REVIEWS Nature Reviews Molecular Cell Biology | AOP, published online 15 August 2005; doi:10.

1038/nrm1713

THE MOLECULAR REPERTOIRE

OF THE ‘ALMIGHTY’ STEM CELL
Craig E. Eckfeldt*, Eric M. Mendenhall* and Catherine M. Verfaillie
Abstract | Stem cells share the defining characteristics of self-renewal, which maintains or
expands the stem-cell pool, and multi-lineage differentiation, which generates and
regenerates tissues. Stem-cell self-renewal and differentiation are influenced by the
convergence of intrinsic cellular signals and extrinsic microenvironmental cues from the
surrounding stem-cell niche, but the specific signals involved are poorly understood.
Recently, several studies have sought to identify the genetic mechanisms that underlie the
stem-cell phenotype. Such a molecular road map of stem-cell function should lead to an
understanding of the true potential of stem cells.

PLURIPOTENT
The ability to give rise to all
embryonic tissues, but not
extra-embryonic tissues.
Totipotent cells can give rise to
all embryonic and extra-
embryonic (trophectodermal)
tissues.
INNER CELL MASS
A mass of pluripotent cells in
the interior of the developing
blastocyst that give rise to all
embryonic tissues. The
blastocyst is part of the pre-
implantation-stage embryo and
consists of a hollow sphere of
cells with a distinct outer
trophectoderm layer and an
inner cell mass.
Department of Medicine
and Stem Cell Institute,
University of Minnesota,
Minneapolis, Minnesota,
USA.
*These authors contributed
equally to this work.
Correspondence to C.M.V.
e-mail: verfa001@umn.edu
doi:10.1038/nrm1713
Published online
15th August 2005
Multicellular organisms such as humans develop from
PLURIPOTENT stem cells that are present in the INNER CELL
MASS (ICM) of the blastocyst and that generate the tril-
lions of mature cells that make up the adult individual.
Such pluripotent stem cells can not only divide to give
rise to daughter pluripotent stem cells — so called
self-renewing cell divisions — but they can also dif-
ferentiate to give rise to all the cells of the mesoderm,
endoderm and ectoderm as well as germ cells (FIG. 1).
During development, pluripotent stem cells from the
ICM become increasingly restricted in their lineage
potential and generate tissue-specific, MULTIPOTENT stem
cells. These multipotent stem cells give rise to progeny
that comprise specific, mature tissue. Although this
hierarchical archetypal model of stem-cell biology is
generally the rule, recent reports have described the
persistence of stem cells with less lineage-restricted
differentiation potential into post-natal life1 BOX 1.
When isolated from the blastocyst in vitro, the
pluripotent stem cells of the ICM can be maintained
in culture as embryonic stem cell (ESC) lines. Murine
(m)ESCs were first isolated in 1981 REF. 2 and are the
most extensively characterized pluripotent cell type,
and human (h)ESCs were isolated and characterized
in the late 1990s3. Primordial germ cells (PGCs), which
are derived from the GENITAL RIDGE of early embryos, are
another type of pluripotent cell4. There are also ESC
lines that have been derived from chicken and rhesus
monkey5,6, however, in this review we will focus on the
pluripotent ESCs from mouse and human.
The self-renewal and multi-lineage differentiation
characteristics of stem cells from embryonic and most
adult sources make these cells uniquely suited for
regenerative medicine, tissue repair and gene therapy
applications. An increasing number of in vitro and
in vivo studies have shown that stem cells can reca-
pitulate embryonic and adult tissue development, and
can therefore repair injured or congenitally defective
tissues. However, the mechanisms that govern the self-
renewal and multi-lineage differentiation potential of
stem cells remain largely unknown. Although stem-
cell fate decisions might have a stochastic component7,
increasing evidence indicates that extrinsic signals from
the stem-cell microenvironment, or niche, can con-
verge on intrinsic cellular signals to regulate stem-cell
proliferation and cell-fate decisions. Various molecu-
lar techniques have been used to reveal the molecular
regulation of stem-cell fate decisions BOX 2.
It has been postulated that, although each unique
stem cell can be characterized by the expression of
a specific set of genes, the defining self-renewal and
multi-lineage differentiation characteristics of stem
cells are encoded by a shared set of genes that are
expressed by all distinct stem-cell populations, and
that therefore represents a conserved stem-cell molec-
ular signature8–10. Several studies have examined the

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Molecular signature of embryonic stem cells

Transcription profiling has revealed that most dif-
ferentiated cell types express only 10–20% of their
genes, consisting of a mix of ubiquitous housekeep-
ing genes and tissue-specific genes11. By contrast,
30–60% of genes are expressed in ESCs11–14. These
data might be consistent with the theory of stem-cell
‘priming’ — the hypothesis that stem cells express many
different lineage-specific genes at low levels15–18. There
is evidence to indicate that much of the chromatin of
embryonic and adult stem cells is in an open, accessible
state, which might allow the promiscuous expression of
lineage-specific genes, and that epigenetic modifications
of chromatin structure and/or methylation of DNA leads
to a more restricted gene-expression pattern concomitant
with lineage differentiation19. It is possible that such low-
level transcription enables the rapid regulation of genes
that is required for differentiation during development
or following injury, by maintaining a transcriptionally
permissive chromatin structure.
Furthermore, the low-level expression of multiple
lineage-specific surface receptors in stem cells might
allow the cell to detect a wide range of extracellular sig-
nals to respond to complex microenvironmental cues.
However, for many lineage-specific transcripts found in
stem cells, no corresponding protein can be detected,
which indicates that gene expression is either post-
transcriptionally repressed or that transcript levels
do not reach a critical threshold until the cell receives
intrinsic and/or extrinsic cues to differentiate. Therefore,
the theory that low-level transcription allows the cells to
sample the microenvironment might not be correct.
) d)
Murine embryonic stem cells. Several studies have
Figure 1 | The stem-cell hierarchy. The totipotent zygote formed by the fusion of egg and
sperm divides to form the inner cell mass (ICM) and the extra-embryonic (EE) tissue of the
evaluated the TRANSCRIPTOME of mESC lines. Most of
blastocyst. When isolated from the blastocyst in vitro, the cells of the ICM can be maintained in these have compared the gene-expression profiles of
culture as pluripotent embryonic stem cell (ESC) lines. During the development of the embryo, single mESC lines with that of adult stem cells, mature
the pluripotent stem cells in the ICM become increasingly restricted in their lineage potential differentiated cells and TROPHECTODERM. For instance,
and generate tissue-specific, multipotent stem cells. These include epidermal stem cells (bulge comparison of the mESC line CCE with adult haemat-
cells) that form skin and hair, haematopoietic stem cells in the bone marrow that give rise to all opoietic stem cells (HSCs), neural stem cells (NSCs)
haematopoietic cells, neural stem cells in the subventricular zone of the brain, gastrointestinal
and mature blood cells identified several transcripts,
stem cells that are located in the crypt of the small intestine, oval cells that give rise to liver (not
shown), and mesenchymal stem cells that reside in the bone marrow and can form bone, including the HOMEODOMAIN TRANSCRIPTION FACTOR Oct4
stromal cells and adipocytes (not shown)88,115. (also known as Pou5F1 or Oct3/4)8, that were enriched
in mESCs, whereas a study that compared the gene-
expression profiles of the mESC line R1 with trophec-
toderm and fibroblasts identified 124 ESC-enriched
gene-expression pattern of both embryonic and adult genes, which also included Oct4 REF. 20. A large scale
stem-cell populations. Although these analyses have EST SEQUENCING project that analysed 19 different tissues
provided some insights into the genetic mechanisms including early mouse embryos, mESCs, newborn
MULTIPOTENT that are responsible for the stem-cell phenotype, there organs and adult stem-cell populations identified 75
The ability to give rise to the are inconsistencies in the resultant lists of ‘stemness’ genes that were expressed specifically by mESCs21.
diverse cell types of one or a few genes. So far, it is not known whether these inconsist- Oct4 and another homeodomain transcription factor,
tissues.
encies are due to technical disparities, or whether these Nanog, are among the functionally characterized genes
GENITAL RIDGE results signify that ‘stemness’ is not defined by a unique that are crucial to the mESC molecular signature22–24. In
The bilateral structures in the set of genes. In this review, we focus on the molecu- the absence of Oct4, mESCs TRANSDIFFERENTIATE into tro-
developing embryo that give lar signature of embryonic and adult stem cells and phectodermal cells22, whereas loss of Nanog results in an
rise to the gonads. their microenvironmental niches as a means of better increase in extra-embryonic endodermal transcripts24.
understanding the stem-cell phenotype. We explain Overexpression of Nanog allows mESC growth in the
TRANSCRIPTOME
The entire transcriptional
how these datasets can be used to further unravel absence of leukaemia inhibitory factor (LIF)23 — a
repertoire of a cell or cell the molecular regulation of stem cells, which will be factor that is required for the maintenance of mESC
population. required to exploit their full therapeutic potential. lines — whereas overexpression of Oct4 induces the

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Box 1 | Adult stem-cell plasticity*
Adult stem cells are thought to be multipotent, but not pluripotent like embryonic
stem cells (ESCs). However, in the past few years, more than 300 reports have
indicated that adult stem cells might possess developmental capabilities that
resemble those of more immature, pluripotent cells, similar to ESCs.
The main criticism regarding the claims of adult STEMCELL PLASTICITY is that most
studies that describe such plasticity do not fulfill the criteria commonly used to
describe stem cells: For instance, most studies published so far have not
definitively proved that the greater potency of adult stem cells can be ascribed to a
single cell that can differentiate into the tissue of origin and one or more
additional tissues. Furthermore, most studies have equated differentiation with
the acquisition of morphological and phenotypic characteristics of a novel cell
type, but have not proven the functionality of the resulting cells. Similarly, few, if
any, studies have shown that the adult stem cell can robustly repopulate not only
the tissue from which it originates but also another tissue.
There are four plausible explanations for the observed plasticity of adult
stem cells:
• The apparent differentiation of an adult stem cell to a cell lineage other than the
tissue of origin could be due to contamination of the population by a stem cell or
progenitor cell from the second tissue of origin.
• Fusion between donor and recipient cells, as occurs in heterokaryons, with
silencing of the genetic programme of one of the two cells. There is evidence that
fusion can occur in vitro and in vivo.
• Stem cells might dedifferentiate and then redifferentiate, or might be
reprogrammed, in a manner similar to that found in other species (that is,
blastema formation in amphibians), during metaplasia, or as occurs in somatic
cell nuclear transplantation.
• Pluripotent stem cells generated before or after gastrulation might persist
during development into adulthood.
*REFS 110–114 specifically address adult stem-cell plasticity.
TROPHECTODERM differentiation of mESCs into endoderm and meso-
The outer portion of the
derm. Nanog, Oct4 and LIF-mediated JAK–STAT3
blastocyst that gives rise to the
embryonic portion of the activation therefore represent independent pathways
placenta. that maintain mESC pluripotency and self renewal25.
Mitsui et al. performed an IN SILICO DIFFERENTIAL DISPLAY
HOMEODOMAIN
and identified 20 genes that were specifically expressed
TRANSCRIPTION FACTOR
A transcription factor that
in mESCs and in mouse pre-implantation embryos24.
contains a homeodomain DNA- These included Oct4 and Nanog; as well as zinc finger
binding domain. protein-42 (Zfp42, also known as Rex1), the transcrip-
tion factor Utf1, the constitutively active Ras protein ES-
EST SEQUENCING
cell-expressed Ras (Eras), and the growth factor Tdgf1
The sequencing of short
segments of expressed genes (also known as Cripto); all these genes were previously
(expressed sequence tags or known to be important for ESC self-renewal24. When
ESTs) present in cDNA libraries OCT4 is found in a complex with the transcription fac-
that can be used for gene tor SOX2, it upregulates its own expression as well as
cloning or to show which genes
are present in a cell population.
the expression of Fgf4, Zfp42 REFS 24,26 and Nanog (S.
Yamanaka, personal communication), which identi-
TRANSDIFFERENTIATE fies Oct4 as a key regulator of ESC genes (reviewed in
Differentiation of one cell type REFS 26,27). The downstream targets of Nanog and Zfp42
directly to another cell type
have yet to be reported. Importantly, none of the genes
without dedifferentiation to a
more primitive intermediate. mentioned above were identified through global gene-
expression profiling. Instead, most were identified as
STEMCELL PLASTICITY downstream targets of Oct4. Ongoing studies are now
The apparent ability of a stem/ using candidate-gene approaches to evaluate the role of
progenitor cell fated to a
particular tissue to acquire a
these and other genes and pathways that are involved
differentiated phenotype of a in the maintenance, cell signalling, metabolism and cell
different tissue. cycle of ESCs25.
4 | ADVANCE ONLINE PUBLICATION
Human embryonic stem cells. Several studies have
compared the transcriptome of hESC lines with differ-
ent populations of more mature cells and mESCs12,13,28.
In the most extensive study, the transcriptomes of six
different hESC lines were compared with universal
RNA29. This study identified 92 genes that showed
increased levels of expression in all 6 hESC lines,
including OCT4, NANOG and TDGF1. Only 15 of the
92 genes were classified as ‘unknown’, which is much
less than other studies12,13,28, perhaps owing to an
under-representation of unknown genes on the arrays
used. By comparing the gene-expression profile of
three independent hESC lines, Abeyta et al. found that
52% of all genes examined were expressed in all three
cell lines11. Most of these 7,385 genes are ubiquitously
expressed, but tissue-specific genes that have been
implicated in stem-cell self-renewal and pluripotency,
such as OCT4, SOX2 and TDGF1, were also expressed
in all three hESC lines11. A comparison of all the gene-
expression profiles of hESC lines described so far iden-
tify LIN28, OCT4, NANOG, DNMT3B, TGIF, TDGF1,
CHEK2, GDF3, GJA1 and FLJ21837 among others, as
being expressed in all these lines11,13,14,28–30.
Cross-species embryonic stem-cell comparisons.
Although human and mouse ESCs show many simi-
larities, these two cell types also have several differ-
ences. For instance, both hESCs and mESCs are
typically cultured in the presence of mouse embryonic
fibroblasts (MEFs), but hESCs, unlike mESCs, do not
require exogenous LIF-mediated JAK–STAT3 activa-
tion to maintain their pluripotency in culture, although
STAT3 might be activated by other means in hESCs25,27.
Maintenance of the pluripotent state of mESC lines also
requires bone morphogenetic protein-4 (BMP4)31; by
contrast, BMP4 induces trophoblast differentiation
in hESCs32. Unlike mESCs, hESCs can be maintained
MEF-free using fibroblast growth factor-2 (FGF2)33.
This indicates that some of the important signal path-
ways that are required for pluripotency differ between
human and mouse ESCs. These differences might
explain the discordance between human and mouse
ESC expression profiles — comparisons of several
important studies show that only 13–55% of transcripts
that are enriched in mESC lines are also enriched in
hESC lines but, by contrast, comparisons between
different hESC lines are 85–99% concordant11,12,25,29.
Furthermore, some mESC-enriched transcripts are
not expressed in all of the hESC lines examined by
expression profiling so far, despite being implicated
in self-renewal and pluripotency. For example, ZFP42
is not expressed in the hESC line HES4, STAT3 is
not expressed in H9 cells, and SOX2 and ESG1 are not
expressed in other hESC lines analysed in these experi-
ments11,29. Therefore, the expression of Zfp42, Oct4 and
Sox2, which form a transcriptional feedback loop, is
upregulated in mESCs, whereas only OCT4 is consist-
ently expressed in hESCs. This might indicate that, in
hESCs, OCT4 does not require the presence of SOX2
to activate transcription. Therefore, the differences in
gene expression that have been noted between mESCs
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diverse array of all mature tissues during embryonic

Box 2 | Common techniques for gene-expression analysis
development. A subset of their progeny, so-called
Northern blotting, RPA, RT-PCR adult or tissue-specific stem cells, retains self-renewal
(Individual mRNA transcripts) and multi-lineage differentiation potential into adult-
hood. Such adult stem cells, however, have much less
self-renewal capacity compared with ESCs and are
Subtractive hybridization Population A Population B Gene-expression arrays
not pluripotent. Adult stem cells have been identified
mRNA mRNA in many tissues, where they continuously generate
Population B Population A
cDNA cDNA Labelled and regenerate mature tissues either as part of nor-
cDNA or mal physiology or in response to injury TABLE 1.
Population A Population B cRNA
mRNA mRNA Haematopoietic, neural, epidermal and gastrointesti-
nal stem cells have been phenotypically characterized,
and this has facilitated the analysis of their gene-
Macroarray Microarray
Serial analysis of
expression patterns. However, complex organs with
cDNA: gene expression numerous cell types such as the lung or kidney might
mRNA not be maintained by a single type of stem cell, but by
hybrids
several stem-cell types that have yet to be identified.
Nylon Glass slide
membrane In general, the lack of phenotypic markers that can
Gene-specific 14mers be used to purify most tissue-specific stem cells and
the lack of appropriate assays to assess the function of
mRNA mRNA Ligation into sequencing Measurement of labelled cDNA many adult stem cells have impeded gene-expression
expressed expressed vectors to determine the or cRNA intensity at a specific
in A > B in B > A analysis.
relative abundance of location on an array to determine
expressed sequences abundance of mRNA transcripts
Haematopoietic stem cells. The HSCs that reside in the
Traditionally, individual transcripts were identified by hybridizing radio-labelled bone marrow microenvironment during adult life are
complementary nucleic-acid probes in Northern blot or RNase protection assays the best-characterized adult stem cells, and therefore
(RPAs). Alternatively, a complementary DNA (cDNA) copy of the entire serve as the model for stem-cell biology (FIG. 2). HSCs
transcriptome can be generated, followed by the identification of specific genes by are also at the forefront of our understanding of the
PCR or direct sequencing of cDNA. These analyses represent a gold standard for gene- molecular regulation of the adult stem-cell phenotype.
expression analysis, but are difficult to perform on a large scale. Investigation of the function and molecular regulation
Subtractive hybridization (SH) (see figure, left) is used to characterize transcripts of HSCs and their progeny, haematopoietic progenitor
that are differentially expressed between two cell populations. The cDNA synthesized cells (HPCs), has been facilitated by the development
from one population is mixed with mRNA from another population, which results in of monoclonal antibodies to cell-surface antigens and
the formation of cDNA–mRNA hybrids of transcripts that are expressed in both
FLUORESCENCE ACTIVATED CELL SORTING, which allow purifi-
populations. More abundant mRNA remains unhybridized, and can then be isolated
cation of murine (m)HSCs to near homogeneity34. The
and characterized. SH does not require specific sequence information and generates a
advent of molecular techniques that make it possible
global picture of differential gene expression, but provides little information
regarding the relative abundance of transcripts.
to perform gene-expression analysis using a few or
Serial analysis of gene expression (SAGE; see figure, centre) involves digesting cDNA even single cells has enabled a detailed analysis of the
to generate short (~14 base pair), gene-specific sequence tags that represent the extrinsic and intrinsic signals that govern HSC-fate
transcriptome of a cell population, followed by sequencing and quantitation of these decisions.
tags. SAGE does not require prior knowledge of target sequences and provides a Most cell-intrinsic genes that define HSCs were
quantitative and global analysis of gene expression. identified before the advent of genome-wide micro-
cDNA macroarray analysis (see figure, right) is performed by hybridizing cDNA array analysis, owing to their involvement in clonal
from a cell population with gene-specific cDNA probes that are immobilized onto genetic aberrations in haematopoietic malignancies.
nylon membranes. cDNA and oligonucleotide microarray analyses are conceptually For instance, recurrent chromosomal translocations
similar to macroarrays, but are typically performed by synthesis and hybridization of in human acute T-cell leukaemias that deregulate
complementary (c)RNA to gene-specific oligonucleotides (25–60mers) that are the expression or function of genes such as NOTCH1
immobilized on glass slides. For each assay, gene-expression data are extracted by REF. 35, TAL1 (also known as SCL)36,37 and LMO2
normalization and quantitation of radioactive or fluorescent tags that have been REF. 38 result in proliferation of primitive haemat-
incorporated into the test cDNA or cRNA to determine the abundance of specific opoietic progenitor cells in the absence of normal
sequences. Global gene-expression profiling using array technology allows assessment differentiation. These observations led to the subse-
of the differential expression of tens of thousands of transcripts from a small number quent characterization of these genes as important
of input cells, but it is limited by the requirement of prior sequence information for transcriptional regulators involved in the initial stages
probe design. of non-malignant haematopoietic development in ani-
mal models39–41. Notch1 is a transmembrane surface
and hESCs might identify differences in the signal receptor that functions as a developmental regulator
pathways that are required for pluripotency in differ- of cell-fate decisions through interactions with sur-
cRNA
ent species. face-expressed Notch ligands on neighbouring cells.
A complementary RNA
molecule that hybridizes with a
On ligand binding, Notch1 is proteolytically processed
specific messenger RNA Molecular signature of adult stem cells thereby liberating its intracellular domain that subse-
sequence. Pluripotent stem cells are crucial for generating the quently translocates to the nucleus and functions as a

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Table 1 | Mammalian adult stem cells

Tissue Stem cell Niche Progeny References
Blood Haematopoietic Endosteal surface of All myeloid and lymphoid 7–10,16–18,34–63,
stem cell bone marrow blood lineages 86,90,91,99,100
Mesenchyme Mesenchymal stem Within bone marrow Bone, cartilage, tendon, 116
cell cavity smooth muscle, adipose
tissue and stroma
Brain Neural stem cell/ Subventricular zone Neurons, glial cells and 8–10,57,64–70,88
neurosphere and hippocampus oligodendrocytes
Gut Crypt cell/gut Gut crypt Enterocytes, 74,75
epithelial progenitor enteroendocrine cells,
goblet cells and Paneth cells
Heart Cardiac progenitor Not determined Cardiac myocytes 117
Liver Oval cell Terminal biliary ductule Hepatocytes and 118–120
cholangiocytes
Pancreas Pancreas-derived Not determined Pancreatic endocrine and 121,122
multipotent acinar cells
precursors
Skeletal muscle Satellite cell Between sarcolemma Myocytes/myofibrils 123
and basil lamina
Skin/hair Bulge cell Bulge in hair follicle Epidermis, hair follicles and 71–73
sebaceous glands
Male germ cells As spermatogonia Basement membrane Sperm 124
of seminiferous tubule

IN SILICO DIFFERENTIAL
DISPLAY
The use of computer algorithms
to determine differential
expression of transcripts from
gene-expression databases.
FLUORESCENCE ACTIVATED
CELL SORTING
Automated, high-speed sorting
of cell populations based on the
presence of intrinsic fluorescent
labels such as GFP expression,
or extrinsic fluorescent labels
such as monoclonal antibodies
conjugated to fluorochromes.
HOMEOBOX HOX GENE
FAMILY
A family of transcriptional
regulators that share a
conserved homeobox DNA-
binding domain, and that are
involved in the regulation of
embryonic and adult
developmental fates.
POLYCOMB PCG GENE FAMILY
Genes encoding a family of
proteins that form complexes
that modify chromatin
structure and selectively repress
gene transcription.
WNT GENE FAMILY
A family of genes that mediate
intercellular signalling through
secreted glycoprotein Wnt
ligands.
transcription factor. Overexpression of a constitutively
active form of Notch1 in murine HSCs creates clonal
multipotent haematopoietic cell lines that can recon-
stitute the haematopoietic system without evidence of
leukaemic transformation39. Likewise, stimulation of
HSCs through the Notch ligands, Delta and Jagged,
results in an expansion of primitive murine and human
in vivo repopulating cells consistent with a role for
Notch signalling in stem-cell self-renewal42–45. Further
insights into the genetic control of HSC function come
from reverse-transcriptase polymerase chain reaction
(RT-PCR)-based screening methods that are designed
to determine the expression patterns of specific genes
and gene families in subsets of human and murine
haematopoietic stem and progenitor cells. Several
RT-PCR-based approaches have focused on crucial
regulators of embryonic development such as the
HOMEOBOX HOX GENE FAMILY, POLYCOMB PCG GENE FAMILY and
46–48
WNT GENE FAMILY . This approach has been effective
in identifying genes that are selectively expressed in
the most primitive subsets of haematopoietic cells and
that functionally regulate HSC-fate decisions. Hoxb4
is one of the most well-characterized genes involved
in the self-renewal of human and murine long-term,
repopulating HSCs49–51. Identified in a similar man-
ner, both the polycomb gene Bmi1, as well as frizzled
receptors that bind secreted Wnt ligands, were found
to be expressed in primitive haematopoietic cells47,48
and were subsequently proven to have crucial roles in
HSC self-renewal52,53. Other genes that are implicated
in the HSC phenotype have been identified on the
basis of their roles in the development of mesoderm
and its derivatives; for example, the morphogens sonic
hedgehog (SHH) and BMP4 REF. 54. And more HSC
genes have been identified on the basis of phenotypic
observations such as quiescence; for example, Cdkn1a
(also known as p21), which was found to be selectively
expressed in the most primitive haematopoietic cells,
and was subsequently shown to be crucial for mainte-
nance of the mHSC pool55.
These studies provide insight into the molecular
regulation of the HSC phenotype. Specifically, long-
term maintenance of the stem-cell pool requires qui-
escence of HSCs, and therefore Bmi1, a transcriptional
repressor, and Cdkn1a, a cell-cycle inhibitor, probably
regulate this aspect of the HSC phenotype. Expansion
of the HSC pool requires symmetric self-renewing cell
divisions that give rise to two daughter cells that retain
HSC function. Hoxb4, Wnt signalling and Notch sig-
nalling represent the best-characterized mechanisms
thought to drive symmetric self-renewal of HSCs.
Early strategies to determine a more global genetic
programme of HSCs used subtractive hybridiza-
tion of highly purified murine fetal liver and murine
adult bone marrow HSCs in combination with high-
density cDNA macroarrays to delineate genes that
are expressed in HSCs56. The presence of previously
characterized HSC-associated genes among those
identified, such as the genes that encode cell-surface
molecules FLT3 and CD34 as well as the transcrip-
tional regulator Runx1 gene, confirmed the validity of
this experimental approach and indicated that genes
and signalling pathways that had not previously been
implicated in haematopoietic development might be
important in HSC behaviour56. To distribute the data
on the HSC-specific transcriptome and to foster the
collaborations that are essential to approach a problem
as complex as the genetic regulation of stem cells, these

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Hoxb4
BMP4 Notch pathway
Bmi1
Cdkn1a
SHH Wnt pathway
Genes/pathways associated with self-renewal
Self-renewal and multi-lineage differentiation potential
Promiscuous gene expression of multiple lineage-specific transcripts
Stem cell Progenitor cells Mature cells
B cell
CLP
T cell
NK cell
MEP
Erythrocyte
HSC CMP Megakaryocyte
GMP
Granulocyte
Monocyte
Genes/pathways associated with haematopoietic cell function
Gene expression of individual lineage-specific transcripts
Globin genes
Gata1
Epor
Mpo
Mpl
Figure 2 | The haematopoietic stem cell. Haematopoietic stem cells (HSCs) supply the
entire repertoire of mature blood cells for the lifetime of an organism. To accomplish this task,
HSCs are endowed with self-renewal capacity that maintains and expands the stem-cell pool,
and multi-lineage differentiation potential that produces the diverse components of the mature
haematopoietic system. As HSCs differentiate, they lose the ability to self-renew and become
increasingly restricted in their lineage potential. Genes and proteins associated with the self-
renewal of HSCs (for example, Hoxb4 REFS 4951, Bmi1 REF. 53, Cdkn1a55, BMP4 REF. 54,
SHH54, the Notch pathway39,42–45 and the Wnt pathway52) are highly expressed in HSCs, but
are downregulated in committed progenitor and mature haematopoietic cells. HSCs also
promiscuously express multiple lineage-specific transcripts at low levels, however,
differentiation is associated with the loss of this promiscuous transcript expression and
selective expression of individual lineage-specific genes17.CLP, common lymphoid progenitor;
CMP, common myeloid progenitor; GMP, granulocyte monocyte progenitor; MEP,
megakaryocyte erythrocyte progenitor; NK, natural killer.
results were compiled in an open access, searchable
stem-cell database (SCDb; see the Online links box)56.
Not long after the establishment of the SCDb,
further expression-profiling studies were performed
using different combinations of phenotypically and
ontogenically distinct murine HSCs and HPCs.
One analysis of transcripts enriched in mHSCs that
were derived from adult bone marrow identified
several genes that were classified in the SCDb as
genes expressed in HSCs derived from murine fetal
liver 56,57. These included angiopoietin-1 (Angpt1)
and the angiopoietin receptor, Tek (also called Tie2).
A functional role for Tek and Angpt1 in enhancing
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primitive cobblestone formation by mHSCs in vitro
(a surrogate readout for HSC-like cells) and in the
maintenance of mHSCs in vivo has recently been
demonstrated58, which supports the validity of this
analysis. The expression of this ligand–receptor pair
in HSCs indicates that HSC-fate decisions might
be regulated in an autocrine or paracrine fashion.
Although comparisons of adult and fetal HSCs have
identified shared patterns of gene expression, there
is only partial concordance between gene expression
in adult and fetal HSCs (reviewed in REF. 59), which
might reflect fundamental differences between
ontogenically distinct HSCs57,60.
A comparison between mHSCs and murine
multipotent progenitors (MPPs), which have only
limited self-renewal potential, found that genes that
are implicated in the development and self-renewal
of mHSCs, such as Bmp4, Bmi1 and Notch1, were
expressed at higher levels in the HSCs61. Almost half
of the genes analysed on a 1,200 probe high-density
cDNA macroarray were expressed at detectable levels
in mHSCs, and differentiation to mMPPs and com-
mitted cells resulted in the upregulation of specific
lineage markers with concomitant loss of global gene
expression16. Similar results were seen in a more exten-
sive analysis of HSCs, MPPs, common myeloid pro-
genitors (CMPs) and common lymphoid progenitors
(CLPs) — more than 40% of haematopoiesis-related
genes analysed, including HSC-specific as well as other
lineage-specific transcripts, were expressed in HSCs,
and this transcriptional promiscuity was lost during
lineage commitment17. Therefore, similar to ESCs,
HSCs have a transcriptionally accessible genome that
becomes progressively more restricted coincident with
lineage differentiation (FIG. 2).
In contrast to mHSCs and murine haematopoetic
progenitor cells (HPCs), hHSCs have not yet been
purified to homogeneity on the basis of their surface
phenotype. Therefore, defining the transcriptome of
hHSCs has been complicated by the greater heteroge-
neity of hHSC and hHPC populations. Nevertheless,
comparison of the transcriptomes of highly puri-
fied mHSCs and the more heterogeneous human
CD34+CD38– cells has identified approximately 40%
congruence. Considering the challenges of hetero-
geneity and comparative genomics8, this is probably
a conservative figure. It is probable that advances in
the ability to prospectively isolate human HSCs using
strategies that enrich for HSCs, for example, the exclu-
sion of rhodamine 123 REF. 62, or aldehyde dehydro-
genase activity63, will show greater conservation of the
molecular signature for murine and human HSCs.
Neural stem cells. Until recently, it was thought that
few, if any, stem cells were present in the adult central
nervous system (CNS). However, several studies have
now shown that NSCs persist into adulthood in the
subventricular zone of the lateral ventricles and the
dentate gyrus of the hippocampus. These NSCs con-
tinuously give rise to new neurons to replace those that
die in the normal aging process or in neurodegenerative
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REVIEWS
disorders64. Although some phenotypic markers have
been identified that allow selection of NSCs65, NSCs are
most commonly isolated by culture as neurospheres
— a heterogeneous population that also contains
more mature cells. Various studies have sought to
define the expression profile of NSCs by comparing
the gene-expression profiles of human and murine
neurospheres66–68, murine retinal progenitor cells69, or
NSC-containing neuroepithelial cells70 with those of
more mature neural progenitors or mature neurons.
Confirming the validity of these studies, several genes
with known roles during CNS development, such as
the transcriptional regulators Sox2, Pax6 and Lhx2,
and the cell-cycle regulator Ccnd1 (also known as
Cyclin D1; REFS 6670) were shown to be enriched in
NSC-containing populations in these studies. And,
consistent with other stem-cell gene-profiling studies,
many genes that have no known role in neural develop-
ment, and transcripts without any known function are
expressed in NSC-enriched populations9,66–70. Further
characterization of the function of these genes might
provide novel insights into the molecular regulation
of NSCs and their progeny. However, because of the
considerable heterogeneity of isolated NSC-containing
cell populations, it is unlikely that the true transcrip-
tome of NSCs is reflected in these studies. Advances
in the methods used to isolate pure populations of
NSCs from the brain or from ESCs will lead to a bet-
ter characterization of the NSC molecular signature.
Towards this goal, human NSCs have been purified to
near homogeneity65, but the transcriptional profile of
such cells has yet to be determined.
Epidermal stem cells. The stem cells that generate skin
epithelium and skin appendages (known as bulge cells)
are found in a niche in the bulge of the root sheath of
hairs (FIG. 1). Bulge cells give rise to both epidermis and
hair follicles in transplant experiments, and develop
into colonies of undifferentiated cells in vitro71. Murine
bulge cells can be isolated using a GFP label that is
expressed under the control of the ‘bulge-preferred’
keratin-15 promoter in combination with the expres-
sion of CD34 and integrin-α6 cell surface antigens, or
by the retention of bromodeoxyuridine in these qui-
escent cells (label retaining cells)71–73. Whether these
approaches purify epidermal stem cells to homogene-
ity, or whether there are several distinct stem cells in
the bulge is not yet clear71. Expression profiling of bulge
stem cells by two independent laboratories has iden-
tified 97–157 genes that are differentially expressed
in bulge cells when compared with differentiated
keratinocytes, with 80–90% concordance between the
different studies71,72. These genes include FGF1 and its
receptor, transforming growth factor-β (TGFβ) activa-
tors, BMP and Wnt pathway inhibitors71,73, which are
all known to be involved in the regulation of epidermal
stem-cell proliferation and differentiation.
Gastrointestinal stem cells. The gene-expression pro-
files of gastric epithelial progenitor (GEP) cells have
been compared with those of more mature zymogen
8 | ADVANCE ONLINE PUBLICATION
cells and acid-secreting parietal cells74, and the expres-
sion profiles of small intestinal epithelial progenitor
cells (SiEPs) have been compared with Paneth cells75
— each cell type was identified on the basis of its
precise location in the crypts of the gastric and small
intestinal mucosa. Of the 147 genes thought to define
GEPs, 11 were also upregulated in SiEPs, and another
22 transcripts that were upregulated in SiEPs encode
functionally related family members or additional
subunits of multi-subunit complexes of transcripts
enriched in GEPs75. This surprising degree of overlap
between the GEP and SiEP datasets indicates that
shared mechanisms probably regulate both SiEP
and GEP function. With advances in the phenotypic
characterization of adult stem cells, it is probable that
more homogeneous stem and progenitor cells from
the gastrointestinal tract will be isolated, and that
fewer background transcripts will obscure potentially
relevant stem-cell-specific transcripts.
Cross-tissue-specific stem-cell comparisons. Despite the
shortcomings in gene-expression evaluation for many
adult stem-cell populations, comparison between the
molecular signatures of HSCs, NSCs, bulge stem cells
and gastrointestinal stem cells have identified several
classes of genes that might be expressed in all tissue-
specific stem cells. For instance, 15–30% of genes that
are expressed specifically in HSCs, but not in HPCs,
are also specifically expressed in NSC-enriched
populations8,9,57; and 14% of genes that are expressed
in bulge stem cells are also expressed in HSCs and
NSCs71. Among these are genes involved in Wnt sig-
nalling (for example, Tcfs and Fzd7), Notch signalling
(for example, Hes1), adhesion (for example, Itga6) and
transcriptional regulation (for example, Bmi1).
The stem-cell niche
Stem cells exist in vivo in a complex microenviron-
ment, or niche, which is composed of differentiated
somatic cells and extracellular matrix, as well as stem
cells and their progeny. The niche provides factors
that maintain the self-renewal ability of stem cells and
prevent their differentiation. Most insights into the role
of stem-cell niches come from studies on Drosophila
melanogaster germ stem cells (GSCs; reviewed in
REFS 76,77), which interact with specialized somatic cells
in the niche — known as cap cells (during oogenesis
in the ovary)78 or hub cells (during spermatogenesis in
the testes)79–81 (FIG. 3). This physical interaction main-
tains the undifferentiated state of the stem cell and is
mediated through a cadherin–catenin pathway82. The
GSC-cap/hub-cell interaction also regulates symmet-
ric versus asymmetric GSC divisions by polarizing the
stem cell and affecting the orientation of the mitotic
spindle and the segregation of differentiation and
stem-cell determinants in daughter cells83–85.
Similar to GSCs, mammalian adult stem cells reside
in niches that protect the ability of the stem cell to self-
renew and that prevent differentiation. Niches have been
identified for epidermal stem cells, gastrointestinal stem
cells, HSCsand NSCs. In common with GSC niches,
www.nature.com/reviews/molcellbio

a Germ stem-cell niche b Haematopoietic stem-cell niche
CXCL12
(secreted by
osteoblasts) CXCL12/CXCR4 Wnts/Frizzled
Terminal
filament
PTH (from Wnts
outside niche) (secreted
Cadherin– HSC by osteoblasts)
catenin
junctions
PTH/PTHR1 Kit Notch1
Cap Integrins
Jag1
KitI
ECM
GSC GSC
BMSC
Endosteal bone surface
Figure 3 | Molecular regulation of stem-cell niches. a | Components of the stem-cell niche
provide cell–cell and cell–matrix interactions that are crucial for protecting the integrity and
function of the resident stem cells. Cadherin–catenin-mediated interactions between somatic
cap cells (cap) and the Drosophila melanogaster germ stem cells (GSCs) in the ovary maintain
the stem cells in an undifferentiated state. They also regulate symmetric versus asymmetric cell
divisions by polarizing the mitotic spindle and facilitating the partitioning of the cellular contents
into the daughter cells. b | The functional interactions between stem cells and their niches have
been conserved from flies to mammals. Within the extracellular matrix (ECM) at the endosteal
surface of the bone marrow cavity, osteoblasts and other bone-marrow stromal cells (BMSCs)
regulate haematopoietic stem cells (HSCs) through secreted signals as well as by cell–cell and
cell–matrix interactions. Chemokines such as CXCL12 provide a signal to recruit CXCR4-
expressing HSCs to the appropriate niche, whereas ECM components interact with HSC-
expressed integrins to retain the stem cells. Niche cells also provide haematopoietic cytokines
such as Kitl. Further regulation of HSCs by the HSC niche depends on activation of Notch
signalling by Jagged ligands (Jag1), and Wnt signalling by secreted Wnt ligands45,48. The HSC
niche itself is regulated by paracrine factors such as parathyroid hormone (PTH) and its
receptor (PTHR1) that can alter the cellular composition and size of the niche, and thereby
regulate HSC numbers45. Part a of the figure was modified with permission from REF. 78 ©
(2000) American Association for the Advancement of Science.
adult stem-cell niches contain extracellular matrix
components and cell-surface ligands that ‘bind’ the stem
cell to the niche by cadherin and integrin interactions71.
Moreover, niche-derived factors that regulate stem-cell
fate are conserved from GSC niches to most adult stem-
cell niches. For instance, in the HSC niche, osteoblasts
express Notch ligands that support HSC self-renewal, as
shown by the promotion of the self-renewal of HSCs45,86
owing to overexpression of activated parathyroid hor-
mone receptors (which upregulates Notch ligand expres-
sion). A similar mechanism operates in NSC niches87.
Other mechanisms through which niches regulate
the fate of adult stem cells is by the secretion of Wnts
and soluble Frizzled receptors48,71,88, by the production
of members of the TGFβ/BMP family71,86 and by the
production of Kitl (also known as stem cell factor)71,89,90
(FIG. 3).
Identification of the precise location of epidermal
and gastrointestinal stem cells has facilitated analy-
sis of the gene-expression profile of the surrounding
niche cells. With the identification of the precise
location of HSCs in the bone marrow, such studies
are now also possible for HSC-niche cells. Another
method to identify niche-derived extrinsic regula-
tors of stem-cell fate is by the creation of stromal cell
lines that support stem-cell self-renewal in vitro. For
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instance, subtractive hybridization was used to com-
pare the transcriptome of a fetal-liver stromal cell
line (known as AFT024) that supports HSC main-
tenance in vitro, with fetal-liver-derived stromal cell
lines that do not support the maintenance of HSCs.
Genes that were expressed in the supportive AFT024
cell line were compiled in an internet-based stromal
cell database (StroCDb; see the Online links box).
Among the genes that were preferentially expressed
in AFT024 were Kitl, Bmp2, Wnt5a and Dll1, as well
as many secreted and surface molecules with no
known haematopoietic function91.
Although adult stem cells can undergo asymmetric
and symmetric divisions, how physical interactions
between somatic stem cells and their niches regulate
these cell divisions, as has been described for GSCs92,
remains largely unknown. As other roles of the
D. melanogaster GSC niche seem to be conserved in
mammalian adult stem cells, the mechanisms that
underly asymmetric versus symmetric cell divisions
will probably also be conserved from fly to man and
from germ stem cell to adult stem cell.
A conserved stem-cell molecular signature?
It has been suggested that the self-renewal and multi-
lineage differentiation characteristics of stem cells is
the result of a genetic programme that is common to
stem cells of all origins, and that stem cells therefore
share a conserved molecular signature. Two seminal
studies have compared the gene-expression profiles of
murine ESCs, HSCs and neurospheres8,9. In one study,
283 genes were expressed by all three stem-cell popu-
lations8 and in the second, 216 genes were expressed
by all three stem-cell sources9. These genes comprised
both known genes as well as genes without an anno-
tated function. However, a comparison of these two
independent studies, which used seemingly compara-
ble input cell populations and methodologies, revealed
only six common stem-cell genes that were identified
in both studies10 TABLE 2. In a third study, investigators
compared the gene-expression profiles of murine ESCs,
neurospheres and retinal progenitor cells, and found
that 385 genes were expressed in all three cell types10.
When compared with the first two studies however,
only one gene, integrin-α6 (Itga6) was expressed in all
stem-cell populations TABLE 2.
Possible explanations for the discrepancies observed
include technical and methodological differences among
the three analyses, such as differences in the stem-cell
isolation methods, the type of gene arrays used and the
type of computational analyses used to identify shared
stem-cell genes. In fact, the number of shared ‘stemness’
genes increased substantially when the data were com-
pared using the same algorithms to define differential
gene expression59. However, as a much higher degree
of congruence was seen when gene expression in ESCs
(n = 332) or NSCs (n = 236) was compared between
the studies (p < 10–8), methodological differences
alone might not fully explain the failure to find more
shared stem-cell genes10. Another explanation might be
that genes that confer stem-cell activity might not be
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REVIEWS

Table 2 | Overlap of stem-cell-specific genes

Gene symbol* Gene description Functional annotation
Fortunel and Ramalho-Santos datasets
1110068L01Rik RIKEN cDNA 1110068L01 gene Not determined
1810009A15Rik RIKEN cDNA 1810009A15 gene Not determined
2410015N17Rik RIKEN cDNA 2410015N17 gene Not determined
2410080P20Rik RIKEN cDNA 2410080P20 gene Not determined
2410091N08Rik RIKEN cDNA 2410091N08 gene Not determined
2700084L22Rik RIKEN cDNA 2700084L22 gene Protein modification
2810436B06Rik RIKEN cDNA 2810436B06 gene Chloride transport
AA407558 Expressed sequence AA407558 Not determined
AA420417 Expressed sequence AA420417 Not determined
C81206 Expressed sequence C81206 Not determined
Cdkap1 CDK2-associated protein-1 Cell growth and/or maintenance
Chd1 Chromodomain helicase DNA binding protein-1 Chromatin assembly/disassembly
eIF4Ebp1 eIF4E binding protein-1 Insulin receptor signalling pathway
Etl1 Enhancer trap locus-1 Transcriptional regulation
Gfer Growth factor, Erv1 (S. cerevisiae)-like Not determined
Nol5 Nucleolar protein-5 Not determined
Rnf4 Ring finger protein-4 Transcriptional regulation
Sfrs3 Splicing factor, arginine/serine-rich 3 (SRp20) mRNA splice-site selection
Tead2 TEA domain family member-2 Transcriptional regulation
Tmk Thymidylate kinase Nucleotide biosynthesis
Trif-pending TRIF gene Not determined
Zfx Zinc finger protein X-linked Transcriptional regulation
Ivanova and Ramalho-Santos datasets
AI643885 Expressed sequence AI643885 Not determined
Cpx1-pending Metallocarboxypeptidase-1 Not determined
Laptm4b Lysosomal-associated protein transmembrane-4B Not determined
Lce-pending Long chain fatty acyl elongase Fatty acid elongation
Pkd2 Polycystic kidney disease-2 Cation transport
Fortunel and Ivanova datasets
Edr1 Early development regulator-1 Development
Tcf3 Transcription factor-3 Transcriptional regulation
Fortunel, Ramalho-Santos and Ivanova datasets
Itga6 Integrin-α6 Integrin signalling pathway
*Transcripts for the corresponding gene symbols were identified as more highly expressed in multiple adult and embryonic stem-cell
populations compared with their progeny using gene-expression microarrays, and they therefore represent conserved stem-cell-
specific genes. Each subheading lists the studies that were included for each section. The stem-cell populations analysed in these
studies were murine haematopoietic stem cells (mHSCs), murine embryonic stem cells (mESCs) and murine neural progenitor cells
(mNPCs) by Ivanova et al.8; mHSCs, mESCs and mNPCs by Ramalho-Santos et al.9; and mHSCs, mESCs and murine retinal
progenitor cells by Fortunel et al.10 This table is adapted from Fortunel et al.10 CDK, cyclin-dependent kinase; elF, eukaryotic
translation-initiation factor; S. cerevisiae, Saccharomyces cerevisiae.

represented on the oligonucleotide-based microarrays
that were used for gene-expression analysis10, therefore
precluding the identification of important genes that
are expressed in all stem cells. The use of subtractive
hybridization and serial analysis of gene expression
(SAGE) that do not share these limitations would
circumvent this problem. It has also been suggested
that shared stem-cell genes might be expressed only
transiently and would therefore be difficult to detect
in a homeostatic stem-cell population10. Furthermore,
stem-cell genes might not be expressed exclusively in
stem cells, but might also be expressed — albeit at lower
or higher levels — in differentiated cells, and therefore
would be more difficult to identify by differential

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 REVIEWS

microRNA
A family of short, non-coding
RNA molecules
(~22 nucleotides) that
post-transcriptionally regulate
target-gene expression
primarily by inhibiting protein
translation.
RNAi
A functional tool that use small
interfering RNAs (siRNAs) to
knock down gene expression
through sequence-specific
decay of target mRNA
molecules.
MORPHOLINO ANTISENSE
OLIGONUCLEOTIDES
Chemically synthesized
oligonucleotide analogues used
to knock down gene expression
by specifically binding to target
transcripts to inhibit RNA
splicing or translation.
CHIP
Technique used to
immunoprecipitate complexes
of DNA with associated
proteins.
expression analysis. Consistent with this notion, only
4 of the 216 genes identified by Ramalho-Santos et al.
as ‘stemness genes’, were not expressed in the differenti-
ated cell populations analysed by differential display9.
Therefore, stem-cell function might not be imparted on
cells by a defined and limited set of master stem-cell-
specific genes or by a small number of pathways, but
by the combined effect of the complex upregulation or
downregulation of many different genes and gene path-
ways. These studies might also indicate that a conserved
stem-cell molecular signature does not exist.
Although no definitive set of genes that defines all
stem cells has been identified, several signalling path-
ways have been identified that seem to regulate dif-
ferent types of stem cell. For instance, canonical Wnt
signalling through β-catenin has been implicated in
the maintenance and self-renewal of ESCs as well as
adult stem cells, such as epidermal, gastrointestinal,
haematopoietic and neural stem cells93. Although acti-
vation of β-catenin induces the self-renewal of various
stem cells, this is not the only mechanism that supports
self-renewal, as HSCs that lack β-catenin maintain
their self-renewal capacity94 and Wnt activators are
downregulated in skin stem cells compared with their
progeny72,73. Notch signalling is another developmen-
tal regulatory pathway that can mediate self-renewal
of stem cells — Notch signals promote the mainte-
nance and self-renewal of neural, haematopoietic,
gastrointestinal and epidermal stem cells by inhibiting
differentiation95. Moreover, interaction between the
Wnt and Notch signalling pathways might provide the
cellular signals that are needed to drive proliferation
(Wnt signals) in the absence of differentiation (Notch
signals), leading to symmetric self-renewing cell divi-
sions required for stem-cell expansion96. Regulation
of stem-cell fate in vivo is undoubtedly more complex,
involving polycomb genes, such as Bmi1, and devel-
opmental morphogens, such as Hedgehog, that have
both been shown to regulate fate decisions for several
stem-cell types95. It should again be noted that few, if
any, of these shared stem-cell regulators were identi-
fied by global gene-expression analysis.
Implications and future directions
The inability to identify a consensus stem-cell gene-
expression signature has led some researchers to ques-
tion the validity of such a strategy97. Despite some of
the caveats regarding the different studies that have
characterized the transcriptome of defined stem cells
discussed above, it is clear that progress is being made
towards defining gene-expression patterns associated
with stem-cell behaviour. The ultimate goal of defining
a molecular signature of stem cells will be advanced
further when it becomes possible to purify stem cells
to homogeneity.
However, expression profiles do not necessarily dic-
tate which of the genes or gene pathways are functionally
involved in self-renewal and pluripotency/multipotency.
There is increasing evidence that the proteome and
transcriptome of cells are only partially overlapping98,
and this complicates elucidation of the functional roles
of expressed genes in stem-cell function. For instance,
highly purified mHSCs express low levels of mRNA for
lineage-specific genes before lineage commitment99, and
fewer than 25% of genes that have been analysed have
consistent levels of both mRNA and protein expression
during myeloid cell differentiation100. Although tech-
niques to define the proteome are less sensitive than
large-scale transcriptional profiling, it is possible that a
proportion of differentially expressed genes identified
in any large-scale transcriptional screen encode proteins
that are subject to post-translational modifications that
alter protein stability, structure or localization. Therefore,
the abundance of mRNA transcripts does not infer
functionality. Furthermore, studies in Caenorhabditis
elegans have shown that microRNAS, small non-coding
RNA sequences, can modify mRNA translation and
therefore have implications for protein synthesis and
gene function101. A large number of microRNAs are
also found in mammalian cells, including stem cells102,103
— such microRNAs could modulate expressed genes to
refine complex cellular functions such as self-renewal
and lineage commitment.
Therefore, a crucial step for determining the implica-
tions of gene-expression profiles is to design gene-target-
ing experiments that functionally characterize the genes
and gene pathways of interest. Currently, several groups
are exploring approaches to functionally validate stem-
cell gene-expression data, which include the creation of
knockout mice or the use of RNAi and gene overexpres-
sion in cell-culture models. An alternative approach is
the use of model organisms to define stem-cell-specific
genes, for example, RNAi in C. elegans and MORPHOLINO
ANTISENSE OLIGONUCLEOTIDES (MOs) in Xenopus laevis and
zebrafish have been used to evaluate loss-of-function
phenotypes in a high-throughput manner104–108. This
comparative genomics approach might increase the
likelihood of identifying pathways that are important
for stem-cell function owing to evolutionary conserva-
tion. In a recent study, the functional roles of genes that
were differentially expressed between human HSCs and
HPCs were evaluated using a zebrafish model of hae-
matopoiesis109. In this system, MO-based knockdown
of 14 out of 61 transcripts (23%) that were differentially
expressed in HSCs compared with HPCs and that had
no previously known roles in early haematopoiesis
resulted in defective haematopoietic-cell development in
MO-injected zebrafish embryos, showing the usefulness
of model organisms for large-scale functional validation
of transcriptional profiles.
As complex transcriptional networks that confer spe-
cialized cellular functions often result from the relatively
simple actions of important transcription factors, studies
will also be needed to determine the protein–DNA inter-
actions that regulate the entire cellular transcriptome. For
instance, chromatin immunoprecipitation CHIP analysis
can be used to gain insight into the proteins that mediate
complex cellular gene-expression patterns. When com-
bined with PCR for focused analysis of transcriptional
regulation or with large promoter arrays, such analyses
might begin to identify the gene networks that regulate
stem-cell behaviour.

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Insights into the genes and gene pathways that regu-
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regenerative medicine, with important implications
for the development of clinically applicable stem-cell
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extrinsic growth factors and small molecules that allow
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Acknowledgements
The authors would like to acknowledge the work of our colleagues
that we have discussed, and apologize to our colleagues whose
work was not discussed due to space limitations.
Competing interests statement
The authors declare no competing financial interests.
Online links
DATABASES
The following terms in this article are linked online to:
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
Angpt1 | Bmi1 | BMP4 | Ccnd1 | Cdkn1a | CHEK2 | DNMT3B |
Eras | FGF1 | GDF3 | GJA1 | Hoxb4 | Lhx2 | LIN28 | LMO2 |
Nanog | NOTCH1 | Oct4 | Pax6 | Runx1 | SHH |SOX2 | TAL1 |
Tek | Tdgf1 | TDGF1 | TGIF | Utf1 | Zfp42
FURTHER INFORMATION
Catherine Verfaillie’s Institute: http://www.stemcell.umn.edu
National Institutes of Health Stem Cell Information:
http://stemcells.nih.gov
The Stem Cell Database (SCDB):
http://stemcell.princeton.edu
The Stem Cell Genome Anatomy Project (SCGAP):
http://www.scgap.org
The Stromal Cell Database (StroCDB):
http://stromalcell.princeton.edu
Access to this interactive links box is free online.
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