of disease
progression involving the establishment
of subclones of genetically, and potentially phenotypically, distinct cells has
required that the field anticipates a broader
range of CSCs within tumours of clinical
significance.
Studies of the events that generate
haematopoietic malignancies have again
historically served as an instructive guide.
A prototypical example of the abnormal
acquisition of self-renewal ability by
neoplastic cells that originally lacked this
ability is represented by the dual types
of blast crisis that characterize disease
progression in CML. Throughout the

chronic phase of CML, the clone is maintained by a rare subset of multipotent, selfrenewing stem cells that have phenotypes
and properties that are only subtly altered
from those of their normal haematopoietic stem cell counterparts. However, if
the continuing generation of B lymphoid
progenitors (BLPs) or granulocytemacrophage progenitors (GMPs) from
these chronic phase CML stem cells is not
therapeutically prevented, these later progenitor cell types can develop mutations
that enable them to acquire CSC properties and hence to generate more aggressive subclones of leukaemic cells. The
result is the emergence of a new dominant
subclone, derived from one of these later
progenitor cell types, that is similar to the
clones that are seen in other forms of Bcell
acute lymphoblastic leukaemia (B-ALL)120
or AML121. Although the treatment of blast
phase CML is rarely effective, transient
suppression of the responsible subclone
is typically accompanied by a transient
resurgence of the chronic phase clone.
These observations are important because
they illustrate that it will probably be necessary to eliminate all CSC subtypes in
a malignancy even those that may be
undetectable in the presence of the most
aggressive subclone.
In human AML, in which many genetic
mechanisms of causation have also been
implicated122, a common origin in a very
primitive cell has been widely inferred
from the repeated demonstration that
cells that are able to regenerate AML when
transplanted into immunodeficient mice
usually have a CD34+CD38 phenotype116.
However, even in human AML, this notion
has been challenged. One recent study
showed that prevalent CD34+CD38+ AML
stem cells may be undetected owing to a
technical problem with the CD38targeted
antibody that was used to isolate the cells117.
A second study found that 80% of all
tested human AML samples contained at
least two additional populations of CSCs.
These additional populations had surface
immunophenotypes and gene expression signatures that most closely matched
normal GMPs and lymphoid-primed
multipotent progenitors (LMPPs) rather
than haematopoietic stem cells123. The
leukaemic GMPs and LMPPs were hierarchically organized, even though they also
displayed self-renewal capacity. Similarly,
a third study confirmed that functionally
defined CSCs from different patients with
AML may exhibit different phenotypes
Total = 25 cells
Assay
Bulk
Overall stem cell
frequency = 11 in 25
Total stem cells = 11
Cell
sorting
Purple phenotype
Blue phenotype
Assay
Assay
Stem cell
frequency = 1 in 2
Stem cell
frequency = 4 in 5
Stem cells
N=7
N=4
Non-stem cells
N=3
N=2
N=7
N=1
N=1
Figure 2 | ProspectiveNature
purification
of CSCs.
Reviews | Cancer
Evidence for the existence of a hierarchy of cell
types in a tumour that are sustained by a biologically distinct subset of cancer stem cells (CSCs) is
commonly provided by the demonstration that
the cells with CSC activity possess a unique and
consistent phenotype. This involves subdividing
the CSCs according to certain phenotypic differences and then defining their functional activity.
Subdivision can be on the basis of differential
expression of various cell surface markers
which can be discriminated using an immunomagnetic approach or fluorescent activated cell
sorting or a differential response to an agent
such as a drug or small interfering RNA. Functional
activity can be demonstrated by tumour production in a xenograft assay, for example. However,
interpreting the results of such experiments can
be problematic if proportionally equal amounts of
all cell subsets are not assessed. The reason is that
a given subset may contain a much higher frequency of CSCs, but a smaller absolute number of
CSCs (calculated by multiplying the CSC frequency by the total cell number; compare purple
to blue in the schema shown). In addition, it
should be remembered that a particular phenotype may enrich for CSC activity but may have no
biological role in CSC functionality. Accordingly,
it may not be a reliable phenotype and cannot be
used to infer the presence or absence of CSCs in
another context.
within the CD34+ subpopulation but the

overall gene expression profile, rather then
the absence of CD38 expression, correlated
most strongly with patient outcome124. It is
thus interesting that direct introduction of
the MLLAF9 fusion gene (which is seen
in some patients with AML) into mouse
PERSPECTIVES
GMPs can produce a mouse leukaemia
without an apparent alteration of the cell
surface markers or gene expression profile
of the target cells125. More recently, similar
results have been reported for mouse common myeloid progenitors that were transduced with MN1, another potent oncogene
in human AML126. Together, these findings
argue against any simple linear progression
model of clonal evolution during human
leukaemogenesis. The pitfalls of drawing
inferences directly from tumour pheno
typing studies are further illustrated by
murine studies of non-haematopoietic cancers (BOX3). Thus, although the cancerinitiating mutation may occur in a stem
cell, the cell that becomes the tumourmaintaining CSC may more closely resemble
a later normal celltype.
In summary, even for cancers that initially arise as genetically homogeneous
clones of malignant cells, more genetically
and phenotypically heterogeneous derivative CSCs will probably have developed by
the time symptoms of disease are produced.
In addition, the cell in which a mutation is
generated may not reflect the cell type upon
which it confers malignant properties.
Instability of the CSC state
Normal stem cell behaviour is modulated by
the external signals that the cells encounter,
and there is growing evidence that this is
generally orchestrated invivo in tissuespecific, niche microenvironments.
Evidence that CSC behaviour may be similarly modulated invivo is also accumulating.
For example, mouse embryonal carcinoma
cells transplanted into blastocysts can form
genetically mosaic mice that develop normally and remain tumour-free, whereas the
same cells injected subcutaneously consistently form tumours within a few weeks127.
Early experiments showed that mouse
leukaemic cells could, in addition to causing leukaemia, sometimes act like normal
haematopoietic stem cells and contribute to
the production of mature myeloid cells when
injected into the placentas of day 1011
mouse embryos128. Examples of malignant
clones producing fully differentiated progeny in humans include chronic phase CML57
and some AML clones in patients in remission129. Examples of malignant cells stimulated to differentiate invitro are abundant.
However, although attempts to exploit this
principle therapeutically have been widely
evaluated, few success stories exist; perhaps
the best is the use of all-trans retinoic acid
(ATRA) to treat human acute promyelocytic
leukaemia (APL)130,131.
The possibility that CSCs can undergo

reversible fluctuations in their stem cell status has also been suggested132135. However,
this possibility is largely based on the behaviour of cell lines as models of malignant
populations and not from studies of primary
isolates of malignant cells. Interestingly,
however, non-tumour-initiating cells from
colon tumours that had low WNT activity
were found to acquire tumour-initiating
properties when co-cultured with myofibro
blasts that secreted hepatocyte growth
factor. The co-culture resulted in the activation of WNT pathway signalling in the
colon cells136, thus reinforcing the idea that
tumour initiation invivo is not a completely
cell-autonomous property and may be
subject to extrinsic regulation.
Also relevant to the issue of the stability
and reversibility of CSCs is the remarkable series of discoveries showing that
normal mammalian cell differentiation is
completely reversible. These began with
the classic experiments in frogs137 and later
sheep138, which demonstrated that whole
animals could be regenerated when single
nuclei that were taken from differentiated
cells were placed in prepared oocytes. Then,
in 2006, the transient expression of four
transcription factors in a differentiated cell
was shown to be sufficient to induce a pluripotent state, similar to that established in
embryonic stem cells (ESCs)139142. However,
unlike ESCs, the progeny of these induced
pluripotent stem cells (iPSCs) proved to
be highly prone to generating tumours in
derived chimeric mice, although this could
be independently modulated143. The fact

that some of the genes that contribute to
iPSC generation are oncogenes makes these
observations particularly intriguing to
the concept of an intrinsically stable
malignant stem cell state that may lie latent
for many generations. The observed expression of reprogramming factors, pluripotency genes and their binding targets in
non-germ-cell cancers has also led to interesting speculations that CSCs, ESCs and
iPSCs may share common mechanisms of
self-maintenance144.
Together, these findings indicate that no
profile of cell behaviour can be assumed
to be completely determined by a fixed
intrinsic state, and highlight the possibility of a greater role for the microenvironment in regulating tumour formation than
might have been previously envisioned.
Although the enforced transient expression of certain genes can make a normal
differentiated cell seem more primitive,
we still have no evidence that such events
contribute to the formation or progression
of humancancers.
Limitations of the CSC model
The utility of the CSC model is based on
the assumption that most of the cells in the
malignant population do not have tumourinitiating activity. The situation of every
cell having stem cell capabilities may arise
when malignant populations accumulate
an overwhelming load of mutations and/or
epigenetic alterations. If this occurs, searching for intrinsic properties of rare subsets of
Box 3 | Experimental evidence that CSCs may emerge from progenitor cells
Multiple mouse models of cancer possess cancer stem cells (CSCs) that do not resemble the stem
cells of the somatic tissue in which they arise. An example is a study by Morrison and colleagues166
of neurofibromas in P0aCre+Nf1fl/ and Nf1+/Cdkn2a/ mice, and peripheral nerve sheath tumours
in Nf1+/Trp53+/- mice. These experiments showed that, despite the presence of these mutations in
the neural crest stem cells, the tumours arose exclusively from their differentiated glial progeny.
Moreover, the ability of these tumours to be propagated either invitro or invivo was not a
function of cancer cells with the phenotype of neural crest stem cells, but rather a population
with glial features. Similarly, directed deletion of Patched1 in either the neural stem cell or granule
neuron precursor compartment of mice has been found to cause the formation of
medulloblastomas. However, a lethal tumour mass did not form until the cells acquired the
identity of granule neuron progenitors167,168.
Another example is the elegant study of Zong and colleagues169. They used a double marker
approach to show that, even when neural stem cells were made deficient in both Trp53 and Nf1,
the CSCs of the resultant gliomas were oligodendrocyte precursor cells (OPCs). Similarly,
homozygous deletion of the DNA-binding domain of p53 in glial fibrillary acidic protein (GFAP)-Cre
mice implicated neural stem cells of the subventricular zone as the origin of the NESTIN+OLIG2+
OPC-like cells, the expansion of which enabled the subsequent hits that led to the emergence of
malignant gliomas170. These results also explain the findings of an earlier study of Nf1fl/flTrp53fl/fl
mice in which the injection of Cre-expressing adenovirus into adults produced diffuse and
infiltrating high-grade gliomas only when targeted to the subventricular zone (where the neural
stem cells are located). This suggests that, in this model, it was the neural stem cells that had to be
mutated but that it was the OPCs that acquired tumour-propagating properties171.
PERSPECTIVES
cells that are responsible for tumour maintenance, metastasis, therapy resistance or
disease relapse may be irrelevant. However,
when the CSC model fits, identifying the cellular and molecular bases of these intrinsic
properties is crucial. Characterizing these
profiles may be challenging because the
genetic instability of most cancers causes
a continuous diversification of the cells
within them. In addition, limiting-dilution
xenotransplant methods for studying CSCs
have caveats, as discussed above. The clinical
assessment of findings from experimental
studies thus remains key to assessing the
relevance of data that are obtained from
characterizing CSCs and their responses in
experimentalmodels.
Clinical relevance of CSCs
The ultimate test of the value of the CSC
concept is how useful it will be for improving disease management. Certainly, the use
of cancer cell lines has generally proven
to have little value thus far for predicting the effectiveness of new therapeutics.
Although efforts to target CSCs date back
to the 1960s in the form of therapies that
were designed to induce differentiation
in cancer cells, this approach gained little
traction (with the exception of ATRA for
APL). Recently, improved methods
for studying CSCs and for demonstrating
the potential relevance of CSCs to tumour
biology and clinical oncology have
profoundly expanded interest in targeting
these cells. However, we still have a minimal understanding of the extent to which
even recurrent genetic changes in particular cancers contribute to their malignant
growth properties. Not surprisingly, an
appreciation of tumour cell hierarchies has
also yet to have a clear impact on patient
management.
Chronic phase CML is an interesting
example of a malignancy for which drugs
that target the pathogenic BCRABL1
oncoprotein have proven to be remarkably
effective clinically, in spite of their failure to
eliminate the neoplastic stem cell population. The downside is that a large proportion
of patients may have to take these drugs permanently, unless the drugs can be combined
with other treatments that have not yet been
devised145. Clearly, a drug that could also target the CML stem cell population, and bring
cures without severe side effects, would offer
a major step forwards.
That many CSCs rely on archetypal
embryonal signalling pathways (such as
SHH, WNT, NOTCH and bone morphogenetic protein (BMP)) has led to inhibitors
of these pathways being considered as

candidate anti-CSC agents146, even though
these pathways are not exclusive to CSCs.
Given the large number of cells in a symptomatic tumour (typically >1011 cells), it
is also unlikely that a single pathway (or
identical pathways) will be operative in
all of the CSCs in a given tumour. Earlier
diagnosis and the simultaneous use of
drugs that can affect multiple targets that
are essential to CSCs are therefore likely to
be required to achieve cures. Therefore, the
development of such drugs will probably
require new ways to rapidly and selectively
screen for agents that can effectively target
primaryCSCs.
The clinical need for better treatments
will continue to fuel efforts to understand
the critical pathways that human cancer cells
depend on for their self-propagation and/or
viability, and to devise new methods to
selectively eliminate or neutralize these cells.
We anticipate that new surrogate strategies
to define and manipulate human CSCs will
accelerate this development on many fronts,
although they may be less useful in some
cases. In the end, clinical trials will necessarily be the ultimate test of evolving concepts
and new agents, thus emphasizing the importance of a continuing multidisciplinary effort
to generate and evaluateboth.
Long V.Nguyen and Connie J.Eaves are at the Terry
Fox Laboratory, British Columbia Cancer Agency and
the University of British Columbia,
675 West 10th Avenue, Vancouver,
British Columbia, V5Z 1L3, Canada.
Robert Vanner and Peter Dirks are at the Department
of Developmental and Stem Cell Biology,
Hospital for Sick Children and University of Toronto,
Toronto, Ontario, Canada.
L.V.N.and R.V.contributed equally to this work
Correspondence to C.J.E.
e-mail: ceaves@bccrc.ca
doi:10.1038/nrc3184
Published online 12 January 2012
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Acknowledgements
The authors acknowledge research support from the British

Columbia Cancer Foundation, the Canadian Breast Cancer
Research Alliance, the Canadian Cancer Society, the Canadian
Institutes of Health Research (CIHR), the Canadian Stem Cell
Network, the Ontario Institute of Cancer Research and the
Terry Fox Foundation. L.V.N. and R.V. are both recipients of
Vanier Canada Graduate Scholarships from theCIHR.
Competing interests statement
The authors declare no competing financial interests.
DATABASES
National Cancer Institute Drug Dictionary:
http://www.cancer.gov/drugdictionary
all-trans retinoic acid
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PERSPECTIVES

Cancer stem cells: an evolving concept

primitive and mature normal cells from the

NATURE REVIEWS | CANCER

that favour self-renewal divisions, whereas

2012 Macmillan Publishers Limited. All rights reserved

to establish the existence of a stem cell is the

In spite of these caveats, functional

The first limiting dilution transplants of murine

Proof of the clonal

The first prospective

The first experimental evidence

Discovery of the Ph chromosome as a

The first demonstration of regenerated

The first use of limiting dilution

The first invivo

134 | FEBRUARY 2012 | VOLUME 12

Leukaemic cells found

malignant structures that contained many

Xlinked RFLPs used to

The first use of limiting

Early in life these medulloblasts form

Demonstration that different

The first clonal assay for non-haematopoietic

The first demonstration

The first success in

(20032004) The first demonstration

(19922000) Demonstration that biologically

NATURE REVIEWS | CANCER

VOLUME 12 | FEBRUARY 2012 | 135

that was described by Levan, Nichols and

Box 1 | Hierarchical versus stochastic models of tumour cell heterogeneity

Normal stem cell

patients. The transient engraftment of these

Nature Reviews | Cancer

field, and they have been very powerful in

Box 2 | Quantitative stem cell assays for haematopoietic stem cells

NATURE REVIEWS | CANCER

VOLUME 12 | FEBRUARY 2012 | 137

Primary immunodecient mouse

The important development of invitro

support the growth of many other types

138 | FEBRUARY 2012 | VOLUME 12

2012 Macmillan Publishers Limited. All rights reserved

progression in CML. Throughout the

NATURE REVIEWS | CANCER

within the CD34+ subpopulation but the

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The authors acknowledge research support from the British