|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online February 24, 2006
doi: 10.1242/10.1242/dev.02286
Department of Genetics, Institute of Life Sciences, The Hebrew University, Jerusalem, Israel
* Author for correspondence (e-mail: nissimb{at}mail.ls.huji.ac.il)
Accepted 12 January 2006
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: NANOG, Human embryonic stem cells, Self-renewal, Primitive ectoderm
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Thomson et al., 1998). These
cells have two distinctive properties: an unlimited capacity for self-renewal
and pluripotency (the ability to differentiate to each of the three embryonic
germ layers, endoderm, mesoderm and ectoderm). Pluripotency has been shown in
vitro by inducing undifferentiated ES cells to form embryoid bodies (EBs)
composed of cells from the three germ layers
(Itskovitz-Eldor et al., 2000;
Schuldiner et al., 2000), and
in vivo by injecting undifferentiated cells to immunodeficient mice where they
form tumors termed teratomas, composed of multiple differentiated cell types
(Reubinoff et al., 2000;
Thomson et al., 1998).
Additionally, numerous studies have described differentiation of HESCs to
specific cell types using growth factors (for reviews, see
Schuldiner and Benvenisty,
2003). Thus, HESCs hold the promise to serve as a source of cells
in transplantation medicine. As they mimic in vitro the onset of early
development they are also viewed as a promising model for early human
development, during stages that are not accessible to research.
The propagation of HESCs requires the presence of feeder cells (e.g. mouse
embryonic fibroblasts, MEFs) or addition of media conditioned by them
(Xu et al., 2001). The factors
secreted by the MEFs and required by HESCs are not fully characterized,
although some factors have been suggested to inhibit the differentiation of
cells (Amit et al., 2004;
Sato et al., 2004;
Xu et al., 2005). One such
factor is bFGF, which has been suggested to enable HESCs propagation in the
absence of conditioned media (Wang et al.,
2005; Xu et al.,
2005).
In addition, the intracellular factors that maintain pluripotency and
self-renewal of HESCs remain elusive. One factor known to be involved in
maintaining pluripotency is OCT4, the downregulation of which in both
mouse and human ES cells causes cell differentiation
(Matin et al., 2004;
Niwa et al., 2000). However,
since the derivation of HESCs, it has become apparent that knowledge
accumulated on mouse embryonic stem cells (MESCs) cannot automatically be
deduced for HESCs. One fundamental difference observed so far is the role of
LIF, which in MESCs substitutes the need for support by feeder cells
(Smith et al., 1988;
Williams et al., 1988). In
MESCs, LIF activates Stat3 signaling, which is sufficient to replace the
requirement for feeder cells (Niwa et al.,
1998). By contrast, in HESCs neither the addition of LIF nor the
activation of STAT3 are able to release the cells from dependence on feeder
cells (Daheron et al., 2004;
Humphrey et al., 2004). BMP4 is
another factor shown to be involved in maintenance of self-renewal in MESCs,
by inhibiting neural differentiation in serum-free media
(Ying et al., 2003). However,
when added to HESCs, BMP4 was shown to actually promote differentiation to
trophectoderm even in the presence of MEF-conditioned media
(Xu et al., 2002). Additional
differences between these cells exist, among them are differences in
expression of cell surface markers (Ginis
et al., 2004) and a different differentiation potential, because
HESCs, as opposed to MESCs, are capable of differentiating to trophoblast
(Xu et al., 2002).
A gene recently shown to be fundamental in maintaining pluripotency in
MESCs is Nanog (Chambers et al.,
2003; Mitsui et al.,
2003). Overexpression of Nanog releases the cells from
LIF dependency, and prevents differentiation upon LIF withdrawal. In addition,
its downregulation leads to differentiation to extra-embryonic endoderm.
Nanog does not function via the Stat3 pathway, but cooperates with it
in maintaining ES cell identity. It was suggested that NANOG may have
a key role in sustaining ES cell identity also in HESCs, and that it may have
taken over the role of STAT3, which does not seem to be involved in HESC
self-renewal.
In this study, we examined the role of NANOG in HESC self-renewal
and pluripotency. NANOG is used as a marker for undifferentiated
HESCs but its role in these cells is not yet fully characterized. Although it
has been shown that its downregulation leads to differentiation of HESCs
(Zaehres et al., 2005), the
result of its overexpression has not yet been examined. We show that
overexpression of NANOG enables the propagation of HESCs for multiple
passages in the absence of feeder cells or conditioned media (CM). These cells
grow as colonies derived from single cells even in the absence of CM, and lose
this ability when the transgene is excised. Additionally, we show that
NANOG expression in wild-type cells is upregulated during early
differentiation, and that its overexpression in HESCs modifies the expression
of marker genes to an expression pattern similar to that of primitive ectoderm
cells. Using microarray analysis, we suggest new marker genes that may
distinguish between the ICM and primitive ectoderm cells in human.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) were
cultured on Mitomycin-C treated mouse embryonic fibroblast (MEF) feeder layer
(obtained from 13.5 day embryos) in 85% KnockOut DMEM medium (GIBCO-BRL),
supplemented with 15% KnockOut SR (a serum-free formulation) (GIBCO-BRL), 1 mM
glutamine, 0.1 mM ß-mercaptoethanol (Sigma), 1% nonessential amino acids
stock (GIBCO-BRL), Penicillin (50 units/ml), Streptomycin (50 µg/ml),
ITSX100 (insulin-transferrin-selenium) in a 1:200 dilution (GIBCO-Invirogen
Corporation), and 4 ng/ml basic fibroblast growth factor (bFGF, PeproTech). To
obtain a feeder-free culture, the cells were plated on laminin (1
µg/cm2, Sigma) or gelatin (0.1%, Merck) coated plates and grown
in media conditioned for at least 24 hours by MEFs. Differentiation in vitro
into embryoid bodies (EBs) was performed by withdrawal of bFGF from the growth
media and allowing aggregation in petri dishes. Differentiation in vivo into
teratomas was performed by injecting undifferentiated ES cells under the
kidney capsule of immunodeficient mice and taken out for analysis 2 months
later.
Transfections and clone establishment
Wild-type ES cells were transfected using the calcium phosphate method as
described previously (Chen and Okayama,
1988). The cells were transfected with a plasmid described earlier
(Chambers et al., 2003) that
contains a human NANOG transgene followed by an IRES and a puromycin
resistance gene. This transcriptional unit is located between two lox-P sites.
Following CRE-recombinase excision there is removal of both the NANOG
gene and puromycin resistance, and the transcriptional activation of a GFP
gene. NANOG overexpressing clones were established by puromycin
selection (0.3 µg/ml, Sigma) following transfection. Revertant clones were
established by transfecting NANOG overexpressing clones with a
CRE-recombinase plasmid. Following transfection, the cells were trypsinzed to
single cells and seeded in low densities (1:1000 split). Cells expressing GFP
were selected, expanded and verified to have lost puromycin resistance.
Growth analysis
Cells were seeded in 96-well dishes coated with 0.1% gelatin in a density
of 2x104 cells per cm2 and medium was changed
daily. Final cell densities were determined by fixating the cells with 0.5%
glutardialdehyde (Sigma) and staining with Methylene Blue (Sigma) dissolved in
0.1 M boric acid (pH 8.5). Color extraction was performed using 0.1 M
hydrochloric acid, and staining (which is proportional to cell number) was
quantified by measuring absorbance at 650 nm. Each experiment was performed in
triplicate.
Colony forming assays
H9 and H13 Cells were trypsinized to a single cell suspension and seeded in
12-well dishes to a density of 500 cells per cm2. After 8 days, the
cells were fixed and assayed for alkaline phosphatase activity (86R kit,
Sigma) according to the manufacturer's instructions. Each experiment was
performed in triplicate and at the end of the experiments the positively
stained colonies were counted.
RNA extraction and RT-PCR analysis
RNA was extracted using TRI-reagent for total RNA isolation according to
the manufacturer's instructions (Sigma). cDNA was synthesized using random
hexamer primers. Ampification was performed on the cDNA using Takara Ex-Taq.
PCR conditions include a first step of 3 minutes at 94°C, a second step of
25-30 cycles of 30 seconds at 94°C, 45 seconds annealing step at
58-64°C, 1 minute at 72°C and a final step of 7 minutes at 72°C.
GAPDH was used as a housekeeping gene to evaluate and compare quality
of different cDNA samples. Primers and product sizes are listed in Table S1 in
the supplementary material. Final products were examined by gel
electrophoresis on 2% agarose ethidium bromide-stained gels. Real-time RT-PCR
analysis was performed using Rotor-gene 2000 (Corbett Research, Sydney). The
reaction was carried out according to the manufacturer's protocol using
Absolute SYBR Green Rox Mix (from ABgene, used according to the manufacturer's
recommendations) with PCR program as follows: 95°C for 5 minutes; a second
step of 35 cycles of 20 seconds at 95°C, 15 seconds annealing step at
60°C, 25 seconds at 72°C and 15 seconds at 82°C; and a final step
of 1 minute at 72°C.
Immunostaining and FACS analysis
For immunostaining cells were washed once with PBS and fixed with 4%
paraformaldehyde. Blocking and permeabilization were performed with 2% BSA,
10% low-fat milk and 0.1% Triton-X in PBS. Staining with primary mouse
anti-human OCT4 was performed for 1 hour (Santa Cruz Biotechnology, used at a
1:50 dilution). As a secondary antibody, Cy3-conjugated goat anti-mouse IgG
(H+L; Jackson ImmunoResearch, dilution 1:200), was used. Nuclear staining was
performed with Hoechst 33258 (Sigma). FACS analysis for TRA-1-60 expression
was performed after trypsinization of the cells. The cells were washed with 3%
BSA in PBS with 0.05% Sodium Azid, incubated with TRA-1-60 antibody (kind gift
from Prof. Peter Andrews) for 1 hour, incubated with Cy3-conjugated rabbit
anti-mouse IgM (Jackson Immunoresearch) and after washes analyzed using the
FACSCalibur system (Becton Dickinson). Analysis was performed on CELLQUEST
software (Becton Dickinson). Forward- and side-scatter plots were used to
exclude dead cells and debris from the histogram analysis.
Western blot analysis
Western blot analysis was performed according to standard protocols. For
NANOG detection a polyclonal rabbit antibody against human NANOG protein
(abcam) in 1:1000 dilution was incubated for 18 hours. A secondary antibody
(peroxidase conjugated Affinipure goat anti-rabbit IgG by Jackson
Immunoresearch) was incubated for 1 hour in 1:10000 dilution. For loading
control we used a mouse antibody against 
-TUBULIN (Sigma).
DNA microarray analysis
Total RNA was extracted according to the manufacturers protocol
(Affymetrix). When extracting RNA from undifferentiated ES cells, the cells
were grown for one passage on gelatin-coated plates with conditioned media in
order to avoid contamination by feeder cells. Hybridization to the U133A DNA
microarray, washing and scanning were performed according to the
manufacturer's protocol, and expression patterns were compared between
samples. Signals were normalized by dividing each probe by the average value
of the chip to avoid differences between different chips and experiments.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). These sequences are enclosed by lox-P sites, and upon
CRE-recombinase excision they are removed, causing a GFP gene to be activated
(Chambers et al., 2003).
NANOG overexpressing stable clones were isolated following selection
with puromycin and expression of the NANOG transgene was verified by
RT-PCR analysis using specific primers to the transgene
(Fig. 1A, part I). As the
puromycin resistant gene lies on the same transcriptional unit as the NANOG
transgene, constant selection with puromycin guaranteed stable expression of
the transgene over time. To asses the overexpression level of the transgene in
different clones, we performed real-time RT-PCR analysis and observed that the
total level of NANOG mRNA in the clones was significantly higher than
in wild-type cells (Fig. 1A,
part II). Concurrently, elevated protein levels were observed in the clones by
western blot (Fig. 1A, part
III). It has previously been shown that Nanog overexpression releases
MESCs from LIF dependency (Chambers et al.,
2003; Mitsui et al.,
2003). However, regulation of self-renewal in HESCs is not
identical to that in MESCs. Thus, we set out to determine the role of
NANOG in HESCs. HESCs are grown on mouse embryonic fibroblasts (MEFs)
or in the presence of media conditioned by MEFs. Therefore, we first examined
whether the requirement of cells for conditioned media (CM) could be replaced
by overexpression of NANOG transgene. When wild-type and
NANOG overexpressing cells were grown in the presence of CM, both
cell types showed similar growth rates
(Fig. 1B, upper panel).
However, when CM was removed, wild-type cells ceased to proliferate, while
NANOG overexpressing cells kept dividing
(Fig. 1B, lower panel) even
though their growth rate was markedly decreased. In the presence of feeder
cells, overall colony morphology of the NANOG overexpressing clones was very
similar to that of wild-type cells, albeit being slightly flatter (see Fig. S1
in the supplementary material). However, in the absence of CM, a striking
difference is observed, as early as a few days after CM withdrawal. While
wild-type cells created only very small and partially differentiated colonies,
Nanog overexpressing clones created much larger colonies, a difference that
was observed as early as 4 days after CM withdrawal (see Fig. S1 in the
supplementary material).
|
NANOG over-expressing cells form colonies in the absence of feeders
As NANOG overexpressing clones proliferate in the absence of CM,
we examined whether the cells can also form colonies from single cells under
these conditions. Cells were trypsinized to single cells, seeded at very low
density, and the ability to form colonies was assayed. Resulting colonies were
verified as undifferentiated ES cells by assaying the activity of the ES cell
marker alkaline phosphatase (AP). When wild type and NANOG
over-expressing cells were seeded in the presence of CM, both were able to
create a large number of AP-positive colonies
(Fig. 3A). When cells were
seeded in medium not conditioned by MEFs and not containing bFGF (basal
media), the number of colonies formed by the wild-type cells was negligible,
while NANOG overexpressing cells were capable of forming a
considerably larger number of AP-positive colonies
(Fig. 3A,B, AP activity shown
as red staining). Therefore, NANOG is capable of maintaining ES cells
undifferentiated independently of CM, even though the frequency of colony
formation is lower than with CM. To confirm that the effect observed upon
NANOG overexpression was not unique to one cell line, NANOG overexpressing
clones were established in another cell line, H13. As in H9 cells, H13 cells
were able to create colonies in the absence of CM only when transfected with a
NANOG overexpression vector (Fig.
3A,B).
|
|
NANOG is upregulated upon early differentiation
In mouse, Nanog acts a stage later than Oct4 during
normal embryonic development. Oct4 inhibits differentiation to
trophoblast and its knockout in MESCs triggers differentiation to trophoblast
(Niwa et al., 2000).
Nanog acts to inhibit differentiation to primitive endoderm and its
knockout in MESCs triggers differentiation to primitive endoderm
(Mitsui et al., 2003). We
examined whether this temporal difference is observed in a human system by
assaying the expression pattern of both OCT4 and NANOG genes
during the differentiation of HESCs. Four time points along their
differentiation were assayed: ES cells, the pluripotent undifferentiated
stage; 2-day-old embryoid bodies (EBs), the early stage of differentiation;
10-day-old EBs, which represent mid differentiation; and 30-day-old EBs, which
represent late differentiation (Dvash et
al., 2004; Leahy et al.,
1999). OCT4 expression is high in ES cells, 2- and
10-day-old EBs, and drops drastically in 30-day-old EBs
(Fig. 4B). However, the
expression pattern of NANOG is different. It is expressed in ES
cells, but its level increases tenfold in 2-day-old EBs, remains high in
10-day-old EBs and drops in 30-day-old EBs
(Fig. 4A,B). This implies that
NANOG may actually be expressed at the highest level in early
differentiating cells and not in undifferentiated cells.
Overexpression of NANOG alters the marker genes expressed by HESCs
Following its formation, the ICM differentiates to primitive endoderm and
primitive ectoderm. We hypothesized that NANOG may be expressed at
higher levels in primitive ectoderm than in the ICM in human. There are
several known marker genes in the mouse that distinguish ICM from primitive
ectoderm (Rathjen and Rathjen,
2003). Rex1 (Zfp42 - Mouse Genome Informatics), Gbx2 and Crtr1
(Tcfcp2l1 - Mouse Genome Informatics) are enriched in ICM, and Fgf5 and PRCE
are absent from ES cells and ICM, and enriched in primitive ectoderm
(Fig. 5A). When compared with
wild-type cells, in NANOG overexpressing cells there is a significant
downregulation of REX1 (fourfold) and GBX2 (twofold) and upregulation of FGF5
(ninefold) (Fig. 5B,C; see Fig.
S2 in the supplementary material). No change in the expression pattern of
CRTR1 and PRCE was observed (data not shown). The magnitude of the change
seemed to correlate with the level of overexpression of NANOG, with the
highest overexpression (as assessed by the results shown in
Fig. 1) leading to the most
significant changes in mRNA levels (clone 9, see Fig. S2 in the supplementary
material) and the clone with the lowest NANOG expression levels
showing less significant changes, and only in two of the markers examined
(clone 4, see Fig. S2 in the supplementary material). To examine if the same
phenomenon exists in MESCs, we looked at the expression pattern of markers
known to distinguish ICM from primitive ectoderm in wild-type and
Nanog overexpressing MESCs. MESCs overexpressing Nanog were
obtained from the laboratories that demonstrated the effect of Nanog
on MESCs (Chambers et al.,
2003; Mitsui et al.,
2003). However, no change was observed upon overexpression of
Nanog in any of the marker genes examined, in the various cell lines
(Fig. 5D).
|
). We therefore examined whether this is also the case in
HESCs. The expression of the differential markers was examined in the
revertant clones derived from NANOG overexpressing clones. However,
the expression of the examined genes remained similar to that of the
overexpressing clones, and no reversion to the expression pattern of wild-type
cells was observed (Fig.
5C).
Transcriptome analysis of NANOG over-expressing cells reveals an increase in similarity to early differentiating cells
DNA microarray analysis was performed on RNA extracted from wild-type and
NANOG overexpressing ES cells, and three stages of differentiating
embryoid bodies (EBs). Dendogram analysis, which clusters samples according to
their degree of similarity, shows that upon overexpression of NANOG
in HESCs, the similarity to early differentiating cells increases
(Fig. 6A). However, the cells
do not appear differentiated as they cluster apart from the samples of 2- and
10-day-old EBs. Four groups of genes differentially expressed between
NANOG overexpressing and wild-type cells were identified
(Fig. 6B).
|
(2) Genes upregulated in NANOG overexpressing cells compared with ES cells and 2-day-old EBs. These may represent transcriptional targets of NANOG.
(3) Genes downregulated in NANOG overexpressing cells and
2-day-old EBs compared with wild-type ES cells
(Fig. 6D). These may be ICM
(and therefore ES cell) markers that are downregulated upon the transition to
primitive ectoderm. Among these genes are LECTIN (LGALS1), a
known marker of HESCs (Dvash et al.,
2004) that is absent from all samples but ES cells, and
HOXA1 (homeo box A1), a transcription factor involved in development.
Among the genes downregulated in NANOG overexpressing cells but to a
smaller extent are genes suggested to be downstream targets of HOXA1
(Shen et al., 2000), such as
GBX2 and BMP2.
(4) Genes downregulated in NANOG cells compared with both
2-day-old EBs and wild-type ES cells. These genes may also be targets of
NANOG, which has been suggested to be both a transcriptional
activator and repressor. In addition, genes reported to be upregulated upon
knock-down of Nanog in MESCs
(Mitsui et al., 2003) are
downregulated upon overexpression of NANOG in HESCs. These include
parietal endoderm markers (like LAMB1, downregulated by 3.5-fold) and
visceral endoderm markers (like BMP2, downregulated by 14-fold). It
has been suggested that NANOG represses expression of primitive
endoderm genes in MESCs, and it seems that the same effect is observed in
HESCs.
The transition to EPL cells from MESCs has been shown to follow treatment
with MEDII medium (a medium conditioned by the human hepatocellularcarcinoma
cell line, Hep G2). This medium has been shown in MESCs to lead to a change in
the marker gene pattern expression from that of ICM to that of primitive
ectoderm. The treatment of HESCs with MEDII
(Calhoun et al., 2004) has also
shown that the resultant cells are pluripotent in nature though with an early
differentiated phenotype. However, unlike MESCs, which show transition to EPL
cells, HESCs treated with MEDII displayed similar gene expression to primitive
streak cells and nascent mesoderm cells, through activation of the
TGFß1/NODAL pathway. We therefore examined NANOG overexpressing
cells to asses whether they show similar changes in gene expression as has
been shown after treatment with MEDII. Our NANOG overexpressing cells did not
fully mimic the effect of MEDII media, as reported for HESCs. Thus, three of
the markers [CRIPTO (TDGF1 - Human Gene Nomenclature
Database), FST and TBX1] examined in the report on MEDII
treatment showed a similar response in our clones, whereas two showed no
effect [GATA6 and ZNF1A1 (ZNFN1A1 - Human Gene
Nomenclature Database)], and one had an opposite effect [LEFTYA
(LEFTY2 - Human Gene Nomenclature Database), see Fig. S3 in the
supplementary material].
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
),
whereas Nanog expression is essential for the development of
primitive ectoderm. Mouse embryos mutated in the Oct4 gene develop up
to the blastocyst stage, but their ICM is restricted to the trophoblast
lineage (Nichols et al.,
1998). Mouse embryos mutated in the Nanog gene also
develop up to the blastocyst stage, but when cultured in vitro, their ICM
creates only primitive endoderm cells and no primitive ectoderm
(Mitsui et al., 2003). This
indicates that in both human and mouse, NANOG may promote the
transition from ICM to primitive ectoderm. However, the idea that NANOG can
actively promote this transition, not only by preventing the transition to
primitive endoderm but by actually driving the transition to primitive
ectoderm, has not been suggested or shown yet, and this is the first report of
such a function. Indeed, a change in marker gene expression occurs upon
overexpression of NANOG in HESCs from that characteristic of ICM to
that characteristic of primitive ectoderm. A protocol for differentiation to
primitive ectoderm was established for MESCs
(Rathjen et al., 1999). These
cells, termed early primitive ectoderm-like (EPL) cells have been defined in
the mouse by three characteristics: different expression of a subset of marker
genes that distinguish ICM from epiblast; different cytokine dependency, which
includes decreased dependency on LIF and creation of undifferentiated colonies
in its absence; and failure to form chimeras upon injection into a blastocyst.
We have shown the first two requirements in HESCs overexpressing
NANOG, while the third cannot be examined in humans. Although we did
not observe a change in all markers known to distinguish ES cells from EPL
cells in mouse, this may result from inherent differences between the two
species, known to exist for several markers. For example, PRCE, which in mouse
is expressed mainly in epiblast and not in ICM or ES cells, is expressed in
wild-type HESCs. As human primitive ectoderm cells are not available for
research, nothing is known about the in vivo expression pattern of the
examined genes, and our analysis was based on the data established in mouse.
However, although the population of NANOG overexpressing cells is
undoubtedly a pluripotent population, a divergence from the normal marker gene
expression pattern of ES cells does occur. NANOG overexpressing cells
also have different cytokine dependency, as observed by the ability to grow in
the absence of CM, another characteristic of mouse primitive ectoderm
cells.
When expression of ICM-specific marker genes was examined in wild-type and
in Nanog overexpressing MESCs, no change was observed in either of
the cell lines examined. One possible explanation is species differences
between mouse and human. Another possibility is that this is a result of
culture conditions or in vitro effects. For example, LIF has been shown to
inhibit the transition from ICM to primitive ectoderm, and the appearance of
primitive ectoderm markers, such as Fgf5
(Shen and Leder, 1992).
Another feature of EPL cells in mouse is that the primitive ectoderm gene
expression pattern can be reverted back to an ICM expression pattern. In MESCs
converted to EPL cells, once MEDII is removed and LIF is returned to the
medium, genes that were differentially expressed are reverted back to levels
distinctive to ICM. However, in HESC revertant clones (after the removal of
the NANOG transgene), although the growth characteristics are
restored to those of wild-type cells, the expression of REX1, GBX2
and FGF5 remains similar to that in the parental clones. This
difference can be attributed to several differences between the experimental
systems. One of these differences may be the difference between mouse and
human ES cells, which could mean that although in mouse this transition is
reversible, in human cells it is not. However, it could also be the result of
the different time-frames used in each experiment. While in the mouse system
the method to derive EPL cells used treatment with MEDII for only a few days
(these cells have been reported to undergo crisis after 8 days, see
Rathjen and Rathjen, 2003), in
our hands the cells were passaged several times (at least five) before
deriving the revertant clones. Therefore, the human mouse discrepancy may not
originate from lack of the ability of human cells to revert from primitive
ectoderm to ICM, but it may be that the longer time frame fixed the fate of
the cells as primitive ectoderm cells, not permitting return to an ICM
state.
When NANOG overexpression in HESCs is compared with what is
published on HESCs treated with MEDII
(Calhoun et al., 2004), two
points seem noteworthy. First, in both cases the cells remain pluripotent and
express markers of pluripotent cells; second, in both cases there seems to be
an activation of the TGFß1/NODAL pathway. Upregulation of CRIPTO
(a co-receptor/ligand of NODAL) and downregulation of FST (which may
function as an activin-binding inhibiting protein and therefore an inhibitior
of this pathway) were observed in both cases. However, in contrast to
treatment with MEDII, in our cells LEFTYA was actually upregulated.
Still, LEFTYA is a transcriptional target of NODAL and therefore this
is consistent with activation of the pathway. Following treatment with MEDII
no change was reported regarding the primitive ectoderm markers, REX1,
GBX2 and FGF5, and some mesoderm markers shown to be upregulated
upon MEDII treatment, did not change upon overexpression of NANOG.
Treatment with MEDII results in the addition of multiple factors to the cells,
factors which may work together or apart to direct cell fate to more than one
direction. By contrast, overexpression of NANOG probably creates a
more unified cell culture, as only the expression of a single gene is altered
compared with wild-type cells. Therefore, this report is the first to describe
the transition from ICM to primitive ectoderm induced by the genetic
manipulation of a specific gene.
Using microarray analysis, we searched for genes differentially expressed
between wild-type and NANOG overexpressing HESCs. NANOG has
been suggested both to repress differentiation to primitive endoderm and to
actively maintain pluripotency. As parietal endoderm markers are among the
genes downregulated upon overexpression of NANOG, it is likely that
human NANOG also inhibits differentiation to primitive endoderm. The
larger number of genes downregulated by NANOG overexpression than
those upregulated might suggest that a significant portion of the effects of
NANOG may come from repressing differentiation into primitive
endoderm. Genes upregulated after NANOG overexpression may have
importance in future research as they may contribute to the release from CM
dependency. One pressing issue in current ES cell research is the
establishment of feeder-free cultures. Therefore, pathways that govern HESC
self-renewal and pluripotency have to be elucidated. Future research of the
group of genes upregulated in NANOG over-expressing clones may
facilitate and optimize this goal, as understanding how NANOG enables
feeder free growth may improve the culture obtained and define the
requirements of the cells. A clue to a possible mechanism of the release from
conditioned media dependency comes from the fact that LECTIN1, one of
the genes downregulated upon overexpression of NANOG, is involved in
promotion of apoptosis (Yang and Liu,
2003). Similarly, HSPA1A, one of the genes upregulated by
NANOG, is known to inhibit apoptosis
(Gabai et al., 2002).
Therefore, the total effect observed upon NANOG overexpression may
result from two activities: the inhibition of differentiation and an increase
in cell survival that results from downregulation of mechanisms that promote
apoptosis.
Finally, using microarrays, new genes expressed differentially between ICM and primitive ectoderm in humans are suggested. Genes expressed differentially between these two closely related populations can be used to identify the primitive ectoderm population. It is likely that at least some of these genes have a role in the transition from one cell population to the other. Most importantly, the recapitulation of the first differentiation step of the ICM in vitro shows that early stages of human development, not accessible otherwise to research, may indeed be mimicked in culture.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/6/1193/DC1
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Amit, M., Shariki, C., Margulets, V. and Itskovitz-Eldor, J.
(2004). Feeder layer- and serum-free culture of human embryonic
stem cells. Biol. Reprod.
70,837
-845.
Calhoun, J. D., Rao, R. R., Warrenfeltz, S., Rekaya, R., Dalton, S., McDonald, J. and Stice, S. L. (2004). Transcriptional profiling of initial differentiation events in human embryonic stem cells. Biochem. Biophys. Res. Commun. 323,453 -464.[CrossRef][Medline]
Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S. and Smith, A. (2003). Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113,643 -655.[CrossRef][Medline]
Chen, C. A. and Okayama, H. (1988). Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. Biotechniques 6,632 -638.[Medline]
Daheron, L., Opitz, S. L., Zaehres, H., Lensch, W. M., Andrews,
P. W., Itskovitz-Eldor, J. and Daley, G. Q. (2004). LIF/STAT3
signaling fails to maintain self-renewal of human embryonic stem cells.
Stem Cells 22,770
-778.
Dvash, T., Mayshar, Y., Darr, H., McElhaney, M., Barker, D.,
Yanuka, O., Kotkow, K. J., Rubin, L. L., Benvenisty, N. and Eiges, R.
(2004). Temporal gene expression during differentiation of human
embryonic stem cells and embryoid bodies. Hum. Reprod.
19,2875
-2883.
Gabai, V. L., Mabuchi, K., Mosser, D. D. and Sherman, M. Y.
(2002). Hsp72 and stress kinase c-jun N-terminal kinase regulate
the bid-dependent pathway in tumor necrosis factor-induced apoptosis.
Mol. Cell. Biol. 22,3415
-3424.
Ginis, I., Luo, Y., Miura, T., Thies, S., Brandenberger, R., Gerecht-Nir, S., Amit, M., Hoke, A., Carpenter, M. K., Itskovitz-Eldor, J. et al. (2004). Differences between human and mouse embryonic stem cells. Dev. Biol. 269,360 -380.[CrossRef][Medline]
Humphrey, R. K., Beattie, G. M., Lopez, A. D., Bucay, N., King,
C. C., Firpo, M. T., Rose-John, S. and Hayek, A. (2004).
Maintenance of pluripotency in human embryonic stem cells is STAT3
independent. Stem Cells
22,522
-530.
Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A., Yanuka, O., Amit, M., Soreq, H. and Benvenisty, N. (2000). Differentiation of human embyronic stem cells into embryoid bodies comprising the three embryonic germ layers. Mol. Med. 6, 88-95.[Medline]
Leahy, A., Xiong, J. W., Kuhnert, F. and Stuhlmann, H. (1999). Use of developmental marker genes to define temporal and spatial patterns of differentiation during embryoid body formation. J. Exp. Zool. 284,67 -81.[CrossRef][Medline]
Matin, M. M., Walsh, J. R., Gokhale, P. J., Draper, J. S.,
Bahrami, A. R., Morton, I., Moore, H. D. and Andrews, P. W.
(2004). Specific knockdown of Oct4 and beta2-microglobulin
expression by RNA interference in human embryonic stem cells and embryonic
carcinoma cells. Stem Cells
22,659
-668.
Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K., Maruyama, M., Maeda, M. and Yamanaka, S. (2003). The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113,631 -642.[CrossRef][Medline]
Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H., Klewe-Nebenius, D., Chambers, I., Scholer, H. and Smith, A. (1998). Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95,379 -391.[CrossRef][Medline]
Niwa, H., Burdon, T., Chambers, I. and Smith, A.
(1998). Self-renewal of pluripotent embryonic stem cells is
mediated via activation of STAT3. Genes Dev.
12,2048
-2060.
Niwa, H., Miyazaki, J. and Smith, A. G. (2000). Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. 24,372 -376.[CrossRef][Medline]
Rathjen, J. and Rathjen, P. D. (2003). Lineage specific differentiation of mouse ES cells: formation and differentiation of early primitive ectoderm-like (EPL) cells. Methods Enzymol. 365,3 -25.[Medline]
Rathjen, J., Lake, J. A., Bettess, M. D., Washington, J. M., Chapman, G. and Rathjen, P. D. (1999). Formation of a primitive ectoderm like cell population, EPL cells, from ES cells in response to biologically derived factors. J. Cell Sci. 112,601 -612.[Abstract]
Reubinoff, B. E., Pera, M. F., Fong, C. Y., Trounson, A. and Bongso, A. (2000). Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat. Biotechnol. 18,399 -404.[CrossRef][Medline]
Sato, N., Meijer, L., Skaltsounis, L., Greengard, P. and Brivanlou, A. H. (2004). Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat. Med. 10, 55-63.[CrossRef][Medline]
Schuldiner, M. and Benvenisty, N. (2003). Factors controlling human embryonic stem cell differentiation. Methods Enzymol. 365,446 -461.[Medline]
Schuldiner, M., Yanuka, O., Itskovitz-Eldor, J., Melton, D. A.
and Benvenisty, N. (2000). Effects of eight growth factors on
the differentiation of cells derived from human embryonic stem cells.
Proc. Natl. Acad. Sci. USA
97,11307
-11312.
Shen, J., Wu, H. and Gudas, L. J. (2000). Molecular cloning and analysis of a group of genes differentially expressed in cells which overexpress the Hoxa-1 homeobox gene. Exp. Cell Res. 259,274 -283.[CrossRef][Medline]
Shen, M. M. and Leder, P. (1992). Leukemia
inhibitory factor is expressed by the preimplantation uterus and selectively
blocks primitive ectoderm formation in vitro. Proc. Natl. Acad.
Sci. USA 89,8240
-8244.
Smith, A. G., Heath, J. K., Donaldson, D. D., Wong, G. G., Moreau, J., Stahl, M. and Rogers, D. (1988). Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336,688 -690.[CrossRef][Medline]
Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M.
A., Swiergiel, J. J., Marshall, V. S. and Jones, J. M.
(1998). Embryonic stem cell lines derived from human blastocysts.
Science 282,1145
-1147.
Wang, G., Zhang, H., Zhao, Y., Li, J., Cai, J., Wang, P., Meng, S., Feng, J., Miao, C., Ding, M. et al. (2005). Noggin and bFGF cooperate to maintain the pluripotency of human embryonic stem cells in the absence of feeder layers. Biochem. Biophys. Res. Commun. 330,934 -942.[CrossRef][Medline]
Williams, R. L., Hilton, D. J., Pease, S., Willson, T. A., Stewart, C. L., Gearing, D. P., Wagner, E. F., Metcalf, D., Nicola, N. A. and Gough, N. M. (1988). Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336,684 -687.[CrossRef][Medline]
Xu, C., Inokuma, M. S., Denham, J., Golds, K., Kundu, P., Gold, J. D. and Carpenter, M. K. (2001). Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19,971 -974.[CrossRef][Medline]
Xu, C., Rosler, E., Jiang, J., Lebkowski, J. S., Gold, J. D.,
O'Sullivan, C., Delavan-Boorsma, K., Mok, M., Bronstein, A. and Carpenter, M.
K. (2005). Basic fibroblast growth factor supports
undifferentiated human embryonic stem cell growth without conditioned medium.
Stem Cells 23,315
-323.
Xu, R. H., Chen, X., Li, D. S., Li, R., Addicks, G. C., Glennon, C., Zwaka, T. P. and Thomson, J. A. (2002). BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat. Biotechnol. 20,1261 -1264.[CrossRef][Medline]
Yang, R. Y. and Liu, F. T. (2003). Galectins in cell growth and apoptosis. Cell. Mol. Life Sci. 60,267 -276.[CrossRef][Medline]
Ying, Q. L., Nichols, J., Chambers, I. and Smith, A. (2003). BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115,281 -292.[CrossRef][Medline]
Zaehres, H., Lensch, M. W., Daheron, L., Stewart, S. A.,
Itskovitz-Eldor, J. and Daley, G. Q. (2005). High-efficiency
RNA interference in human embryonic stem cells. Stem
Cells 23,299
-305.
This article has been cited by other articles:
|
|
 |
|
  G. Cauffman, M. De Rycke, K. Sermon, I. Liebaers, and H. Van de Velde Markers that define stemness in ESC are unable to identify the totipotent cells in human preimplantation embryos Hum. Reprod., September 29, 2008; (2008) den351v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B.V. Johnson, N. Shindo, P.D. Rathjen, J. Rathjen, and R.A. Keough Understanding pluripotency--how embryonic stem cells keep their options open Mol. Hum. Reprod., September 1, 2008; 14(9): 513 - 520. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Yu and J. A. Thomson Pluripotent stem cell lines Genes & Dev., August 1, 2008; 22(15): 1987 - 1997. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Chen and J. S. Khillan Promotion of Feeder-Independent Self-Renewal of Embryonic Stem Cells by Retinol (Vitamin A) Stem Cells, July 1, 2008; 26(7): 1858 - 1864. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Mayshar, E. Rom, I. Chumakov, A. Kronman, A. Yayon, and N. Benvenisty Fibroblast Growth Factor 4 and Its Novel Splice Isoform Have Opposing Effects on the Maintenance of Human Embryonic Stem Cell Self-Renewal Stem Cells, March 1, 2008; 26(3): 767 - 774. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Yu, M. A. Vodyanik, K. Smuga-Otto, J. Antosiewicz-Bourget, J. L. Frane, S. Tian, J. Nie, G. A. Jonsdottir, V. Ruotti, R. Stewart, et al. Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells Science, December 21, 2007; 318(5858): 1917 - 1920. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Assou, T. Le Carrour, S. Tondeur, S. Strom, A. Gabelle, S. Marty, L. Nadal, V. Pantesco, T. Reme, J.-P. Hugnot, et al. A Meta-Analysis of Human Embryonic Stem Cells Transcriptome Integrated into a Web-Based Expression Atlas Stem Cells, April 1, 2007; 25(4): 961 - 973. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-y. Yasuda, N. Tsuneyoshi, T. Sumi, K. Hasegawa, T. Tada, N. Nakatsuji, and H. Suemori NANOG maintains self-renewal of primate ES cells in the absence of a feeder layer. Genes Cells, September 1, 2006; 11(9): 1115 - 1123. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||
