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EMBO reports 5, 3, 241–244 (2004)
doi:10.1038/sj.embor.7400098
Published online: 20 February 2004
Signalling in stem cells
Meeting on Signal Transduction Determining the Fate of Stem
Cells
Lynn E. Heasley1 & Bryon E. Petersen2
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1 Department of Renal
Medicine, C-281, University of Colorado Health Sciences Center,
4200 E. Ninth Avenue, Denver, Colorado
80262, USA
2 Department of Pathology,
Immunology and Laboratory Medicine, College of Medicine, University of Florida,
PO Box 100275, Gainesville, Florida
32610-0275, USA
To whom correspondence should be addressed
Lynn E. Heasley Tel: +1 303 315 6065; Fax: +1 303 315 4852;
lynn.heasley@uchsc.edu
Received 21 November 2003; Accepted 12 January 2004; Published online 20 February 2004.
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The meeting "Signal Transduction Determining the Fate of Stem
Cells" was held at Montana State University in Bozeman, Montana, between
August 9 and 12, 2003. The meeting was sponsored by the American Society for
Cell Biology and was organized by G.L. Johnson and N. Terada.
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Introduction
Many scientific meetings have been organized to present research
findings on stem-cell sources, their pluripotent nature and potential
therapeutic applications for treating disease. The theme of this meeting was
the signalling pathways that control stem-cell maintenance and differentiation.
In his keynote address, A. Spiegel (Bethesda, MD, USA) emphasized the high
therapeutic potential of stem cells, and also the need to understand more about
their unique cell and molecular biology to harness their therapeutic
application. Although embryonic, haematopoietic and hepatic stem cells from
humans are the obvious therapeutics, stem-cell research in various organisms
from flies to humans was presented at this meeting.
Signalling of stem-cell proliferation and survival
A common feature of stem cells, regardless of origin and type, is
their ability to undergo self-renewal. Cultured stem cells, especially
embryonic stem (ES) cells, exhibit a high rate of proliferation and a short
cell cycle time (10–12 hours). Studies by S. Dalton (Athens, GA, USA)
showed that ES cells have a unique cell cycle that lacks complete G1 and G2 gap
phases (Stead et al, 2002). The activity of
cyclin-dependent kinases (Cdks) is constitutively high relative to their
activity in somatic cells such as mouse embryo fibroblasts. Genes that are the
target of the E2F transcription factor are also constitutively active,
consistent with the negligible activity of the retinoblastoma (Rb) protein
pathway in these cells. Analysis of Cdk activity in mouse ES cells reveals a
novel, constitutively active Cdk6–cyclin D3 complex that is rapidly
downregulated following ES cell differentiation. Importantly, this
Cdk–cyclin complex is insensitive to the defined Cdk inhibitors such as
p16. Taken together, these results suggest that rapid cell division in ES cells
is driven by the high activity of novel Cdk–cyclin complexes. Primordial
germ cells (PGCs) are multipotent precursors of the gametes of the adult
animal. They are also the cell of origin for testicular teratomas. Culturing
PGCs with a cocktail of growth factors including fibroblast growth factor 2
(FGF2), leukaemia inhibitory factor (LIF) and the c-Kit ligand gives rise to
pluripotent embryonic germ (EG) cells. P. Donovan (Philadelphia, PA, USA) used
retroviral-mediated gene transfer to demonstrate an important role for the Akt
kinase in PGC proliferation and survival (De Miguel et
al, 2002). Using a genetic approach with mice bearing a
PGC-specific deletion of the phosphatase and tensin homologue (PTEN) gene, T.
Nakano (Osaka, Japan) showed that PTEN-null PGCs exhibited an increased ability
to proliferate and enhanced formation of EG cell colonies. Thus, components of
the phosphatidylinositol-3-OH-kinase (PI(3)K) pathway, including a key effector
pathway (Akt) and a negative regulator of the pathway (PTEN), are important for
controlling the survival and proliferation of EG cells in a similar way to
their role in non-stem-cell systems. The in vivo microenvironment that
controls the self-renewal and maintenance of stem cells is termed the
'stem-cell niche'. It has been difficult to identify stem-cell niches for
tissue stem cells in mammalian systems, but they have been elegantly modelled
in the Drosophila ovary and testis by H. Lin (Durham, NC, USA;
Lin, 2002). Within the Drosophila ovary,
specific support cells, such as CAP cells and inner sheath (IS) cells, provide
key instructional cues for the maintenance of germline stem cells and somatic
stem cells (Fig 1). Studies so far have highlighted the
requirement for E-cadherin and  -catenin-containing adherens junctions
between the support cells and the stem cells for the maintenance of the latter.
Lin presented research on the Piwi family of proteins, which have an
evolutionarily conserved role in stem-cell maintenance from plants to humans.
The piwi gene encodes a highly basic protein the precise biochemical
function of which remains ill-defined. The mouse homologue, miwi, is a
protein that is associated with the endoplasmic reticulum and has RNA-binding
activity. It may be involved in RNA silencing and translational regulation of
specific mRNAs.
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Figure 1
Putative role of -catenin in stem-cell renewal and maintenance
through cadherin-containing adherens junctions and Wnt signalling. Support
cells such as CAP cells, inner sheath cells or spindle-shaped
N-cadherin+CD45- osteoblastic (SNO) cells
form adherens junctions with germline stem cells, somatic stem cells or
haematopoietic stem cells (HSCs), respectively, through E-cadherin or
N-cadherin and -catenin (Lin, 2002). In
addition, -catenin can be stabilized as a result of Wnt signalling
through the frizzled (Fzd) receptors leading to the inhibition of the
APC–axin–GSK3- complex, allowing transcriptional activation
of target genes by T-cell factor (TCF)/-catenin complexes.
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Haematopoietic stem cells (HSCs) are multipotential, self-renewing
stem cells that can form all blood cell types (Kondo et
al, 2003). The precise in vivo regulatory environment
within the bone marrow and the cues that control HSC proliferation and renewal
have remained poorly defined. Using mice with a conditional disruption of the
bone morphogenetic protein (BMP) receptor type IA, L. Li (Kansas City, MO, USA)
showed that these animals had increased numbers of HSCs. This observation was
not due to increased HSC self-renewal or an inhibition of differentiation, but
rather to change in the microenvironment or niche size (Zhang et al, 2003). Further experiments revealed
that the HSCs are found attached to spindle-shaped
N-cadherin+CD45- osteoblastic (SNO) cells.
Interestingly, the junction between the HSC and the SNO cells contained
N-cadherin and -catenin (Fig 1). Thus, SNO cells
lining the bone surface function as a key compartment of the microenvironment
that supports HSCs. In another presentation about HSC self-renewal, I. Weissman
(Stanford, CA, USA) showed that the Wnt pathway has an important role in HSC
renewal and proliferation. Overexpression of activated -catenin, a key
mediator of Wnt signalling, expands HSCs in long-term in vitro culture
(Reya et al, 2003). In addition, the
lymphoid enhancer factor 1 and T-cell factor (LEF1/TCF) mediate Wnt-inducible
transcription and a transfected LEF1/TCF-dependent reporter is activated in
HSCs that are present in their normal niche. Finally, transfection of molecular
inhibitors of the Wnt pathway reduced HSC growth in vitro. Taken
together, these two talks suggest that -catenin function is regulated
through an N-cadherin-dependent, SNO cell-specific interaction as well as
through secreted Wnt proteins that regulate HSC renewal within the niche. The
requirement for adherens junctions comprised of -catenin and cadherin
proteins in the fly germline stem cell and HSC niches is intriguing. The
signalling pathways that are engaged through adherens junctions have been
actively investigated (Juliano, 2002), but not yet
applied to these stem-cell systems. The increased molecular understanding of
the niches for the fly germline stem cells and mammalian HSCs may provide a
template for exploring the nature of the niches for neural, hepatic and
epithelial tissue stem-cell systems.
Regulation of cellular plasticity and pluripotency
The pluripotency of stem cells, both embryonic and somatic, renders
them highly suited for producing diverse differentiated cell types. As a
remarkable example of this property in cultured ES cells, H. Schöler
(Philadelphia, PA, USA) presented findings that mouse ES cells can develop into
oocytes that enter meiosis, recruit adjacent cells to form follicle-like
structures, and subsequently develop into blastocysts (Hubner
et al, 2003). Developmental potential is ultimately dictated
by gene expression patterns that are regulated by specific transcription
factors, but also by epigenetic processes including DNA methylation and
chromatin acetylation. A. Müller (Würzburg, Germany) showed that
treating neural stem cells with inhibitors of DNA methylation or histone
deacetylases increases their haematopoietic potential, supporting the role of
epigenetic processes in the regulation of cell plasticity. As discussed by I.
Wilmut (Edinburgh, UK), cloning by nuclear transfer from adult somatic cells is
a striking demonstration of developmental plasticity, which is the ability of a
cell to switch from one committed lineage to another. His presentation
highlighted the achievements and present limitations of cloning by nuclear
transfer. Significant advances have occurred but there has also been a high
failure rate associated with frequent and severe defects in cloned animals.
Wilmut suggested that the failings probably reflect epigenetic phenomena
(Wilmut & Paterson, 2003). Identifying methods
to enhance the ability of the oocyte cytoplasm to appropriately remodel the
chromatin of the transferred nucleus is a high priority.The identification of
the factors and signals that maintain pluripotency, a property of most stem
cells, is a focus of many laboratories. LIF signalling through the signal
transducer and activator of transcription 3 (Stat3) and the appropriate
expression of the transcription factor Oct4 is well established for the
maintenance of the pluripotent, undifferentiated state of mouse ES cells
(Smith, 2001). The meeting highlighted the role of
two recently defined transcription factors, Nanog, a homeobox transcription
factor, and Foxd3, a winged-helix transcription factor of the forkhead family
of transcription factors, in ES cell function (Fig 2). P.
Labosky's (Philadelphia, PA, USA) presentation revealed the importance of Foxd3
for the maintenance of embryonic cells of the early mouse embryo (Hanna et al, 2002). Foxd3-null embryos die soon after
implantation with a marked loss of epiblast cells, which are the cells within
the inner cell mass (ICM) of the blastocyst that will form the three somatic
germ layers. Significantly, Foxd3-/- ES cells
could not be derived from blastocysts or by double-targeting approaches in ES
cells, indicating the crucial requirement of Foxd3 for ES-cell maintenance. In
a search for additional factors that promote pluripotency, S. Yamanaka (Nara,
Japan) identified the homeoprotein, Nanog, which can maintain ES-cell
self-renewal independently of LIF and Stat3 (Mitsui et
al, 2003). Moreover, ICMs from Nanog-deficient blastocysts
produced only parietal endoderm and Nanog-/-
ES cells lost pluripotency and differentiated into extraembryonic endoderm.
Thus, Nanog is another key transcription factor that is required for
pluripotency. T. Tanaka (Baltimore, MD, USA) described another ES-cell-specific
protein, Esg1, that is expressed in the ICM and the trophectoderm of the early
embryo (Tanaka et al, 2002). The function
of Esg1 is presently ill-defined, although it has a KH domain, a feature of
many RNA-binding proteins. Reduction of Esg1 expression in blastocysts
following siRNA injection into fertilized oocytes decreased the number of cells
in the ICM of mouse blastocysts. Identifying the genes that are dependent on
Oct4, Foxd3 and Nanog for their expression and dissecting Esg1 function at the
molecular level will provide additional insight into pluripotency.
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Figure 2
Signalling pathways and transcription factors involved in the
maintenance, proliferation, survival and differentiation of embryonic stem (ES)
cells and embryonic germ (EG) cells. LIF,leukaemia inhibitory factor; PTEN,
phosphatase and tensin homologue; PI(3)K, phosphatidylinositol-3-OH-kinase;
Stat3, signal transducer and activator of transcription 3.
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Signals regulating stem-cell differentiation
The identification of dominant signal pathways that regulate
stem-cell differentiation will facilitate the generation of distinct cell types
in vitro. Three examples in which loss of specific signalling enzymes in
ES cells leads to alterations in the potential for differentiation were
presented. The first concerned SHP2, an SH2-domain-containing protein tyrosine
phosphatase that, paradoxically, functions to promote signalling from receptor
tyrosine kinases to the Ras/Raf/MEK/ERK pathway. G.-S. Feng (La Jolla, CA, USA)
presented findings that ES cells bearing homozygous SHP2 mutations exhibited
decreased potential for haematopoiesis (Chan et al,
2003). This defect is associated with decreased differentiation to
mesoderm lineages and to hemangioblasts. In another example, L. Heasley
(Denver, CO, USA) presented findings with mouse ES cells in which specific cJun
amino-terminal kinases (JNKs) were disrupted. Although the
jnk-/- ES cells and wild-type ES cells
exhibited equivalent abilities to form embryoid bodies in culture, microarray
analysis revealed a decreased expression of a panel of marker genes for the
extraembryonic endoderm lineages in jnk2-/-
embryoid bodies. J. Chen (Urbana, IL, USA) discussed the role of the mammalian
target of rapamycin (mTOR) pathway in skeletal muscle differentiation modelled
in cultured C2C12 cells (Erbay & Chen, 2001).
Interestingly, rapamycin-resistant mTOR rescues rapamycin-inhibited myogenesis
in a novel kinase-independent manner. Target genes involved in mTOR-regulated
C2C12 differentiation are likely to include insulin-like growth factors. It
will be interesting to confirm these studies in muscle satellite cells, which
are a self-renewing pool of stem cells that give rise to myogenic precursor
cells.
A. Miyajima (Tokyo, Japan) presented studies in which fetal
hepatocytes were differentiated with oncostatin M (OSM), a cytokine of the
interleukin 6 family that stimulates expression of tyrosine aminotransferase
and glucose 6-phosphatase and that represses D-type cyclins through a Stat3
signalling pathway (Matsui et al, 2002). By
contrast, OSM induces, in a K-Ras-dependent manner, the formation of
E-cadherin-based adherens junctions, which are essential structures for the
organogenesis of epithelial tissues such as the liver. Apart from hepatic
differentiation, the OSM signalling pathway is also likely to be a key
contributor to liver regeneration.
A novel example of the determination of the fate of a cell through a
specific transcription factor was presented by L. Sussel (Denver, CO, USA). The
homeobox transcription factor Nkx2.2 is required to direct pancreatic
progenitor cells to become insulin-producing beta cells as well as a subset of
glucagon-producing alpha cells. As a result, the pancreatic islets of
Nkx2.2-null mice contain a large number of cells that fail to synthesize any of
the four known islet hormones. Gene profiling studies of mutant islets revealed
marked induction of the ghrelin gene that encodes an appetite-stimulating gut
peptide. A small number of ghrelin-positive cells are found in normal islets,
but loss of Nkx2.2 results in the majority of the islets cells becoming
ghrelin-positive. These results reveal that Nkx2.2 is required for
specification of pancreatic progenitors to beta cells and suggests that a lack
of Nkx2.2 allows differentiation to ghrelin cells.
Interface between stem-cell research and cancer
research
The interface between stem-cell research and cancer research was
much discussed at this meeting. If stem cells are defined as clonigenic cells
that are capable of self-renewal as well as able to differentiate into mature
cells appropriate for the specific tissue in which they reside, then cancer may
represent the consequence of poorly regulated self-renewal of mutated stem
cells with a limited potential for differentiation. In fact, cancer stem cells
have long been suspected to be a source of leukaemias. More recently, it has
been suggested that they may also be a source of cancer cells in solid tumours,
including breast and brain cancers (Marx, 2003;
Reya et al, 2001). I. Weissman (Stanford,
CA, USA) presented studies revealing that leukaemias are generated by a limited
number of leukaemia stem cells (LSCs) of heterogeneous origin (Passegue et al, 2003). LSCs can be either HSCs that
have accumulated mutations to become leukemic or they may derive from more
restricted progenitors that have reacquired self-renewal through mutation. C.
Kim (Cambridge, MA, USA) presented findings that bronchiolar epithelial cells
positive for markers of both bronchial Clara cells and alveolar type II cells
might represent a putative lung stem cell that is the target for murine
adenocarcinoma. Compared to normal murine lung, increased numbers of
double-positive cells are observed in early adenomas and adenocarcinomas. The
finding that double-positive cells can be sorted with Sca1, a stem-cell marker,
will allow the mutation status of these cells to be defined relative to the
cells that comprise the adenoma and adenocarcinoma. However, as a cautionary
tale when considering cancer stem cells, M. Applebury (Boston, MA, USA)
described the derivation of self-renewing human neural cell lines from
retinoblastomas that exhibit some of the properties of multipotent retinal cell
precursors. Although it is tempting to speculate that these cells represent
retinoblastoma stem cells, the retinal cell precursors do not carry mutations
in the Rb gene, indicating that the retinal precursor cells may simply
persist in close association with the tumour cells that bear mutant
Rb.
Many of the signalling pathways required for normal stem-cell
function are subverted in cancer cells, thus stem-cell research and cancer
research share common interests. For instance, constitutive Cdk/cyclinD
activity leading to the inactivation of the Rb pathway, a common
occurrence in various cancer cells, is observed in ES cells (S. Dalton). H. Lin
noted that overexpression of the human homologues of Drosophila piwi
genes, required for normal germline stem-cell function, is associated with
human testicular tumours of germ cell origin (Qiao et
al, 2002). Also, specific deletion in mice of the Bmp receptor
type IA in hair follicles yielded an increased number of stem cells in this
tissue and eventually led to tumours of the hair follicle (trichofolliculoma)
(L. Li). Finally, mice in which PTEN was deleted specifically in primordial
germ cells developed testicular teratomas (Kimura et
al, 2003). This is consistent with the enhanced proliferation of
PTEN-null primordial germ cells (T. Nakano).
Perspectives
Researchers with a wide variety of interests in stem-cell biology,
developmental biology and basic signal transduction gathered for this meeting.
If stem cells are truly destined to become therapeutic agents for the treatment
of disease, we will continue to require a wide range of different specialists
to address the key questions that remain. For example, can the emerging
molecular and cellular description of niches for germline stem cells and HSCs
enhance the characterization of the specific niches for other tissue stem
cells? If so, can tissue stem-cell niches eventually be manipulated in
vivo to mobilize specific stem-cell populations? In many ways, the ES cell
seems a more tractable system for the in vitro production of specific
differentiated cell types. However, are the signalling pathways and cues that
induce specific cell types in mouse ES cells similar in human ES cells?
Finally, if various cancers do arise from rare cancer stem cells, can they be
selectively targeted with therapeutics without destroying the normal tissue
stem cells that are required for the maintenance of healthy tissues? Addressing
these questions will certainly depend, to a large degree, on continued
exploration of the basic properties of stem cells.
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