1 Monash Institute of Reproduction and Development, Monash University, Clayton,
Victoria, Australia
2 Department of Physiology, Monash University, Clayton, Victoria,
Australia
* Author for correspondence (e-mail: martin.pera{at}med.monash.edu.au)
SUMMARY
It is widely anticipated that human embryonic stem (ES) cells will serve as an experimental model for studying early development in our species, and, conversely, that studies of development in model systems, the mouse in particular, will inform our efforts to manipulate human stem cells in vitro. A comparison of primate and mouse ES cells suggests that a common underlying blueprint for the pluripotent state has undergone significant species-specific modification. As we discuss here, technical advances in the propagation and manipulation of human ES cells have improved our understanding of their growth and differentiation, providing the potential to investigate early human development and to develop new clinical therapies.
Introduction
Human embryonic stem (hES) cells have been the subject of many review
articles since the first report of their derivation over five years ago
(Thomson et al., 1998), and
the characteristics of the cells, their potential use in regenerative
medicine, and the ethical issues surrounding their provenance, have been
widely discussed in the scientific literature. However, few reviews have
focused on hES cells from an embryological standpoint. We now have an
experimental system that provides us with routine access to stages of the
human life cycle that were previously out of reach to experimentation. How
useful a tool will these cells be for studying these developmental stages?
Conversely, will our understanding of the molecular regulation of mammalian
development based on studies in the mouse provide us with a framework to
control hES cells in vitro, enabling us to develop the many important clinical
applications that are envisioned for these cells in the future?
This second question is particularly important because the answer
profoundly affects how we devise strategies to manipulate ES cell
differentiation. If differentiation in vitro is a reflection of events in the
embryo, as exemplified by the conversion of mouse ES cells into motoneurons in
vitro (Wichterle et al.,
2002), then our efforts should be informed and strongly influenced
by developmental studies in model systems. If hES cell differentiation does
not closely resemble mouse embryonic development, a more empirical approach
will be needed to identify the signaling pathways that control hES cell
differentiation, as we discuss later.
In this article, we review aspects of primate embryology that are relevant
to ES cell biology, survey the similarities and differences between mouse and
primate ES cells, and then discuss recent advances in hES cell technology, and
in understanding primate ES cell differentiation. We do not discuss here a
very promising and relatively neglected alternative source of human
pluripotent cells, the embryonic gonad
(Shamblott et al., 1998);
recent encouraging work confirms that pluripotent cell lines can be derived
from this source, although there are substantial challenges in maintaining and
manipulating these cells (Turnpenny et
al., 2003
).
Primate embryonic development
Our understanding of mammalian embryology, and of pluripotent stem cells, is based chiefly on studies in the mouse. However, there are significant differences between mouse and primate development (Box 1). We need to take these differences into account when we consider the embryological context in which the embryological counterparts of ES cells from the two species first appear (pre-implantation blastocyst) and then differentiate (early postimplantation period).
There is very little information about the molecular control of development
in primates, and on gene expression patterns during postimplantation primate
development, which makes comparison with the extensive data from the mouse
very difficult. Comparison of the mouse and human genome shows that genes
involved in reproduction are amongst those subject to the strongest
evolutionary pressure, and that various types of non-coding regulatory
elements are under strong selective pressure
(Waterston et al., 2002).
Thus, the comparison of coding sequences alone may overlook important species
differences in gene expression control during development.
A recent study that compared gene expression patterns in rhesus monkey and
mouse embryos found differences in the abundance of certain transcripts
between the two species, in the oocyte, and at the cleavage, morula and
blastocyst stages of development (Zheng et
al., 2004). Although there is little information on gene
expression in the immediate postimplantation period in human development,
compared with mouse, some studies have found differences in the timing and
expression patterns of certain developmental regulatory genes, such as
SOX9 and SRY, between the two species, at later stages of
development (Fougerousse et al.,
2000
; Hanley et al.,
2000
). Conversely, gene expression surveys of placental tissue,
which is readily available, have shown that the human and mouse trophoblast
(the postimplantation derivatives of the outer epithelial layer of the
blastocyst, which make up most of the fetal part of the placenta) do share
certain regulatory pathways (Loregger et
al., 2003
). Overviews of the genetics of human developmental
anomalies, for example of the heart (Ryan
and Chin, 2003
) or of the limb
(Tickle, 2002
), similarly
suggest the conservation of the role of key genes. There is certainly a need
for a more extensive analysis of the expression of developmental regulatory
genes in the primate, particularly in the early postimplantation embryo. Until
we have such data, the use of transcription factors or other genes as markers
of progenitor cell populations in hES cell cultures, based on their expression
in the mouse embryo, should be regarded as being presumptive.
The postimplantation period is when most key commitment and differentiation
events take place, and is also the phase of primate development that is least
accessible to study. Nevertheless, histological and ultrastructural analyses
have provided some insights into the differences between primate and murine
development (Fig. 1). It is
important to remember that the primate embryo devotes its first two weeks
almost exclusively to the formation of extraembryonic membranes
(Enders and Schlafke, 1981;
Enders et al., 1986
;
Luckett, 1978
). Thus, in the
time it takes the mouse embryo to develop past midgestation, the primate
embryo has just begun germ layer formation.
|
The origin of extraembryonic mesoderm in the primate embryo is unclear.
Enders and King (Enders and King,
1988) obtained ultrastructural evidence that the extraembryonic
mesoderm appears prior to overt formation of the primitive streak, which is
observed at the caudal end of the embryonic disc. These authors in fact
concluded that this tissue might arise from the hypoblast or extraembryonic
endoderm (Bianchi et al.,
1993
; Enders and King,
1988
). This extraembryonic mesoderm continues to expand upward and
laterally to form part of the chorionic villi and to line the entire inner
aspect of the trophectoderm, the outer epithelial layer of the blastocyst that
gives rise to most of the embryonic part of the placenta. The formation of a
secondary yolk sac between the embryonic disk and the primary yolk sac is a
characteristic feature of primate embryos. The visceral endoderm shows
asymmetry in the early embryo as it is thickened over the future cranial end
of the epiblast. The secondary yolk sac undergoes considerable expansion, and
only near the end of the third week of development is yolk sac hematopoeisis
underway.
Structural studies of early postimplantation development in the baboon
(Enders et al., 1990) and
human (Hamilton and Mossman,
1972
; Luckett,
1978
) show that they closely resemble that in the rhesus macaque
monkey. The human embryo undergoes interstitial implantation (it becomes
entirely surrounded by endometrial tissue). Unlike in the rhesus, in the human
and in some species of lemur, a mesh of endodermal cells forms within the
blastocoel cavity, perhaps the equivalent of an ingrowth of parietal endoderm.
The differentiation of the primitive streak is evident early in development in
the form of loosely associated cells on the ventral surface of the epiblast.
This earliest mesoderm is thought to be extraembryonic, and its subsequent
development is similar to that of the rhesus embryo; in the mouse,
extraembryonic mesoderm is not prominent at the equivalent stage. By around
day 14 of development in human embryos, the embryonic disk with its three germ
layers is evident.
Comparing pluripotent mouse and human stem cells
To a certain degree, the use of hES cells as a developmental model, and the
success of applying embryological principles to manipulate their growth and
differentiation, depends upon understanding their relationship to the
pluripotent cells of the embryo. Mouse ES cells can be derived with highest
efficiency from the epiblast of the peri-implantation embryo
(Brook and Gardner, 1997).
However, in many respects, including their developmental capacity, mouse ES
cells seem to resemble the inner cell mass (ICM)
(Pelton et al., 2002
).
Human and other primate ES cells are also derived from the ICM of the
pre-implantation embryo, and share with mouse ES cells the key biological
properties of pluripotentiality and immortality
(Reubinoff et al., 2000;
Thomson et al., 1998
).
Unfortunately, to date, no studies have examined the ability of rhesus
(Thomson et al., 1995
),
marmoset (Thomson et al.,
1996
) or cynomolgus (Suemori
et al., 2001
) monkey ES cells to participate in chimera formation
following blastocyst injection, and so the assessment of the developmental
capacity of primate ES cells is limited to data from teratoma formation in
xenografts in immunodeprived mice. These studies show that such grafts contain
a wide variey of cell types, often with a considerable degree of histotypic
differentiation. However, the finding of multilineage differentiation in
teratomas provides no information about the functional status of the
differentiated cells and, of course, gives no indication of the ability of the
ES cells to participate in normal embryogenesis.
Human and mouse ES cells also appear to express a set of genes that are
found in pluripotent cell populations in the mouse embryo, including some that
are known to be important in establishing or maintaining the pluripotent
lineage. Many studies of the hES cell transcriptome
(Bhattacharya et al., 2004;
Richards et al., 2004
;
Sato et al., 2003
;
Sperger et al., 2003
) have
identified genes (see Table 1)
that are expressed at a higher level in hES cells relative to differentiated
ES cultures or to other human cell types. Although the results of these
studies vary appreciably (depending on the technology platform used, the cell
populations to which the ES cells were compared, and the ES cell lines that
were used), there are consistent findings. The overall pattern indicates that
there may be a molecular blueprint for the pluripotent state, which is
conserved across species (Pera et al.,
2000
). The most extensive conservation of gene expression between
mouse and human is most likely to be amongst genes encoding transcriptional
regulators or DNA-modifying enzymes. By contrast, a direct comparison of human
and mouse ES cells identified many differences in the expression of cytokines
between the two species (Ginis et al.,
2004
). It is noteworthy that the various studies did not all
identify these genes as stem cell markers. Also, some molecules are notable
for their absence; CD9, which is expressed by mouse and hES cells
(Oka et al., 2002
;
Carpenter, 2004
), was not
reported as a prominently expressed gene in any of these assays. The embryonic
markers defined by antibodies SSEA-1, -3 and -4 against cell-surface
glycolipids are expressed differently in mouse and hES cells. The cell-surface
proteoglycan recognised by several monoclonal antibodies reactive with hES
cells, including TRA-1-60, TRA-1-81 and GCTM-2, is not detected on mouse ES
cells by these reagents, but it is unclear whether these monoclonal antibodies
show any cross-reactivity with mouse tissue.
|
There are likely to be other differences in the extrinsic control of stem
cell maintenance between the species. The MEK kinase pathway promotes
differentiation in mouse ES cells (Nichols
et al., 2001), but in the human, fibroblast growth factor 2 (FGF2)
(Amit et al., 2000
), which
activates this pathway, can maintain hES cells in the undifferentiated state.
Bone morphogenetic protein (BMP) 2 or BMP4, under serum-free culture
conditions and in cooperation with an active gp130 signaling pathway, will
inhibit neural differentiation and thereby assist in the maintenance of
pluripotentiality in mouse ES cells (Ying
et al., 2003
). In the human, BMPs will induce differentiation into
extraembryonic lineages, either in the presence
(Pera et al., 2004
) or absence
(Xu et al., 2002b
) of
serum.
Are the differences between mouse and hES cells related to genuine species
differences in pluripotent cell phenotype, or do they reflect that the ES
cells correspond to a different stage of embryonic development in the two
species? Some data suggest that the same pattern of antigen expression is seen
in the ICM of the human blastocyst and in hES cells. SSEA-3, SSEA-4 and
TRA-1-60 were all found to be expressed in the ICM in a limited series of
human pre-implantation blastocysts cultured in vitro
(Henderson et al., 2002). As
these markers are not expressed in the mouse ICM, this finding suggests that
there are actual species-specific differences in the expression of these
markers in pluripotent cells. Moreover, a general argument in favor of the
existence of species-specific differences in pluripotent stem cell phenotype
is that pluripotent cells in mice and humans show these differences
irrespective of whether they originate directly from embryos or from
primordial germ cells through the protracted process of teratocarcinogenesis
(Pera et al., 2000
). Thus,
human embryonal carcinoma stem cells resemble hES cells, mouse embryonal
carcinoma stem cells resemble mouse ES cells, and the two cell types show the
same interspecies differences.
Human ES cell technology
Although our understanding of the regulation of human pluripotent stem cells is still limited, substantial recent advances in ES cell technology will facilitate their use in studying the cellular and molecular control of human development.
Deriving and maintaining human ES cells
Human ES cells were first successfully derived using mouse embryonic
fibroblast feeder cells and serum-containing medium
(Reubinoff et al., 2000;
Thomson et al., 1998
), in a
culture method that has since been widely used
(Cowan et al., 2004
;
Hovatta et al., 2003
;
Mitalipova et al., 2003
;
Park et al., 2003
;
Reubinoff et al., 2000
;
Richards et al., 2002
).
Recently, one group described the derivation of a hES cell line from a
blastocyst developed through somatic cell nuclear transfer
(Hwang et al., 2004
), a
technique that might prove useful for generating histocompatible ES cell
lines, or ES cell lines from individuals with known genetic predisposition to
disease. Interestingly, in contrast to mouse ES cell work, hES cells with a
diploid XX genotype can be readily established and maintained. A lack of
X-inactivation and X-chromosome dosage compensation could account for the
difficulty in maintaining XX mouse ES cells, but the status of X-inactivation
in hES cells is unknown.
Technical advances have partially overcome some of the limitations of the
original systems for culturing hES cells, such as the spontaneous
differentiation of the cells and the need to mechanically dissect ES colonies
for subculture. A serum-free system based on combining a proprietary serum
substitute and FGF2 enables the propagation of cultures with a higher
proportion of stem cells. This system removes the need to mechanically isolate
stem cells, which can instead be dissociated enzymatically
(Amit et al., 2000). This
technique has been widely adapted for the routine growth of hES cells.
However, it has only modestly improved the cloning efficiency of hES cells,
and because the proprietary serum replacement used has an undefined protein
component, it is possible that it may modulate the effects of added
differentiation inducers in an unknown fashion. In one modification of this
technique, the feeder cell component is replaced with Matrigel, an
extracellular matrix (ECM) preparation, and conditioned medium from the feeder
cell layer (Xu et al., 2001
).
This system enables the long-term maintenance of the stem cell phenotype, with
strong suppression of the spontaneous differentiation observed at high passage
levels (Carpenter et al.,
2004
). Amit and co-workers
(Amit et al., 2004
) have
reported that the combination of FGF2, TGFß, LIF and a proprietary serum
replacer can achieve serum-free, feeder-free maintenance of hES cells on a
fibronectin ECM. A recent report also suggests that Wnt signaling modulation
can support the short-term maintenance of some stem cell markers in hES cell
cultures in the absence of a feeder cell layer
(Sato et al., 2004
). However,
new hES cell culture methods must undergo testing to ensure that the key
properties of pluripotent stem cells are maintained and that the system does
not select for karyotypically altered cells
(Draper et al., 2004
). No
culture method to date enables high-efficiency clonal propagation of hES
cells.
Manipulating human ES cells
Developing improved technology for the genetic manipulation of hES cells
will also be crucial for their effective application in research. Although hES
cells can be modified by transgenesis and gene targeting, there are still
questions over the efficiency of the techniques in different cell lines. The
generation of stable transformants of hES cells has been achieved using
conventional DNA delivery systems (Eiges
et al., 2001), or through the use of lentiviral
(Gropp et al., 2003
;
Ma et al., 2003
) or adenoviral
(Smith-Arica et al., 2003
)
vectors. One group (Zwaka and Thomson,
2003
) used gene targeting via electroporation to obtain homologous
recombination in hES cells at frequencies similar to those observed in mouse
ES cells. The use of short interfering (si)RNA to knockdown gene expression is
another methodology that holds promise for use in ES cell research, and
several recent reports have shown that this technique can be used to knockdown
gene expression in ES cells (Hay et al.,
2004
; Vallier et al.,
2004
).
ES cell differentiation: a model for embryonic cell commitment
It seems unlikely that hES cells, at least as we understand them currently, will yield information that is of direct relevance to the mechanisms of patterning, axis formation or segmentation in the primate embryo. There is no evidence to date that differentiating ES cells can reproducibly generate the spatial organisation of embryonic and extraembryonic tissue that is seen in the embryo in vivo.
However, hES cells might provide important new information about the cellular and molecular basis of commitment and differentiation events during human development. To enable mechanistic studies of the events controlling the formation of a specific cell lineage, the differentiation system should meet specific criteria, as outlined in Box 2. Well-defined differentiation methodology, the ability to manipulate gene expression through siRNA, transgenesis or targeted genetic modification, and the use of engineered lineage-specific reporters, will enable the study of gene expression and function in a human developmental context.
Spontaneous differentiation
Most early reports of hES cell differentiation studied spontaneous
differentiation in vitro, either during long-term maturation of adherent cell
layers in situ (Reubinoff et al.,
2000), or after the formation of embryoid bodies
(Itskovitz-Eldor et al., 2000
)
(see Fig. 2). The formation of
embryoid bodies, three-dimensional multicellular structures formed by
non-adherent cultures of differentiating ES cells (see
Fig. 2B), is thought to mimic
the environment of the peri-implantation embryo, where interactions between
various cell types facilitate inductive events. The spontaneous
differentiation of adherent ES cell cultures in situ has not been widely
studied, but it is likely that the interactions between the various cells is
similar to that which occurs in embryoid bodies.
|
Investigators have used various means to influence the outcome of
differentiation in embyroid bodies, such as treating them with growth factors
or with differentiation inducers, such as retinoic acid
(Schuldiner et al., 2000).
Although these treatments can influence the outcome of differentiation, they
still generally result in a mixed population of cells that is enriched only to
a limited degree for the cell of interest. Nonetheless, when combined with the
selection for certain cell types, based on their expression of surface markers
(Levenberg et al., 2002
), by
using lineage-specific promoters to drive selectable marker genes, or by using
selective culture methodology (Reubinoff
et al., 2001
), spontaneous differentiation does enable the
isolation and analysis of lineage-committed human progenitor cells from ES
cultures.
Box 2. Desirable features of an in vitro differentiation system
|
Directed differentiation
Both co-culturing ES cells with inducing cells and treating cultures with
growth factors have been used to direct the differentiation of hES cells. The
effect of these two treatments can be based on one or both of two mechanisms:
inducing differentiation along a lineage of interest, or the enhanced growth
or survival of a spontaneously differentiating cell population. It is not
always easy to delineate these effects in the experiments described to date,
and, although in principle it should be possible to identify the key factor(s)
produced by the inducing cell lines in co-culture experiments, the effects of
co-culture are complex and often involve multiple factors.
Human ES cells have now been shown to differentiate into the following cell
types in vitro: neural progenitors and cells differentiated thereof
(Carpenter et al., 2001;
Reubinoff et al., 2001
;
Schuldiner et al., 2001
;
Schulz et al., 2003
), blood
cell precursors (Chadwick et al.,
2003
; Kaufman et al.,
2001
), endothelial cells
(Gerecht-Nir et al., 2003
;
Levenberg et al., 2002
),
osteogenic cells (Sottile et al.,
2003
), cardiomyocytes (Kehat
et al., 2001
; Mummery et al.,
2003
; Xu et al.,
2002a
), insulin producing cells
(Assady et al., 2001
),
hepatocytes (Rambhatla et al.,
2003
), keratinocytes (Green et
al., 2003
) and trophoblast cells
(Xu et al., 2002b
). The
different ways in which these cell types have been induced, and the various
inducing factors that have been identified and used in these experiments, are
discussed below.
Differentiating lineages from ES cells in vitro
Extraembryonic lineages
Two studies have shown that hES cells can be used to study the formation of
extraembryonic tissues from pluripotent cells, thus modelling the first
commitment events in mammalian development.
In the first study, hES cells grown under serum-free conditions with BMP4
were induced to form flat epithelial cells that express many of the genes
associated with trophoblast or placental development
(Xu et al., 2002b). In this
study, early trophoblast-lineage-specific genes, such as MSX2, were
switched on very rapidly and expression remained elevated. With prolonged BMP4
treatment, markers of fully differentiated trophoblast cells, such as human
chorionic gonadotrophin, were activated, and human chorionic gonadotrophin,
estradiol and progesterone were secreted into the culture medium. Some cells
underwent fusion to form syncytial giant cells that expressed human chorionic
gonadotrophin. The time-lapse studies carried out by Xu et al. suggested that
BMP4 directly effects differentiation, rather than cell survival
(Xu et al., 2002b
).
Although there is no evidence that BMPs are involved in trophoblast differentiation in the mammalian embryo, this culture system could be used to analyse the early stages of commitment to the trophoblast lineage.
By contrast, in the second study, hES cells treated with BMP2 or related
molecules, in the presence of serum and a feeder cell layer, differentiate
into flattened epithelial cells that express genes characteristic of the
extraembryonic endoderm (Pera et al.,
2004), as reported previously in human embryonal carinoma cells
treated with BMP2 (Pera and Herszfeld,
1998
).
Thus, the differentiation of two main extraembryonic lineages in the human
embryo, trophoblast and extraembryonic endoderm, can be analysed using hES
cell cultures. The controlled differentiation of ES cells into these two
lineages will provide important information on the molecular regulation of
these crucial events in peri-implantation development
(Hay et al., 2004).
Ectodermal lineages
The neural differentiation of hES cells has been studied by several groups
(Carpenter et al., 2001;
Reubinoff et al., 2001
;
Schuldiner et al., 2001
;
Schulz et al., 2003
;
Zhang et al., 2001
). The first
three studies (Carpenter et al.,
2001
; Reubinoff et al.,
2001
; Schuldiner et al.,
2001
) used the spontaneous differentiation of ES cells as a
starting point for the isolation and culture of highly purified populations of
neural progenitors using selective serum-free culture conditions. These
progenitors could be cultivated for
25 population doublings
(Reubinoff et al., 2001
) as
neurospheres in suspension culture, and they expressed markers of the early
neuroectoderm, such as nestin, polysialylated N-CAM, musashi and Pax6. The
neural progenitor cells could differentiate into neurons and astrocytes, and,
to a minor degree, into cells expressing oligodendrocyte markers. However,
rigorous proof that the progenitor cells were actually stem cells (such as
studies of the differentiation of progenitor clones into neural lineages) was
not obtained. Following engraftment into newborn mouse brains, the precursor
cells survived, migrated out from the injection site, and underwent regionally
appropriate differentiation (Reubinoff et
al., 2001
; Zhang et al.,
2001
). Electrophysiological studies showed that the ES-derived
neurons could also respond to neurotransmitters in vitro
(Carpenter et al., 2001
). The
use of retinoic acid and nerve growth factor
(Schuldiner et al., 2001
), or
conditioned medium previously shown to induce ectoderm differentiation of
mouse ES cells (Schulz et al.,
2003
), enhanced the yield of neuronal cells from hES cell embryoid
bodies.
As noted above, we have recently reported a paracrine loop involving
BMP-driven differentiation of hES cells into the extraembryonic endoderm
(Pera et al., 2004). This
study showed that a blockade of BMP signaling by noggin, a BMP antagonist,
caused hES cells to differentiate into an intermediate cell type that lacked
neural markers, but that could be easily converted into neural progenitor
cells upon transfer into suspension culture in basal medium supplemented with
FGF2. This effect of noggin is consistent with its role in neurogenesis in the
embyro.
In several interesting studies of cynomolgus monkey ES cells
(Kawasaki et al., 2002;
Mizuseki et al., 2003
), neural
differentiation has been induced by co-culturing ES cells with a PA6 cell line
that produces an undefined, cell-associated differentiation inducer known as
SDIA (stromal cell derived inducing activity). The co-culture of cynomolgus
monkey ES cells with PA6 cells results in 35% of the ES cells forming tyrosine
hydroxylase-positive cells, which also express the transcription factors NURR1
and LMX1b, thus confirming their identity as midbrain neuronal cells. SDIA
induces neural differentiation of mouse ES cells in about the same time scale
as that in the embryo, whereas the time that it takes to induce monkey ES
cells to form midbrain neurons is longer than in cultures of mouse ES cells,
but far more rapid than during monkey development and neurogenesis in vivo.
Further experiments showed that these neural precursors could be committed to
regionally specific fates. The authors suggested that SDIA induced the ES
cells to form ectoderm, which spontaneously gave rise to rostral CNS
precursors; further treatment with appropriate inducers, such as sonic
hedgehog (SHH), can yield cells that express markers characteristic of the
full range of dorsal and ventral neural fates.
In studies of the neural differentiation of primate stem cells, it has therefore been possible to identify factors (such as SDIA and noggin) that predispose ES cells to undergo transition to neuroectoderm, to isolate early neural precursors, to convert them into neurons or astrocytes, and to direct regionally specific patterns of CNS differentiation. These model systems thus provide a means to study the cellular and molecular basis of the early stages of neurogenesis in the human embryo, and enable these events to be directly compared with those in the mouse.
Recently, Green and co-workers (Green
et al., 2003) described the stepwise differentiation of
keratinocytes, representing another ectodermal lineage, from hES cell-derived
embryoid bodies that were replated onto monolayers following suspension
culture. The transcription factor p63, required in the mouse for the
development of the epidermis and its appendages, was first expressed, followed
by markers of more mature stages of the keratinocyte lineage, including
cytokeratin 14 and basonuclin.
Mesodermal lineages
Hematopoietic precursors have been obtained from hES cells by either
co-culturing or by inducing them with growth factors. In the first study to
isolate blood cell progenitors from hES cells, Kaufman et al.
(Kaufman et al., 2001)
cultured ES cells on marrow stromal or yolk sac-derived cell lines, and
monitored the culture for the expression of blood cell lineage markers, such
as CD34. The appearance of CD34+ cells peaked at 17 days, at a
level of 1-2% of the total cells, and declined thereafter; these cells were
also CD45 (a general marker for hematopoetic cells), but
many were CD31+ (a marker of the endothelial lineage). These
progenitors could form both erythroid and myeloid colonies in agar. The
expression of adult and fetal hemoglobin, but not embryonic globin, was
observed during erythroid differentiation.
Chadwick et al. (Chadwick et al.,
2003) used a combination of embryoid body formation and treatment
with various hematopoietic cytokines, plus BMP4, to induce the formation of
hematopoietic progenitors from hES cultures. The combination of BMP4 and the
cytokines SCF, FLT3 ligand, IL3, IL6 and granulocyte colony stimulating factor
(GCSF) enhanced the yield of CD34+CD45+ cells from
embryoid bodies cultures by almost sixfold. The
CD34+CD45+ phenotype is similar to that of hematopoietic
precursors identified from the dorsal aorta of humans. The precursors could
form erythroid and myeloid colonies in agar, and the yield of colony-forming
units, which showed some capacity for self renewal, was greatly enhanced by
BMP4. The results were consistent with the direct induction of hematopoietic
differentiation by the cytokines, or with an action of the factors on the
multiplication and survival of an early precursor cell (hemangioblast) in the
embryoid body.
Human ES cells have also been shown to differentiate into endothelial cells
in embryoid bodies (Levenberg et al.,
2002). At 13-15 days following embryoid body formation,
transcripts of CD31 (P-selectin) and VE-cadherin were detected. Expression of
CD34 and GATA2 was also elevated in the differentiating cultures, as expected
during the early phases of endothelial cell differentiation. Curiously, unlike
mouse ES cells, hES cells themselves expressed VEGF-R2, as well as the
receptor TIE2 and AC133; the expression of these genes is characteristic of
differentiating endothelial cells. CD31 expression was used to isolate the
putative endothelial cell precursors by flow cytometry, with positive cells
comprising about 2% of the population. The further culturing of sorted cells
produced cells that expressed markers of the mature endothelium, such as von
Willebrand factor; they could also uptake low-density lipoprotein.
Furthermore, they formed tube-like structures in vitro, and functional
microvessels when grafted into immunodeprived mice on artificial matrices in
vivo.
Cynomolgus monkey ES cells have also been shown to differentiate into
endothelial precursors by culturing them on a feeder cell layer of OP9 cells
(Sone et al., 2003). A
VEGF-R2+ VE-cadherin cell population was isolated
by flow cytometry from these cultures, and under different conditions
differentiated into either CD31+VE-cadherin+ endothelial
cells, or into vascular mural cells (vascular smooth muscle cells or
pericytes).
Cardiac muscle has also been derived from either spontaneously
differentiating hES cells or from co-culture systems. Kehat and colleagues
(Kehat et al., 2001) isolated
beating cardiomyocyte foci from spontaneously differentiating human embryoid
bodies, and showed that the cells had properties of fetal or neonatal
cardiocytes, as evidenced by their subcellular distribution of gap junctions,
their myofibrillar organisation, and their electrical activity. Xu et al.
(Xu et al., 2002a
) also
exploited the spontaneous generation of beating foci in embryoid bodies to
study cardiomyocyte differentiation. Beating foci were observed in up to 70%
of embryoid bodies in this study. The cells expressed specific myocardial
markers, including cardiac troponin 1 and alpha myosin heavy chain, as well as
the transcription factors Nkx2.5, GATA4 and MEF2. The expression of atrial
natriuretic factor characterised the ventricular myocytes as developing cells,
and the study showed that the expression of adrenergic receptors underwent
maturation patterns in culture that were similar to those seen in the
developing heart in vivo. Recent studies have closely analysed the phenotype
of the immature cardiac cells formed in human embryoid bodies
electrophysiologically (Amit et al.,
2003
), and have documented the maturation of these cells by the
ultrastructural analysis of sarcomere development
(Snir et al., 2003
).
By contrast, the hES cell lines studied by Mummery et al.
(Mummery et al., 2003) showed
no tendency to spontaneously differentiate into cardiac muscle. This group
co-cultured ES cells with the END-2 murine cell line, which is thought to
resemble visceral endoderm and has been shown previously to induce cardiac
differentiation of mouse ES and EC cells. The END-2 line caused hES cells to
differentiate into two lineages, one exemplified by cysts that express genes
characteristic of the visceral endoderm, and the other consisting of foci of
beating muscle. Sarcomeric organisation, the expression of atrial natriuretic
factor and the L-type calcium channel, and an action potential similar to
fetal ventricular cells indicated that the cells were immature
cardiomyocytes.
Thus, several mesodermally derived cell types can be isolated from hES cells and their maturation studied in vitro. The cells involved in the earliest stages of ES commitment to mesoderm formation are unknown, and the factors that induce mesodermal progenitors and control their subsequent development remain largely uncharacterised. However, with further study, it should prove possible to isolate cells at early stages of mesoderm differentiation and to identify factors that drive their commitment to particular mesodermal lineages.
Endodermal lineages
The differentiation of hES cells into embryonic endodermal lineages has
been more difficult to achieve. As in the mouse, studies are hampered by an
incomplete understanding of the commitment and differentiation of embryonic
endoderm in the embryo proper, by a lack of specific markers for the early
progenitors of this lineage, and by an overlap of gene expression patterns
between extraembryonic and embryonic cell lineages. One study
(Assady et al., 2001) has
demonstrated that genes characteristic of the pancreatic lineage are switched
on during human embryoid body formation, and cells stained by anti-insulin
antibodies have been found in human embryoid bodies. However, little is known
about what controls the appearance of these cells or their precursors. Another
group (Rambhatla et al., 2003
)
have shown that hES cells grown as embryoid bodies and treated with sodium
butyrate, or adherent hES cultures treated with dimethyl sulphoxide followed
by sodium butyrate, differentiate into cells that express various hepatocyte
markers.
Future prospects
The first five years of hES cell research have achieved some significant objectives. The original observations on the derivation and properties of hES cells have been repeatedly confirmed, and the technology has been successfully disseminated to a number of research groups. The ethical debate over the use of human embryos in research is unlikely ever to be fully resolved, as it is driven by religious and philosophical considerations of the nature of human existence, in addition to scientific and practical considerations. However, in most jurisdictions, extensive discussion, debate, and, ultimately, compromise have led to the formulation of laws and regulations that enable the work to progress on an ethical basis. It is most important for the future that researchers can generate and gain access to new hES cell lines derived using improved technologies.
Several key questions remain to be answered in the coming years. For example, what are the functions of the genes that maintain the pluripotentiality of hES cells? And what do the early differentiated progeny of hES cells represent in relation to the primate peri-implantation embryo, and why are there species-specific differences in the biology of cultured pluripotent cells? Our ability to grow and manipulate the cells has improved markedly, but there are several technical challenges that have still not been met, such as the large-scale propagation of pure ES cell cultures in defined media in the absence of feeder cells, their clonal growth, and easy and efficient ways to genetically manipulate them.
We also need to better understand the events that control ES cell
commitment and differentiation. The appearance of a low proportion of
differentiated cells in a complex mixture complicates the interpretation of
the currently available data, and obscures whatever cell lineage relationships
and cell interactions may have led to the final outcome. However, it has been
possible to isolate and identify progenitors, to study their gene expression,
and to assess the effect of exogenous factors on their generation,
proliferation and differentiation. As in the mouse
(Loebel et al., 2003), there
is some evidence that the behaviour of hES cells can be predicted from what we
know of mammalian embryogenesis, but the data on hES cell differentiation are
very limited as yet. In the future, it is likely that high throughput
screening approaches (Ding et al.,
2003
) will complement embryological studies in the effort to
discover new means of manipulating ES cells. This approach could identify
synthetic or natural small molecules that could serve as lead compounds for
pharmaceutical development in the field of regenerative medicine.
It is important to recognise that ES cell differentiation events and the extrinsic factors that control them may prove to be extremely context dependent. ES cells may have the ability to respond to normal developmental cues, but the cells are in a totally different environment to the embryo. These considerations notwithstanding, progress during the first five years of hES cell research strongly suggests that these versatile cells will provide an important resource for understanding human development, alongside their anticipated roles in regenerative medicine.
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