Monash Immunology and Stem Cell Laboratories, Faculty of Medicine, Monash University, Wellington Road, Clayton, Victoria, 3800, Australia
E-mail: jill.mcfadyean{at}med.monash.edu.au
SUMMARY
Stem cell research is a vibrant and rapidly moving field of science that investigates self-renewing cells in the adult and embryo. Two debates currently exist in the stem cell field concerning the transcriptional redirection of stem cell differentiation and fate, and the reorganization of cell commitment through nuclear reprogramming caused by cell fusion and nuclear transfer. The recent Keystone Symposium in Colorado on stem cells, organised by Fiona Watt and Leonard Zon, brought together both leading and upcoming researchers in the field to explore stem cell biology and these issues.
Stem cell research is one of the most controversial fields of science
today. Controversies in the field include: access to early human embryos from
infertility treatment clinics for the derivation of human embryonic stem (hES)
cells (Reubinoff et al.,
2000); the production of ES cells from embryos formed by nuclear
transfer of somatic cells into oocytes
(Munsie et al., 2000
); the
transdifferentiation or de-differentiation of adult stem (AS) cells of one
tissue type to another; and the clinical applications of stem cell
therapies.
The focus of many presentations at the Keystone Symposium earlier this year was the control of stem cell renewal, and the molecular and cellular mechanisms of stem cell differentiation, particularly the role of growth factors and extracellular inducers, the regulation of gene expression and the reoccurring activation of specific messenger pathways during differentiation.
The nature of pluripotential ES cells
Pluripotent stem cells produce all or most cell types in the body, whereas
multipotential stem cells produce several cell types of a particular lineage.
The important questions of how pluripotential ES cells are maintained and
directed to differentiate into the primary embryonic lineages of endoderm,
ectoderm, mesoderm and extraembryonic endoderm were addressed in several
presentations. Austin Smith (University of Edinburgh, UK) demonstrated how
important regulators of stem cell renewal and pluripotentiality can be
identified in mouse (mES) cell cultures in the absence of serum or feeder
cells. Leukaemia inhibitory factor (LIF) inhibits mES cell differentiation by
activating the JAK-STAT pathway, but requires serum. However, LIF together
with bone morphogenetic protein 4 (Bmp4) can block mES cell differentiation in
serum-free medium, enabling the de novo derivation and maintenance of mES
cells. Bmp4 works through the Smad pathway to increase the expression of the
Id1, Id2 and Id3 genes (which encode negative
HLH proteins that inhibit differentiation), thus blocking mES cell
differentiation into ectoderm, whereas activation of the LIF-Stat3 pathway
blocks mesodermal differentiation (Smith,
2001).
There is considerable interest in the factors that govern
pluripotentiality. If key regulators can be found, can these be manipulated to
enable stem cells to be derived from a broader range of mouse strains and
other species than is presently possible? The transcription factors Oct4 and
Nanog are expressed in all mouse and human ES cells, and are characteristic of
undifferentiated pluripotential cells. Decreasing Oct4 expression
levels in mouse and human ES cells generally results in the loss of
pluripotentiality (Buehr et al.,
2003). As Shawn Burgess (National Human Genome Research Institute,
Bethesda, USA) discussed, microarray analysis has identified over 500 human,
mouse and zebrafish genes that undergo significant changes in expression when
Oct4 levels are altered.
During mES cell formation, the blastocyst-stage pre-implantation embryo is allowed to attach to the culture dish, and the inner cell mass outgrowths form mES cell colonies. However, not all the cells of these outgrowths express Oct4. Joanna Maldonado-Saldivia (The Wellcome Trust/Cancer Research UK Gurdon Institute, Cambridge, UK) and colleagues in Azim Surani's laboratory have been examining the expression profiles of Oct4+ and Oct4- cells. The Oct4-expressing (Oct4+) cells inside the outgrowth retain the expression of other pluripotency genes, such as embryonal stem cell-specific gene 1 (Esg1). Preliminary experiments using RNAi suggest that transient loss of Esg1 expression results in increased mES-cell differentiation and the promotion of ectodermal differentiation. Hence, there appears to be an organised microenvironment required for the generation of mES cells.
hES cells are derived similarly to mES cells, but their maintenance and renewal does not appear to involve LIF (Alan Trounson, Monash University, Australia). Sphingosine-1-phosphate (S1P) and platelet derived-growth factor (PDGF) maintain hES cells under serum-free conditions (A. Pebay, R. Wong, S. M. Pitson, E. J. Wolvetang, G. S.-L. Peh, A. Filipczyk, K. L. L. Koh, I. Tellis, L. T. V. Nguyen and M. F. Pera, unpublished). hES cell maintenance appears to involve signalling pathways that are activated by tyrosine kinase receptors acting synergistically with those that are downstream of lysophospholipid receptors.
In the human, BMPs derived from hES cells drive the formation of
extraembryonic endoderm in an autocrine manner that can be blocked by the BMP2
antagonist noggin (Pera et al.,
2004). The apparently homogeneous colonies of noggin-treated hES
cells are neuroectodermal in character, and form neurons and glia very
efficiently under appropriate culture conditions. As Alan Trounson discussed,
this is one of the first examples of the directed differentiation of hES
cells. Treating hES cells with specific growth factors and embryonic tissues
can also enrich for specific lineages. For example, cardiomyocytes are formed
when hES cells are cultured with mouse visceral endoderm
(Mummery et al., 2003
), and
respiratory precursors and prostate tissue form from hES cells that are
co-cultured with embryonic mesenchyme (R. Mollard, A.T., M. Denham, R. Jarred
and G. Risbridger, unpublished).
Epithelial stem cells and cancer
There is increasing interest in the origins of cancer in adult stem cell populations and their derivatives. Mutations may arise in the slowly renewing stem cells that are retained in their niche; their oncogenic properties are then exposed when they are recruited for differentiation. Loss of regulatory control in early differentiation stages may also lead to tumours and could explain the heterogeneity of cell types encountered in cancers (see Fig. 1). Interesting observations from several stem cell types (particularly epithelial cells) that supported this idea were discussed.
|
Using fluorescence-activated cell sorting (FACS), it is clear that LRCs can
also be isolated as a side-population (SP) of mammary cells. Jeffrey Rosen and
colleagues (Baylor College of Medicine, Texas, USA) have been examining gene
expression in normal and breast cancer cells that are derived from LRC stem
cells. The SP and LRC cells represent 0.5% of mammary epithelial cells,
and 75% of the SP cells are positive for Sca1 (stem cell-associated antigen 1,
which is present in many adult stem cell populations). Sca1+ cells
localize to the terminal end buds of growing ducts. When transplanted, these
cells produce outgrowths, whereas Sca1- cells do not. Mammary
hyperplasias and Wnt1-expressing tumours in MMTV-Wnt1 transgenic mice are
Sca1+ and express the transcription factor and master regulator of
mammary epithelial cell fate, keratin 6 (K6). Mammary tumours in transgenic
mice that express ß-catenin and c-Myc, downstream components of the
canonical Wnt signalling pathway, also have cells expressing K6. This
indicates that breast tumour heterogeneity appears to derive from the
activation of specific oncogene and/or tumour suppressor-regulated signalling
pathways in specific mammary progenitors.
Mutations in Wnt pathway genes represent the initiating mutation in nearly
all colorectal cancers. Tcf4 null mice have no proliferating cells in
the intervillus region, where crypts that contain intestinal stem cells form.
Hans Clevers and colleagues (Hubrecht Laboratory, Utrecht, The Netherlands)
have shown how active transcription factor formed from ß-catenin and TCF
is found in low concentrations associated with APC
(Roose and Clevers, 1999),
which suppresses ß-catenin/TCF signalling. Constitutively active
ß-catenin/TCF complexes are present in APC-/- colon
carcinomas. The ephrin B2 (Ephb2) and Ephb3 receptor tyrosine kinases are
ß-catenin/TCF target genes that help direct localization of crypt cells
and are associated with early neoplastic events in these cells. EphB
expression is lost in malignant colorectal carcinomas, and very large
adenocarcinomas form in Ephb2-/- mice. Therefore, epithelial crypt
stem cells that escape from EphB regulation are associated with carcinoma
formation.
The origins of leukaemia in the stem cell cascade are also being investigated by Irvine Weissman (Stanford University, CA, USA). Leukaemia cells avoid apoptosis, and in haematopoietic cells isolated by FACS from leukaemic bone marrow, high levels of CD47 expression are observed (high levels of CD47 are indicative of immunity to macrophage attack). In the chronic phase of leukaemia, Wnt signalling is not increased, but in a blast crisis, ß-catenin is activated. Weissman's laboratory are now studying the expression profile of the tumour stem cell, and where it appears in the haematopoietic stem cell cascade.
Stem cell differentiation and commitment
There has been considerable debate about the transdifferentiation of adult
stem cells, particularly of haematopoietic stem cells (HSCs), into other
tissue types. If stem cells can repopulate a variety of tissue types (that is,
be multipotential), or if tissue repair can be induced by the mobilisation or
delivery of harvested stem cells, the clinical options for repairing damaged
tissues are increased. However, there is considerable controversy about the
contributions that HSCs can make to non-haematopoietic tissues, except in rare
cases where cell fusion has occurred
(Balsam et al., 2004;
Wagers et al., 2002
). Diane
Krause (Yale University, NY, USA) believes that bone marrow-derived cells
might be short-term proliferating cells that contribute to the lung and other
tissues, without cell fusion, as seen in mice with irradiated bone marrow.
However, Markus Grompe (Oregon Health and Science University, OR, USA)
discussed how adult bone marrow HSCs (defined as c-Kit+,
Lin-, Thylo, Sca1+) can give rise to cells
that express hepatocellular markers when transplanted into lethally irradiated
hosts, and can reverse hepatic dysfunction in a mouse model of hereditary
tyrosinaemia type 1 (HT1) liver disease. Transplants of female bone marrow
expressing the lacZ reporter gene
(Fah+/+xRosa26+/-) into HT1
affected (Fah-/-) male recipients, and then to
Fah-/- female recipients, resulted in hepatocytes with
fusion karyotypes (see Fig. 2).
However, the fusion of putative transdifferentiated hepatocytes with recipient
hepatocytes does not occur. Transplanted granulocyte-macrophage progenitors
themselves were also able to generate hepatocytes by fusion. Margaret Goodell
(Baylor College of Medicine, TX, USA) also postulated that muscle repopulation
by HSC in acute muscle injury is by cells of the macrophage lineage. Grompe
also noted that failed cytokinesis can result in mono- and bi-nucleated (2n),
4n and 8n cells in the liver, and that fused cells can undergo tetraploid
reduction division to diploidy (e.g. 80 XXXY to 40XY and 40XX). Therefore
diploidy in donor-derived cells cannot be used to demonstrate
transdifferentiation and to rule out cell fusion as the underlying
mechanism.
|
Another adult cell type with multipotential capacity is the mesoangioblast;
mesoangioblasts are bone marrow progenitor cells that can enter the
circulation and contribute to skeletal muscle regeneration
(De Angelis et al., 1999;
Ferrari et al., 1998
;
Minasi et al., 2002
). As
Giulio Cossu (University La Sapienza, Italy) presented, clones derived from a
single cell taken from a mouse embryonic aorta express early endothelial
markers and differentiate into endothelium and skeletal muscle. These cells
are also able to differentiate into endothelium, smooth, cardiac and skeletal
muscle, and cartilage and bone. The mesoangioblasts express various cell
markers, including Sca1, Thy1 and transient Flk1, but are Oct4-.
Research by Cossu and colleagues suggests that Flk1+ progenitor
cells may differentiate into haemoangioblasts or mesoangioblasts
(Cossu and Bianco, 2003
;
Motoike et al., 2003
).
Injecting wild-type mesoangioblasts into
-sarcoglycan knock-out mice
also corrects their dystrophic phenotype
(Sampaolesi et al., 2003
).
Haematopoiesis
Identifying the factors that direct pluripotential stem cells into becoming HSCs, and that maintain their renewal and control their multipotential differentiation is a priority for several research groups, who are making important contributions towards establishing the key regulators and pathways.
Len Zon (Harvard Medical School, Boston, USA) has shown that zebrafish are
an excellent model for studying embryonic haematopoiesis and stem cell
differentiation. He discussed the kgg mutant, which has a defect in
the caudal-like homeobox gene cdx4 and a deficit in
scl-expressing haematopoietic precursors. Erythropoiesis in
kgg mutants is rescued by the overexpression of hoxb7a and
hoxa9a, but not by hoxb4 or hoxb8a (hoxb6b
rescued poorly), whereas scl overexpression induces ectopic
haematopoiesis but does not rescue erythropoiesis. In addition, cdx4
overexpression induces ectopic erythroid cells in the midline of embryos and
increases hoxb4 expression dramatically. These data, together with
the finding that mouse Cdx4 expressed in mES cells expands the
multipotential haematopoietic progenitor cell compartment, show that the
Cdx-Hox pathway is necessary and sufficient for haematopoietic stem cell
formation (Davidson et al.,
2003).
Neural stem cells
The application of cell therapies in the treatment of neurodegenerative disease depends on neural stem cell researchers discovering how to recapitulate the functions of neuronal and glial cells in the central and peripheral nervous systems. Progress in this field is providing a range of cell types that are giving encouraging results in various animal models.
Small populations of neurones are formed in the adult olfactory bulb and hippocampus of vertebrates from putative stem cells. Stem cells can be harvested from brain and spinal cord, and will differentiate into mature glia and neurones depending on their local environment. Fred Gage and colleagues (Salk Institute, CA, USA) are studying the cellular, molecular and environmental influences that regulate neurogenesis in the adult mouse brain. The gene Tlx (Nr2e1 - Mouse Genome Informatics) appears to be involved in the maintenance and renewal of adult-derived neural stem cells. Mutant Tlx cells (which are Gfap+) do not propagate, and the mutant can be rescued by lentivirus-transfected Tlx. Tlx maintains the nestin+ state of neural stem cells and inhibits Gfap and differentiation into the glial lineage. Hence, single factors, such as Tlx, can have multiple functions in neurogenesis.
The remarkable capacity of ES cells to generate functional tissues was
described by Ron McKay (National Institute of Neurological Disorders and
Stroke, Bethesda, USA). His studies focus on cell cycle control, choice of
cell fate, and the differentiation of stem cells into electrophysiologically
functional neurones (Vicario-Abejon et
al., 2000). Mouse ES cells can be efficiently directed into
neuronal and glial fates, and can integrate into the neonatal and adult brain
(Brustle et al., 1999
;
Kim et al., 2002
;
Lee et al., 2000
;
Studer et al., 1998
). These
studies show that mES cells generate functional midbrain dopamine neurones
that function in vivo. Similar strategies have been used to generate neurones
that secrete dopamine from nestin+ cells, which have been selected
from spontaneously differentiated hES cell-derived embryoid bodies. Neurones
generated from hES cells have synaptic and action potentials that can be
measured in vitro. Embryoid bodies also form endoderm precursors and
endodermal organ rudiments that can form many hepatocytes.
Yoshiki Sasai and colleagues (RIKEN Center for Developmental Biology, Japan) have identified a stromal cell-derived inducing activity (SDIA) that directs mES cells to differentiate into neural cells, including midbrain TH+ dopaminergic neurones. The identification of bioactive neural-differentiating factors is crucial for producing sufficient numbers of cells for therapeutic purposes. SDIA also induces TH+ neurones in partially disassociated primate ES cell colonies that will colonise the substantia niagra in non-human primate models of Parkinson's disease. This induction requires E-cadherin-mediated cell-cell contact. SDIA also induces co-cultured ES cells to differentiate in CNS tissues (both ventral and dorsal cells). Early exposure of SDIA co-cultured ES cells to Bmp4 suppresses neural differentiation and promotes epidermogenesis, whereas late Bmp4 exposure (>fourth day) induces differentiation into neural crest cells and dorsal-most CNS cells. Shh suppresses the differentiation of the neural crest lineages and promotes motor neurone formation.
Plasticity and multilineage potential
It is apparent that some adult stem cells, such as mesenchymal stem cells,
have a greater plasticity than others and are able to contribute to a range of
different tissues (Pittenger and Marshak,
2001). The identification of a pluripotential adult stem cell with
the properties of ES cells is of particular biological and clinical
interest.
Multipotential adult cells (MAPCs) that appear after the long-term culture
of bone extracts were identified by Catherine Verfaillie (University of
Minnesota, MN, USA), who believes that they are a mesenchymal stem cell but
with a greater potency (Jiang et al.,
2002). These cells have been isolated in mouse, rat, monkey, pig
and human. In mouse, they can be grown for >200 population doublings, and
in human the for >80 population doublings. Single MAPCs differentiate in
vitro into most mesodermal cell types, and engraft haematopoietic and
epithelial tissues in response to local cues after postnatal transplantation.
These cells may be closely related to the CD45-, Sca1lo,
c-Kit- and Thy1lo quiescent cells of bone marrow, and
could be culture induced or a remnant of a pluripotential population. They
express ES cell specific genes, such as Oct4 and Nanog, and
contribute to all germ layers when injected into mouse blastocysts.
A skin-derived precursor (SKP) cell has been derived from adult mammalian dermis, which can differentiate into neural and mesodermal progeny (Freda Miller, University of Toronto, Canada). These cells share many characteristics with neural crest stem cells, and occupy a distinct niche within developing and adult dermis. SKP spheres grown from neonatal and adult mouse skin express some neural precursor markers. They generate cells with smooth muscle morphology and express nestin, fibronectin and vimentin, but not P75, GAD, tyrosinase and c-Kit. SKPs differentiate into peripheral nerves and glia (Schwann) cells. They are found in the dermal papillae of hair follicles, and appear to be neural crest stem cell-derived cells.
Lineage restriction during the differentiation of pluripotential cells is
probably determined by the availability of specific transcription factors and
by changes to chromatin structure, which presumably affects the accessibility
of particular genes to transcription factors. Veronique Azuara (Medical
Research Council, Clinical Sciences Centre, Hammersmith Hospital, UK) has
shown that DNA replication timing can be used as an indicator of chromatin
accessibility (Azuara et al.,
2003). Genes that encode neural commitment factors frequently
switch from being early replicating (as in ES cells) to being late replicating
(as in HSCs or mature lymphocytes). Interestingly, Azuara proposed that this
`chromatin profiling' could be a way of discriminating stem cells from
progenitors, or of predicting the developmental potential of stem cell
populations isolated from different sources.
ES cell applications
Clinical applications of hES cells may depend on their compatibility for
transplantation. As they are derived from embryos with their own genomic and
histocompatibility profile, they may need to be tissue matched to avoid
rejection. One approach to this problem is to form `patient specific' ES cells
by nuclear transfer (see Munsie et al.,
2000). This technique involves the substitution of the oocyte
nucleus with that of an adult somatic cell. The method is used to produce
cloned offspring in animals. Unfortunately, many embryos, foetuses and
offspring generated by nuclear transfer are abnormal
(Rhind et al., 2003
) because
of abnormal genomic reprogramming in the early embryo. Ian Wilmut (Roslin
Institute, Edinburgh, UK) described cloned offspring as, ironically, being
more variable than siblings. However, ES cells produced from nuclear-transfer
embryos do not show the abnormal phenotypes of cloned animals, indicating that
stem cells with normal embryonic epigenetic characteristics have been
selected, or that they undergo further reprogramming as stem cells in vitro or
in vivo when incorporated as tissue progenitors. However, the success rate of
producing hES cells by nuclear transfer is very low
(Hwang et al., 2004
).
The inherent difficulty of reprogramming cells of a committed lineage by
nuclear transfer will potentially limit the application of ES cell
technologies. Rudi Jaenisch (Whitehead Institute, Boston, USA) posed the
question: can a postmitotic cell be reprogrammed for development? Olfactory
neurones express specific receptors (1 of 1000) and specific receptor genes,
which can be labelled with GFP. mES cells formed from nuclear transfer show a
low proportion (1%) of GFP expression, and cloned mice showed no
difference in olfactory receptor repertoire. Hence, the choice of receptor in
this population remained epigenetic, and terminally differentiated cells can
be reprogrammed to a pluripotential state. The implications of this work for
stem cells is that even end-differentiated adult stem cells can be manipulated
into pluripotentiality, and that future research will provide a means of doing
this successfully for tissue repair and regeneration.
Conclusion
The key issues for stem cell research are the determination of the inducers and directors of their differentiation. The degree of plasticity of adult stem cells and the demonstration of function in their tissue of final residence is very important for clinical application. The function of ES cell-derived tissue progenitors needs to be confirmed, and strategies are needed to enable compatibility for transplantation. All of these areas are actively being researched.
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