Download Regulation of human embryonic stem cell differentiation by BMP

Research Article
1269
Regulation of human embryonic stem cell
differentiation by BMP-2 and its antagonist noggin
Martin F. Pera1,*, Jessica Andrade1, Souheir Houssami1, Benjamin Reubinoff1,‡, Alan Trounson1,
Edouard G. Stanley1, Dorien Ward-van Oostwaard‡ and Christine Mummery2,‡
1Monash Institute of Reproduction and Development, Monash University, 246 Clayton Road, Clayton, Victoria
2The Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
3168, Australia
*Author for correspondence (e-mail: [email protected])
‡Present address: Hadassah University Hospital, 91120 Jerusalem, Israel
Accepted 5 November 2003
Journal of Cell Science 117, 1269-1280 Published by The Company of Biologists 2004
doi:10.1242/jcs.00970
Summary
Human embryonic stem cells differentiate spontaneously in
vitro into a range of cell types, and they frequently give rise
to cells with the properties of extra-embryonic endoderm.
We show here that endogenous signaling by bone
morphogenetic protein-2 controls the differentiation of
embryonic stem cells into this lineage. Treatment of
embryonic stem cell cultures with the bone morphogenetic
protein antagonist noggin blocks this form of
differentiation and induces the appearance of a novel cell
type that can give rise to neural precursors. These findings
indicate that bone morphogenetic protein-2 controls a key
Introduction
The development of human embryonic stem (ES) cells
(Reubinoff et al., 2000; Thomson et al., 1998) has opened up
exciting new opportunities for basic research and regenerative
medicine (reviewed by Pera et al., 2000). To exploit the
potential of human ES cells, it will be essential to understand
the molecular control of their growth and differentiation. Under
certain conditions in vitro, human ES cells differentiate
spontaneously into a wide range of somatic and extraembryonic cell types (Itskovitz-Eldor et al., 2000; Reubinoff et
al., 2000). It is logical to assume that ES cell differentiation in
vitro mimics in a chaotic way the inductive events seen in the
peri-implantation embryo in vivo. While our understanding
of these processes in mammals is still incomplete, studies
of mouse development have identified several secreted
polypeptide factors which mediate critical events in cellular
commitment and development (reviewed by Beddington and
Robertson, 1999).
Members of the transforming growth factor beta superfamily
play a prominent role in driving cell commitment events
in early development across the animal phyla. Bone
morphogenetic protein-2 (BMP-2) is a member of the
transforming growth factor beta superfamily implicated by
gene ablation studies in several critical processes in early
mouse development (Ying and Zhao, 2001b; Zhang and
Bradley, 1996). Studies of mouse ES cell differentiation in
vitro adduced evidence for a critical role for BMP-2 in the
differentiation of extra-embryonic endoderm, and in the
cavitation of the embryo, the process whereby programmed
cell death in a subpopulation of the pluripotent stem cells of
early commitment step in human embryonic stem cell
differentiation, and show that the conservation of
developmental mechanisms at the cellular level can be
exploited in this system – in this case, to provide a facile
route for the generation of neural precursors from
pluripotent cells.
Key words: Human embryonic stem cell, Noggin, Bone
morphogenetic protein, Extra-embryonic endoderm, Neural,
Differentiation
the inner cell mass leads to the formation of the egg cylinder
(Coucouvanis and Martin, 1999). This study further showed
that BMP-2 transcripts were expressed in the extra-embryonic
endoderm, a finding that has recently been confirmed in a study
which defined a role for this molecule in the induction of
primordial germ cells (Ying and Zhao, 2001b).
Before the development of human ES cells, human
embryonal carcinoma (EC) cells were used as a model to study
cell differentiation in early human development. Human EC
cells resemble primate ES cells in their morphology, surface
marker and gene expression, and growth properties (Pera et al.,
2000). A screen for factors affecting growth and differentiation
of human pluripotent EC cells revealed that BMP-2 induced
differentiation of the cells into a flattened, epithelial squamous
cell displaying an immunophenotype and gene expression
profile similar to extra-embryonic endoderm (Pera and
Herszfeld, 1998). Treatment with retinoic acid had previously
been shown to induce EC cells to undergo a very similar
program of differentiation (Roach et al., 1994), and cells with
similar morphology and marker expression commonly appear
in EC cell cultures undergoing spontaneous differentiation.
Recently, Xu et al. (Xu et al., 2002b) reported that BMP-4
induced the differentiation of human ES cells grown in serumfee medium in the presence of FGF-2 into a different extraembryonic lineage, the trophoblast.
Cells that morphologically resemble the flat epithelial
cells induced in human EC cultures by BMP-2 also appear
spontaneously in human ES cultures grown under standard
conditions and are different in appearance to the cells described
by Xu et al. (Xu et al., 2002b); under suboptimal conditions
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Journal of Cell Science 117 (7)
this differentiated cell grows rapidly and often overtakes the
culture, leading to the elimination of stem cells and other
differentiated progeny. We speculated that the commitment to
undertake primitive endoderm differentiation might be driven
by a positive feedback loop involving BMP-2, and that
modulation of this pathway might facilitate ES stem cell
renewal or differentiation into embryonic lineages, as
suggested by studies in the mouse (Lake et al., 2000; Niwa et
al., 2000). In this study we sought to characterize this form of
differentiation and assess the role of BMP-2 in directing ES
differentiation along this lineage.
Materials and Methods
Cell culture and treatment with growth and differentiation
factors
Human EC cells and ES cells were cultured as described in previous
publications (Pera et al., 1989; Reubinoff et al., 2000). All
experiments were carried out at least twice on cell lines HES-2 and
HES-3. The mouse embryo fibroblasts used throughout most of this
study came from two different lots, and they were plated at a density
of 7.5×104/cm2 for routine ES cell maintenance and experimental
protocols. With one lot of embryo fibroblasts, BMP-2 effects were
seen in ES cells plated onto feeder cell layers at this density, but with
the second lot, effects were only apparent when ES cells were cultured
on a feeder layer prepared at a lower density (see Results). Effects of
noggin were apparent using either lot of feeder cells at the density
normally employed for ES cell maintenance. Human EC cell
differentiation was induced by all-trans-retinoic acid or BMP-2
treatment as described elsewhere (Pera and Herszfeld, 1998; Roach et
al., 1994). Treatment of ES cells with noggin or bone morphogenetic
proteins was begun one day following routine subculture and
continued for 5-14 days. The medium used for conversion of noggin
monolayer cells to neural progenitors (neural progenitor medium) was
the same as that used previously to grow neural progenitors from
spontaneously differentiating ES cell cultures (Reubinoff et al., 2001).
Recombinant human BMP-2, BMP-4 and recombinant mouse
noggin were obtained from R&D Systems. Mammalian expression
plasmids containing DAN or Cerberus cDNA were described
elsewhere (Biben et al., 1998; Stanley et al., 1998). These proteins
were expressed in 293 T cells and purified by affinity chromatography
using a FLAG affinity column.
The coding sequences of BMP-2, BMP-4 and BMP-7 were
amplified from a mouse embryonic day 7 cDNA library using a PCRbased approach. This process yielded two fragments for each of BMP4 and -7, representing the pro-domain and cystine knot. For BMP-2,
only the cystine knot region was amplified. The primers used to
generate these fragments were: BMP-4 Pro-domain (CATGGCGCGCCTGATGATTCCTGGTAACCGAATG and ACGCGTCTTGGGACTACGTTTGGCCCT), BMP-7 Pro-domain (ACGCGTATGCACGTGCGCTCGCTGCGCGCTG and ACGCGTCCAAAGAACCAAGAGGCCCTG), BMP-2 cystine knot (GGGACGCGTAAGCGCCTCAAGTCCAGCTGC and CAACGCGTTGCTGTGCTAACGACACCCGCAG), BMP-4 cystine knot (CTGACGCGTAGGAAGAAGAATAAGAACTGC and TCCGCCCTCCGGACTGCCTGATCTC), BMP-7 cystine knot (ACGCGTCCAAAGAACCAAGAGGCCCTG
and
GACGCGTGAAGAGCTAGTGGCAGCCACAGG). MluI digested DNA fragments encompassing sequences
encoding the pro-domains of BMP-4 and BMP-7 were ligated into the
AscI site of pEFBOS (Mizushima and Nagata, 1990) that had been
previously modified to include myc or glu/glu epitope tags.
Recombinant plasmids resulting from this ligation contained the prodomains N-terminal to the epitope tag. These plasmids were subsequently digested with MluI and ligated to MluI fragments representing
the cystine knot regions of BMP4, 2 and 7 to yield a series of vectors
of the configuration BMP4-Pro-domain-myc-BMP-4, BMP-4-Pro-
domain-myc-BMP-2 and BMP-7-Pro-domain-glu-BMP-7. Western
blot analysis of supernatants from 293T cells transfected with these
vectors contained myc- and glu-tagged proteins of approximately 20
kDa (data not shown), the predicted size of processed monomeric
BMPs. These supernatants also possessed BMP activity as adjudged
by their ability to induce alkaline phosphatase activity in C2C12 cells
(Katagiri et al., 1994) (data not shown). To generate supernatants
containing BMP-2/-7 heterodimers, 293T cells were transfected with
the vectors BMP-4-Pro-domain-myc-BMP-2 and BMP-7-Prodomain-glu/glu-BMP-7, whereas supernatants containing BMP
homodimers were derived from cells transfected with only one of the
above expression vectors. Formation of heterodimers was confirmed
by affinity copurification of myc- and glu-tagged proteins and by the
much higher biological activity of the heterodimers in the C2C12
bioassay compared with homodimers. Purified follistatin (native
protein from follicular fluid) was obtained from David Phillips of this
Institute.
Noggin-treated cells were harvested after 10-14 days using dispase
and were further subcultured on monolayers of mouse embryo
fibroblasts, as described for human ES cells, or under similar
conditions in the absence of a feeder cell layer, or as neurospheres
(Reubinoff et al., 2001). To determine the proportion of noggin or
control ES cell cultures that could be converted to neural progenitors,
colonies were grown in control or noggin containing medium for 5
days, dissected into pieces approximately 0.5 µm in diameter and
transferred to neural progenitor medium. After 1 week growth in
suspension culture, the embryoid bodies or neurospheres were
replated as described previously (Reubinoff et al., 2001) on to
laminin-coated dishes in neural precursor medium lacking growth
factors, allowed to attach and grow for 2 days and then stained for
nestin. The proportion of colonies showing uniform positive staining
for nestin was determined by inspection and counting using indirect
immunofluorescence.
Gene expression
RNA isolation on magnetic oligo dT beads or on oligo dT Separose
was carried out as described elsewhere (Reubinoff et al., 2000; Roach
et al., 1994). RT-PCR was carried out as described previously
(Reubinoff et al., 2000). Product sizes, annealing temperatures and
primers for PCR reactions for all gene products examined in this study
are listed in Table 1. Thirty cycles of PCR were carried out for all
reactions. All products were sequenced, and identity with the expected
human cDNA was confirmed in all cases. All RT-PCR reactions were
repeated on three separate occasions with consistent results.
Northern analysis of human EC cell RNA for BMP-2 transcripts
was carried out as described (Roach et al., 1994) using a random
primed 32PcDNA probe consisting of a partial cDNA clone
corresponding to the region from 677-1333 bp of the cDNA sequence
for human BMP-2 (GI4557368).
Immunoblot analysis of BMP-2 expression
ES cell cultures 7-14 days old containing stem cells and differentiated
cells were extracted in organ culture dishes in situ for 30 minutes into
buffer containing 150 mM NaCl, 50mM Tris and 1% Nonidet P-40,
pH 8. The lysate, and the nuclei and cytoskeletal material detached
with it, were removed from the dishes, and the remaining proteins on
the monolayer were harvested into 100-200 µl of SDS-PAGE sample
buffer, and 20 µl was added to each lane of a gel. Four to six organ
culture dishes were used in each experiment. The samples were run
on 6% or 12% polyacrylamide gels under reducing and denaturing
conditions, and the proteins were transferred to Immobilon
membranes, which were probed with mouse anti-BMP-2 (12% gel)
or GCTM-2 (6% gel). Detection was carried out using rabbit antimouse immunoglobulin conjugated to horseradish peroxidase and
enhanced chemiluminescence.
BMP-2 and noggin effects on human ES cells
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Table 1. Primers used for RT-PCR
Gene
Oct-4
FoxD3
Cripto
AFP
Sox17
HNF3-α
HNF4
GATA4
GATA6
SPARC
Transferrin
Vitronectin
BMPR-IA
BMPR-2
Activin receptor IIB
Gremlin
Chordin
Noggin
Actin
Nestin
Pax6
Brachyury
OCT15:
OCT26:
GENF480:
GENR785:
CRIPTOF484:
CRIPTOR668:
AFPF736:
AFPR1173:
SOXF17:
SOXR198:
HNF3-α F1939:
HNF3-α R2328:
HNF4F:
HNF4R:
GATA4F:
GATA4R:
GATA6F:
GATA6R:
SPARCF:
SPARCR:
TRFF1197:
TRFR1765:
VNF34:
VNR336:
BMP2RIAF:
BMP2RIAR:
BMP2RIIF:
BMP2RIIR:
ActivinRIIBF:
ActivinRIIBR:
Grem259F:
Grem500R:
ChdF3:
ChdR3:
NOGF1029:
NOGR1059:
ACTINFOR:
ACTINBAC:
Nest856F:
Nest1064R:
PX6F1368:
PX6R1642:
BrachyF:
BrachyR:
CGT
ACA
GCA
CTG
CAG
GTA
CCA
CTC
CGC
GGA
GAG
GAG
GCT
CAG
CTC
AGT
GCC
TCA
CTG
CTG
CTG
CCA
TTG
TGT
GGA
CAG
TCC
AGT
CCG
CCG
AAT
TTC
AAC
CTG
CTC
GCA
CGC
TTC
CAG
AGG
AAC
CGG
GTG
TGT
TCT
CTC
GAA
TAA
AAC
GAA
TGT
CAA
ACG
TCA
TTT
GGC
TGG
GAG
TAC
GTG
TCA
GAT
CAG
CAG
ACC
TCA
CAG
TCA
CAT
ACC
TCT
TAC
GGA
CTC
ACC
TTG
ACA
TGG
GGG
CGA
ACC
TCC
CTG
GAA
AGA
GAA
ACC
TCC
Primer sequence
CTT TGG AAA GGT
GGA CCA CGT CTT
GAA GCT GAC CCT
GCG CCG AAG CTC
CTG CTG CCT GAA
ATG CCT GAG GAA
ACA TGA GCA CTG
TAA CTC CTG GTA
GAA TTT GAA CAG
GGG ACC TGT CAC
ACA GGC TTG TGG
AAT TCC TGA GGA
TTC TCG TTG AGT
CTT ATA GGG CTC
CAC AAG ATG AAC
CTC GTG CTG AAG
CTC CAC TCG TGT
CAG CCA CAC AAT
GGA GTG GAT TTA
ACC ATG AGG GCC
TCA CCT GGG ACA
AGG CAC AGC AAC
GCA CTC AGC TAG
TGG ACA GTG GCA
TGC TTT GCC ATC
CAC TAC CAG AAC
CAT CAG CCA TTT
TAC ACA TTC TTC
TCC TAC GGC CAT
GAG ATG CAG GTA
TGA AGC GAG ACT
GTA GGT GGC TGT
TGC TTC TTC GAG
TTC CCA GAG GTA
GGC CAC TAC GAC
GCA CTT GCA CTC
ACT GGC ATT GTC
TTG ATG TCA CGC
GCG CAC CTC AAG
GTT GGG CTC AGG
CAC AGC CCT CAC
CTT GAA CTG GAA
AAG AAC GGC AGG
GAT GAG CAT AGG
GTT
TC
GA
T
TG
ACG
TTG
TCC
TA
AC
CA
TT
GG
AGA C
GG
CT
ATG
GAT
TGG
AT
TC
AA
TT
ATA
TTT
GTC
ATA
GTG
TGA
GGT
AG
G
GTG
Product size (bp)
350
305
185
338
181
390
762
750
541
A
CAC AAG
ATC
700
367
300
424
CTT TC
G
GAC ATC CA
GAG GCC TCG TGA
457
407
241
900-1 kb (60°C)
488 (60°C)
G
AT
AC
ATG
ACT GG
AAA CA
CTG AC
AGG
GGC
200
208
274
706 (60°C)
The PCR conditions were as follows: 94°C/4 minutes, 94°C/1 minute, 55°C/1 minute (except where specified otherwise), 72°C/1
minute, 72°C/7 minutes.
Indirect immunofluorescence
The sources and methods for indirect immunofluorescence
microscopy with antibodies GCTM-2, anti-desmin and neural
markers, were as described previously (Reubinoff et al., 2000). The
same methods were used with these antibodies: TG343, reactive with
the surface proteoglycan recognized by GCTM-2 and TRA1-60, from
this laboratory (Cooper et al., 2002); TG42.1 reactive with a 25 kDa
surface protein found on stem cells (this antigen copurifies with the
GCTM-2 antigen and is identical to the tetraspannin CD9) (L. Stamp,
A. Laslett and M.F.P., unpublished); Oct-4, mouse monoclonal against
a human Oct-4 sequence from Santa Cruz; rabbit antiserum against
Sox-2 from Robin Lovell-Badge at the National Institute for Medical
Research, London; GCTM-5 reactive with a surface protein found on
differentiating cells of human ES and EC cultures and with fetal
hepatocytes, from this laboratory (L. Stamp, H. A. Crosby, S. M.
Hawes, A. J. Strain and M.F.P., unpublished); antibodies to Notch-1
and Notch-2, from the laboratory of Professor Spiros Artavanis; antihuman BMP-2 from R&D Systems; antibody PHM-4, reactive with
Class I HLA surface antigens, from the Department of Nephrology,
Monash Medical Centre (Hancock et al., 1982); mouse monoclonal
H17 against placental alkaline phosphatase from Jose Luis Millan at
the Burnham Institute, La Jolla Ca.; mouse monoclonal antibody
against SPARC from Hematologic Technologies, Essex, Vermont.
Assays for the beta subunit of human chorionic gonadotrophin were
performed as described elsewhere (Reubinoff et al., 2000).
Quantitative analysis for GCTM-2 staining in control, BMP-2 and
noggin-treated cultures was carried out by harvesting colonies first
with dispase, then dissociating them to single cells with trypsin. Cells
were stained in suspension using the primary antibody and anti-mouse
immunoglobulins conjugated to FITC, after which they were counted
under the fluorescence microscope to determine the proportion of
positive cells. Five hundred cells from each group were counted.
Alternately, when larger numbers of colonies were harvested, analysis
was carried out by flow cytometry (below).
For staining with antibodies to phosphorylated Smad1, ES cell
suspensions were plated onto glass coverslips coated with 0.1%
gelatin, grown overnight then transferred to ES medium supplemented
with 0.5% FCS for 4 hours, after which 50 ng/ml BMP-2 was added
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Journal of Cell Science 117 (7)
for 1.5 hours. Cells were fixed in 2% paraformaldehyde, rinsed three
times in PBS, permeabilized in 0.1% Triton, rinsed again, then
incubated in anti-phosphorylated-Smad1 (1:50) (Persson et al.,
1998)or GCTM-2 (1:10) in PBS with 4% normal goat serum for 1
hour at room temp. Secondary antibodies (goat anti-rabbit-cy3-IgG
1:250 and goat anti-mouse IgM-FITC 1:100, respectively) were added
for 1 hour after washing three times in PBS/0.05% Tween. Coverslips
were mounted in Mowiol and viewed in a confocal laser scanning
microscope.
Flow cytometry
Control cells or cells treated with 25 ng/ml BMP-2 were harvested
after 5 days of treatment following subculture. The cells were
carefully trypsinized to yield single cell suspensions, which were
incubated with monoclonal antibody GCTM-2 or an isotype-matched
control antibody on ice for 30 minutes, followed by several washes in
phosphate buffered saline, then a 30 minute incubation with antimouse immunoglobulins conjugated to fluorescein isothiocyanate.
The cells were rinsed with phosphate buffered saline and fixed in 0.4%
paraformaldehyde before analysis. Cells were also incubated with
isotype matched control primary antibody followed by secondary
antibody.
Results
BMP-2 treatment of human ES cells
BMP-2 treatment of human ES cell cultures grown in medium
containing fetal calf serum in the presence of a feeder cell layer
induced differentiation into flat, squamous epithelial cells (Fig.
1A, control; B, treated cells); these cells sometimes formed
fluid-filled cysts within the culture dish. They were very similar
in appearance to those commonly observed during spontaneous
differentiation of human ES cells (Fig. 1C). The appearance of
a sheet of spontaneously differentiating cells that was scraped
from the culture surface and processed using routine
histological methods is shown in Fig. 1D (BMP-treated cells
had a similar appearance). The flattened cells with abundant
eosinophilic intracellular material resembled cells found in the
primary yolk sac of the primate embryo and in certain
morphological variants of yolk sac carcinomas; in the presence
of BMP-2 the entire culture eventually took on this appearance.
By contrast, under optimal growth conditions, spontaneously
differentiating ES cell cultures grown in monolayer gave rise
not only to these cells but also to a wide range of additional
cell types.
Fig. 1. Spontaneous or BMP-2induced extra-embryonic
differentiation of human ES
cells. (A,B) Phase contrast
morphology of control (A) or
BMP-2 (B, 25 ng/ml) cells 7
days after treatment. A shows
typical stem cell morphology.
(C) Spontaneously
differentiating ES cell colony.
(D) Hematoxylin and eosin
stained section of cells similar
to those shown in C after
removal from culture dish.
(E,F) Phase contrast (E) and
indirect immunofluorescence
(F) image of BMP-2-treated
cells stained with antibody to
cytokeratins 8, 18 and 19.
(G,H) Phase contrast (G) or
indirect immunofluorescence
(H) micrographs of BMP-2treated cells stained with
antibody to laminin.
(I) Double-label staining of
BMP-2-treated cells stained
with GCTM-2 recognizing a
stem cell surface proteoglycan
(red) and SPARC (green).
(J,K) Phase contrast (J) or
indirect immunofluorescence
(K) micrographs of a cystic
structure and cells at its base
stained with antiserum to
alphafetoprotein. Wall of cyst
(at left) and cells at base (at
right) are stained. Bars,
A-C, 50 µM; D, 10 µM; E-K,
20 µM.
BMP-2 and noggin effects on human ES cells
Indirect immunofluorescence examination revealed that the
BMP-2-treated cells or spontaneously differentiating cells
resembling them expressed low molecular weight cytokeratins
and abundant extracellular matrix proteins (Fig. 1E,F,
cytokeratin; G,H, laminin; I, SPARC staining at an early
stage of differentiation). Particularly strong staining for
alphafetoprotein was observed in cystic cells and cells at the
base of cysts (Fig. 1J,K). These flat epithelial cells were not
stained with the ES stem cell antibody GCTM-2, nor were they
reactive with antibodies to class I major histocompatibility
complex (MHC) molecules. The effect of BMP-2 on the
expression of the stem cell marker GCTM-2 could be shown
quantitatively by flow cytometric analysis, with a lower
proportion of any cells showing staining after 5 days of BMP2 treatment compared with control cultures (negative cells
increased 1.6-fold relative to controls, Fig. 2).
Xu et al. (Xu et al., 2002b) recently showed that treatment
of the human ES cell line H-1 with BMP-4 induced
differentiation into trophoblast cells. Under our conditions of
treatment, very few cells expressing placental alkaline
phosphatase or human chorionic gonadotrophin were observed
in control or treated cultures, and the morphology of the BMP2- or BMP-4-treated cells was very different to that observed
by Xu et al. (data not shown). Nevertheless, we occasionally
observed foci of cells resembling the trophoblast precursors
described by these workers; these cells appeared in less than
5% of colonies treated with BMP-2, BMP-4 or BMP-2/-7
under our culture conditions.
Initially our experiments used BMP-2 only and were
performed with ES cells grown on a specific lot of mouse
embryo feeder cells. When another lot of feeder cells was used,
the effects of BMP-2 and other bone morphogenetic proteins
were minimal or nonexistent. Antagonism of the action of the
exogenous bone morphogenetic proteins appeared to account
for the lack of a BMP effect on ES cells cultured using the
second lot of feeder cells, as further examination showed
clearly that the outcome of treatment was entirely dependent
on feeder cell density. When the second batch of feeder cells
was used at the density employed for routine ES maintenance,
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the effect of BMP-2 was variable, but a threefold reduction in
feeder cell density revealed a strongly inhibitory effect of the
protein on stem cell maintenance (Fig. 3A; the experiment was
repeated three times on HES-2 and HES-3 with similar results
and each experiment analyzed duplicate wells containing at
least four colonies per treatment). Using this density of the
second lot of mouse embryo feeder cells, we observed that
other bone morphogenetic proteins, including BMP-4, BMP-7
and BMP-2/BMP-7 heterodimers, could induce the same effect
as BMP-2. To determine the possible mechanism whereby
feeder cell layers might inhibit the action of bone
morphogenetic proteins, we carried out RT-PCR analysis for
expression of known antagonists of BMP in this system.
Examination of embryo fibroblast feeder cells for transcripts
encoding known inhibitors of bone morphogenetic protein
revealed that the antagonist gremlin was transcribed in these
cells (Fig. 3B).
Analysis of gene expression in the BMP-2-treated cultures
consisting mainly of the flat epithelial cells by RT-PCR (Fig.
4A) showed reproducible loss of stem cell markers, and
upregulation of a range of markers characteristic of endoderm,
including transcription factors (HNF3-α, HNF-4, GATA-4 and
GATA-6) and genes encoding secreted products and
extracellular matrix molecules expressed in parietal endoderm
in the mouse and visceral endoderm in mouse and human.
RNA from stem cells and BMP-2-treated cells yielded similar
amounts of RT-PCR product for beta actin. Some transcripts
characteristic of endoderm differentiation were observed at
lower levels in control ES cell cultures, indicative of low levels
of spontaneous differentiation into this lineage.
Expression of BMP-2, its receptors and signal
transduction machinery in human ES and EC cell
cultures
The response of human ES cells to BMP-2, and the presence
of cells resembling those in BMP-2-treated cultures
in untreated ES cell cultures undergoing spontaneous
differentiation, suggested that endogenous BMP-2 production
Fig. 2. Flow cytometric analysis of GCTM-2 stem cell
surface proteoglycan staining in control cells and cells
treated for 5 days with 25 ng/ml BMP-2. Left panels
show side scatter versus forward scatter; middle panels
show histograms of cell counts versus fluorescence
intensity of cells stained with isotype matched control;
right panels show histograms of cell counts versus
fluorescence intensity of cells stained with antibody
GCTM-2 against stem cell surface proteoglycan.
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Journal of Cell Science 117 (7)
Fig. 3. Feeder cell antagonism of BMP action. (A) The effect of feeder cell density on the response of human ES cells to BMP-2. ES cells were
plated onto feeder cells prepared at a density of either 6.6×104/cm2 (high density) or 1.3×104/cm2 (low density) and were grown for 5 days
with or without treatment with 50 ng/ml BMP-2. The wells were then fixed and stained with monoclonal antibody GCTM-2 followed by
detection with anti-mouse immunoglobulin conjugated to alkaline phosphatase; red staining against blue counterstain indicates activity. (B) RTPCR analysis for transcripts of three BMP antagonists in ES cells, differentiating cultures of ES cells and mouse embryo fibroblasts. Gremlin
transcripts are strongly expressed in mouse embryo fibroblasts.
might be modulating the spontaneous differentiation observed.
We carried out RT-PCR analysis for BMP-2, and its receptors
BMPR1A, BMPR2 and the activin receptor II beta, and showed
expression of transcripts for all these genes in stem cells and
in cultures consisting predominantly of flat squamous
epithelial cells (Fig. 4B).
We speculated that BMP-2 production might be activated at
early stages of ES cell differentiation, driving a positive
feedback loop towards extra-embryonic differentiation. To
assess this possibility we studied BMP-2 expression in
differentiating cultures of human EC cells. Pluripotent human
EC cell line GCT 27X-1 resembles ES cells in morphology,
marker expression and growth requirements. However, EC
cells are easier to grow in large quantities as pure stem cell
population. Cell line GCT27X-1 was cultured in the absence
of a feeder cell layer and differentiation was induced by
treatment with all-trans retinoic acid or BMP-2 as described
previously (Pera and Herszfeld, 1998; Roach et al., 1994).
Spontaneously differentiating cultures (controls) and retinoic
acid treated cultures both show increased levels of transcripts
for BMP-2, but a very striking increase in these transcripts is
seen 12-48 hours after BMP-2 treatment (Fig. 4C). Thus,
treatment of human pluripotent cells with BMP-2 leads to the
accumulation of transcripts for this factor, consistent with a
positive feedback model.
We also determined whether or not BMP-2 protein was
present in ES cell cultures. Attempts to identify the protein in
culture supernatant or cell lysates prepared using nonionic
detergents failed. Following lysis of spontaneously
differentiating ES cell monolayers with nonionic detergent and
removal of the lysate, we extracted the remaining protein on
the monolayer with reducing SDS-PAGE sample buffer.
Immunoblotting of this material revealed the presence of BMP2 protein of the expected size, and a strong band corresponding
to a doublet (Fig. 4D); despite the use of reducing sample
buffer, it was difficult to convert all this material to monomeric
form. BMP-2 protein was also detected by immunostaining in
differentiating colonies of human ES cells (Fig. 4E-G, cells
grown in the absence of a feeder cell layer). Recent cell
biological (Suzawa et al., 1999) and genetic (Arteaga-Solis
et al., 2001) studies have highlighted the activity of bone
morphogenetic proteins bound to pericellular matrix.
Examination of feeder cell layer by RT-PCR or by
immunostaining failed to detect BMP-2 or BMP-4 transcripts,
or BMP-2 immunoreactivity (data not shown).
To determine whether BMP-2 addition to human ES cell
cultures resulted in activation of the Smad signal transduction
pathways, and to evaluate the extent to which this signaling
operated spontaneously in ES cells, we analyzed cells for the
presence of phosphorylated Smad-1 protein in the nucleus.
Before BMP2 treatment, phosphorylated Smad-1 staining was
found at low levels in stem cell nuclei (Fig. 5A-D), whereas
after BMP2 addition, much brighter staining was detectable
in the nuclei of undifferentiated cells (Fig. 5C-I). Thus, stem
cells appeared to activate the Smad-1 pathway under
basal conditions, and BMP-2 treatment enhanced nuclear
localization of this signal transduction molecule.
Results of noggin treatment of human ES cells
The results above suggested that stem cell maintenance might
be a dependent on a balance between expression of the BMP
antagonist gremlin by the fibroblast feeder cell layer and the
endogenous production of BMP-2 by the differentiating stem
cells. We postulated that by strongly interfering with
endogenous BMP-2 action, it would be possible to prevent the
differentiation of the cells into the extra-embryonic phenotype,
leading either to enhancement of stem cell renewal, or to
BMP-2 and noggin effects on human ES cells
1275
Fig. 4. Gene expression in spontaneously differentiating and BMP-2-treated ES cells, spontaneously differentiating and BMP-2- or retinoic
acid-treated EC cells, and noggin-treated ES cells. (A) RT-PCR for transcripts for stem cell markers (Oct-4, Cripto and FoxD3) or markers of
extra-embryonic endoderm differentiation (alphafetoprotein (AFP), HNF3-α, HNF-4, GATA-4, GATA-6, transferring (TRF), vitronectin (Vn)
and SPARC) and beta actin in control ES cells (C2, HES-2, and C3, HES-3) or cells treated with BMP-2 (B2, HES-2 treated with BMP; B3,
HES-3 treated with BMP) at 25 ng/ml for 5 days. Positive control for ES cell markers, human EC cell line GCT 27X-1 and for extra-embryonic
endoderm markers yolk sac carcinoma cell line GCT 72. (B) RTPCR for BMP-2 and BMPR1-A, BMPR-2, β-actin and activin receptor β in
undifferentiated control and spontaneously differentiating ES cell cultures. Controls are on the right, and differentiated cells are on the left, for
each PCR pair shown. (C) RNA blotting analysis for BMP-2 and glyceraldehyde-3 phosphate dehydrogenase transcripts in human EC cells at 0,
12, 24, 48, 96 hours and 7 days after plating in the absence of a feeder cell layer (controls) with or without treatment with BMP-2 or retinoic
acid. (D) Immunoblot analysis for BMP-2 in spontaneously differentiating ES cells. Tracks from left to right show 10 ng recombinant BMP-2,
25 ng recombinant BMP-2, black bars indicating the position of 19, 24 and 36 kDa marker standards, cell lysate. (E-G) Phase contrast (E)
image of differentiating ES cell colony showing staining by indirect immunofluourescence of BMP-2 (F) and GCTM-2 (G). Bar in E-G, 50 µM.
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Journal of Cell Science 117 (7)
Fig. 5. Smad1 phosphorylation induced by BMP
addition to hES cells. Undifferentiated hES cells
were deprived of fetal calf serum for 4 hours then
used as controls (A-D) or treated with BMP2 (50
ng/ml) for 1.5 hours (E-H). After fixing and
permeabilization, cells were stained with GCTM2
(green, A and E) and anti-PSmad1 (red, F).
Comparison of B and F and the overlays of
control and treated cells in C and G indicate that
BMP treatment leads to nuclear translocation of
Smad1. (I) Nuclear fluorescent intensity
quantified as pixel density in ten serial z-sections
in a confocal laser scanning microscope.
an enhancement of differentiation into
embryonic lineages. We tested several
natural and synthetic antagonists of BMP for
activity on human ES cell cultures.
Follistatin, DAN, and Cer-1 were without
any obvious morphological effect, as was a
soluble form of the BMPRIA ectodomain.
However, treatment with a recombinant form
of mouse noggin had a profound effect on
human ES cell morphology in the dose range
100-500 ng/ml. After approximately 5 days
in culture, noggin-treated cultures consisted
of colonies of small round cells, which were
different in appearance from ES cells, and,
in contrast to control cultures, colonies in
noggin-treated dishes contained no flat
squamous epithelial cells or cystic structures
similar to those seen after BMP- 2 treatment
(Fig. 6A-C). The size of the noggin-treated
colonies was smaller than those of controls,
although the same number of cells was
present in each (not shown). In noggintreated cultures, the percentage of cells
positive for the stem cell marker GCTM-2
was much lower than controls (Fig. 6D).
The immunophenotype of the noggintreated cells was distinguished by their
lack of expression of several markers
characteristic of ES cells or differentiated
cells found spontaneously at early time
points (approximately 5-7 days) following
ES cell subculture under standard
conditions (Table 2). Some markers of early
neural differentiation were found in noggintreated colonies.
RT-PCR analysis confirmed that the
noggin-treated cells expressed Oct-4
transcripts at low levels, if at all (Fig. 6E,
comparison of noggin-treated culture with
ES cells). The noggin-treated cells did
express
transcripts
for
markers
characteristic of early neuroectoderm such
as Pax-6 or nestin. The noggin cells did not
express brachyury, characteristic of early
mesoderm, and finally, they did not express
gene products characteristic of extraembryonic endoderm, which are found
in spontaneously differentiating control
cultures or in BMP-2-treated cultures.
Fig. 6. Effects of noggin on human ES cells.
(A) Area of differentiation in ES cell colony; (B)
noggin-treated cells. (C) Cells from a colony
similar to B after replating onto fresh feeder cell
layer. Bar, 50 µm. (D) Proportion of GCTM-2positive cells in human ES cultures after 5 days
of growth under control conditions or in the
presence of 200 ng/ml noggin. (E) RT-PCR
analysis of gene expression in human ES
cultures after 5 days of growth under control
conditions or in the presence of 200 ng/ml
noggin.
BMP-2 and noggin effects on human ES cells
Table 2. Expression of antigens in ES cells and noggintreated cells
Antibody
Transcription factors
Oct-4
Sox-2
Specificity
Human Oct-4
Sox-2
Stem cell surface markers
GCTM-2
Stem cell
protoeoglycan
TG343
Stem cell
protoeoglycan
TG42.1
CD9
Differentiated cell surface markers
GCTM-5
Embryonic liver
surface marker
PHM-4
HLA class I
UJ13A
Polysialylated N-CAM
Intermediate filament markers
Cam 5.2
Cytokeratin 8,18
AMF 17
Vimentin
Anti-desmin
Desmin
NF-68
Low molecular weight
neurofilament
NF-160
160 kDa neurofilament
protein
Control
Noggin
+++
+++
–
+++
+++
–
+++
–
+++
–
–
–
–
–
–
–
+
+
–
–
–
–
–
+
–
–
+++, expression in 80% of cells or more; +, expression in 10-20% of
cells; –, expression in less than 5% of cells
1277
reattached to the culture surface (Fig. 7A). By contrast, noggintreated cells formed spheres which remained floating in
suspension and could be serially cultivated (Fig. 7B). The fate
of either control ES cells or noggin-treated ES cells following
culture in serum-free medium was examined by allowing them
to reattach to a poly-L-lysine-coated surface, then staining
them with nestin (Fig. 7C,D). The proportion of structures
forming neurospheres after 5 days of noggin treatment varied
between 40-70%, but, in any given experiment it was at least
fivefold higher than that of untreated ES cells (Fig. 7E). Nestinnegative colonies from noggin-treated cells retained the
appearance of noggin cells grown on feeder layers, whereas
nestin-negative colonies from control cultures had a varied
appearance typical of mixed embryoid bodies allowed to
reattach to the culture surface. After 2 weeks of treatment, the
proportion of noggin-treated colonies that could be converted
to nestin-positive spheres rose to 90% or higher (B.R.,
unpublished; G. Peh, S. Hawes and M.F.P., unpublished).
When the spheres derived from noggin-treated cells were
allowed to attach to the dish, cells forming elongated processes
migrated out onto the monolayer (Fig. 8A). These cells
displayed an immunophenotype consistent with their
identification as mature neurons, including expression of 200
kDa neurofilament protein and MAP2-a,b (Fig. 8B-E).
However, if noggin-treated cells were cultivated as monolayers
in the absence of a feeder cell layer and in the presence of
serum, they gave rise to cultures consisting of cells with
fibroblastoid morphology. At least 50% of the cells in these
cultures stained with antibodies against glial fibrillary acidic
protein and vimentin (Fig. 8F,G).
The
noggin-treated
cells
were
further
characterized in biological studies. The addition of
25 ng/ml recombinant human BMP-2 to ES cell
cultures along with 250 ng/ml noggin led to the
appearance of squamous cells and cysts
characteristic of spontaneously differentiating ES
cell cultures or BMP-2-treated cultures, indicating
that BMP-2 could antagonize the noggin effect (not
shown). The noggin-treated cells could be
subcultivated under standard conditions for ES cell
culture in the presence of a feeder cell layer, and
retained their distinctive morphology under these
conditions for at least several passages (Fig. 6C). To
compare the neurogenic potential of control and
noggin-treated ES cells, colonies of either cell type
were dissected under microscopic control and
transferred to medium designed to support the
growth of neural stem cells (Fig. 7A-D). Control
cells formed embryoid bodies that displayed variable
morphology, were often cystic and frequently
Fig. 7. Differentiation of noggin cells into neural precursors. (A) Phase contrast micrograph
of control cells after transfer to neural progenitor medium; (B) phase contrast appearance of
noggin-treated cells after transfer to neural progenitor medium; (C) phase contrast
appearance of noggin-treated cells following transfer to neural progenitor medium and
attachment to culture surface; (D) same field as C stained with antibody to nestin. (E) Graph
showing proportion of control and noggin-treated cells forming nestin-positive colonies after
5 days of growth on a feeder cell layer in the absence or presence of 200 ng/ml noggin
followed by 1 week of growth in neural progenitor medium. Bars, A-D, 50 µM.
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Journal of Cell Science 117 (7)
Fig. 8. Neural derivatives of noggin-treated cells. (A) Outgrowth of cells from a sphere similar
to that shown in 7B; (B,C) staining of outgrowth similar to that shown in A with antibody to 200
kDa neurofilament protein (B, phase contrast; C, indirect immunfluorescence); (D,E) staining of
outgrowth similar to that shown in A with antibody to Map 2 a,b (D, phase contrast; E, indirect
immunofluorescence); (F,G) cells similar to those shown in Fig. 6C following transfer to
monolayer culture in the presence of serum without feeder cell support (F, phase contrast;
G, indirect immunofluorescence for glial fibrillary acidic protein). Bar shown in B for A-E,
50 µM.
Discussion
Several groups have now reported the spontaneous
differentiation of human ES cells (Itskovitz-Eldor et al., 2000;
Levenberg et al., 2002; Reubinoff et al., 2000; Schuldiner et
al., 2000; Xu et al., 2002a), and most of these have used
embryoid body formation to begin the initial process of
differentiation. ES cells allowed to differentiate on plastic
surfaces or embryoid bodies give rise to a mixture of cells, a
significant number of which express markers of extraembryonic endoderm. The regulation of this process of
spontaneous differentiation is poorly understood. Schuldiner et
al. (Schuldiner et al., 2000) showed that growth factor
treatment could influence some of the outcome of spontaneous
differentiation of ES cells but, like other studies to date, this
one did not directly elucidate the factors involved in
spontaneous differentiation. BMP-2 has a role in
visceral endoderm differentiation in the mouse
ES cell system, and it is expressed in the
extra-embryonic endoderm in this species
(Coucouvanis and Martin, 1999; Ying and Zhao,
2001a). We previously observed that BMP-2
could induce differentiation of human EC cells
into a cell with patterns of gene expression
resembling extra-embryonic endoderm (Pera and
Herszfeld, 1998).
Here we showed that BMP-2 has a similar
effect in human ES cell cultures, and that this
cell type is also seen during spontaneous
differentiation of ES cells. The identification of
these differentiated epithelial cells is based
chiefly on their patterns of gene expression
and on immunostaining. Thus, the cells are
epithelial cells expressing low molecular
weight cytokeratins, laminin, alpha-fetoprotein,
transferrin, SPARC and vitronectin but not class
I HLA molecules. Morphologically, the BMPtreated cells resemble a cell found in the human
peri-implantation embryo, which forms a meshlike network within the blastocoel cavity. This
cell was known classically in the human
embryology literature as a mesoblast, but on
comparative anatomical grounds Luckett
(Luckett, 1978) argued that these cells were
more likely to represent extra-embryonic
endoderm. Several further morphological studies
of the primate embryo have documented the
development of cells resembling parietal
endoderm and visceral endoderm from flattened
cells formed below the epiblast (Enders et al.,
1990; Enders and Schlafke, 1981; Enders et al.,
1986). Although detailed studies of marker
expression in these cells in the primate embryo have not been
carried out, the properties of the BMP-induced cell are
consistent with their identification as extra-embryonic
endoderm. Formation of this cell in vitro may provide a source
of multiple signals that influence human ES cell differentiation,
and controlling these signals may be critical to directing
differentiation of the ES cells themselves.
Xu et al. (Xu et al., 2002b) have shown that a different type
of extra-embryonic cell, the trophoblast, can be induced by
BMP-4 treatment of ES cells cultured in serum-free medium
in the presence of FGF-2 (Fig. 9). Although we have not
routinely observed cells of this phenotype in our experiments,
we have occasionally observed small foci of cells resembling
those shown in the study of Xu et al. (Xu et al., 2002b). It is
possible that differences between the cell lines studied by our
BMP-2 and noggin effects on human ES cells
Fig. 9. Early differentiation events in human ES cell cultures. ES
cells undergo spontaneous differentiation in a BMP-dependent
fashion to extra-embryonic tissues; the choice of extra-embryonic
endoderm or trophoblast (Xu et al., 2002b) depends on
environmental factors. Extra-embryonic tissues can produce factors
that either drive stem cell renewal or various differentiation
pathways. Gremlin produced in feeder cells partially offsets extraembryonic differentiation locally; addition of noggin to medium
completely inhibits this differentiation and allows the formation of
neural progenitor through a default mechanism. Lack of extraembryonic endoderm in noggin-treated cultures results in a loss of
signals for stem cell renewal and differentiation into other cell
lineages.
two groups could account for differences in response to these
factors. It is also possible that serum components influence the
outcome of differentiation, but it is interesting that activation
of the same signal transduction pathway can result in the
formation of either extra-embryonic endoderm or trophoblast.
It has been shown that mouse ES cells undergoing
differentiation can follow either of these pathways depending
on whether Oct-4 levels rise or fall (Niwa et al., 2000).
The data reported here strongly suggest that there is a
positive feedback loop involving BMP-2, which acts to
drive extra-embryonic endoderm differentiation of human
pluripotent stem cells. Our data show that BMP-2 can induce
its own expression in human EC cells. Both human EC and ES
cells, and the cells that differentiate from them, express
transcripts for receptors that enable them to respond to the
factor. Consistent with the RNA analyses, BMP-2 protein
is present in the pericellular matrix of spontaneously
differentiating ES cells. Data on nuclear localization of
phosphorylated Smad-1 show that under standard culture
conditions some nuclear staining is observed in ES cells; the
intensity of the nuclear staining is greatly increased following
treatment with BMP-2. All these data are consistent with an
endogenous signaling system based on BMP-2 that drives
extra-embryonic endoderm differentiation.
There are several natural and synthetic antagonists to the
BMPs. We showed that transcripts for the antagonist gremlin
are produced by mouse embryo fibroblasts, and previous
studies have shown that rat embryo fibroblasts produce gremlin
protein, much of which is cell associated and not soluble
(Topol et al., 2000). Gremlin may be one of a number of factors
produced by feeder cells that are required for stem cell renewal.
We speculate that high local concentrations of cell-associated
1279
gremlin or other feeder cell products may have a spatially
limited effect in blocking stem cell differentiation. We show
elsewhere (S. M. Hawes and M.F.P., unpublished) that the
zones of human ES cell colonies at some distance from the
feeder cell layer undergo differentiation first.
We hypothesize that stem cell fate may be determined in part
by a balance between feeder cell inhibition of differentiation,
mediated by gremlin, and by the production of BMP-2, which
drives differentiation. We tested the effect of the addition of
several BMP antagonists in the human ES cell system and
obtained a specific effect with noggin. This effect was evident
after a short period of treatment of ES cell colonies, and
appeared to affect most cells in the culture. Noggin is known
to play a role in the induction of the nervous system in several
vertebrate model systems by antagonizing BMPs (Bachiller et
al., 2000; Smith and Harland, 1992) (reviewed by Streit and
Stern, 1999). The noggin-treated cells can give rise to both
neuronal and glial lineages, but they themselves do not appear
to be equivalent to the neural progenitors that we and others
have previously derived from human ES cells. Noggin-derived
neural cells have properties expected of mature neurons and
glial cells, and the noggin neural progenitors have been shown
to engraft into the nervous system of experimental animals
and undergo appropriate differentiation (B.R., T. Ben-Hur, E.
Reinhartz, A. Itzik, M. Idelson and M.F.P., unpublished), as
shown previously for neural progenitors derived from
spontaneously differentiating ES cell cultures (Reubinoff et al.,
2001).
Because antagonism of BMP signaling blocks extraembryonic differentiation, it is interesting that noggin
treatment does not enhance stem cell renewal. One plausible
hypothesis to account for our results is that extra-embryonic
cells, in addition to secreting bone morphogenetic proteins and
other factors that can induce differentiation, also produce
factors that help maintain ES cells. In the mouse embryo, the
extra-embryonic endoderm produces a variety of factors that
act locally; some are postulated to induce ectoderm, others
mesoderm, and some are known to induce germ cell formation.
Thus, this tissue may have multiple effects on embryonic stem
cells. If the extra-embryonic factors are not present to maintain
stem cells, then the stem cells may default towards
neuroectoderm differentiation as depicted in the scheme shown
in Fig. 9. In this scheme, feeder cells produce gremlin or other
factors that act locally to maintain stem cell renewal but allow
for some degree of extra-embryonic differentiation. Complete
blockade of extra-embryonic differentiation by exogenous
noggin removes a source of factors that can support stem cell
renewal and factors that drive other differentiation pathways,
resulting in default differentiation into the neural lineage.
Further work will be required to determine the
developmental potential of the noggin cells isolated here. It
appears clear that they have neurogenic potential. By bypassing
extra-embyonic endoderm differentiation, it may be possible to
direct the fate of human ES cells into specific somatic lineages,
as suggested for mouse ES cells (Lake et al., 2000). The
results here indicate that BMP-controlled extra-embryonic
differentiation is an important regulatory node in ES
differentiation, show that addition of polypeptide regulators of
early mammalian development can direct the fate of human ES
cells, and provide a facile route to the generation of neural
precursors from ES cells.
1280
Journal of Cell Science 117 (7)
At Monash University, this work was supported by grants from the
National Health and Medical Research Council, and the National
Institutes of Health (NIGMS GM68417), and by a sponsored research
agreement with ES Cell International Pte. We gratefully acknowledge
the expert assistance of Jacqui Johnson, Irene Tellis, Karen Koh and
Linh Nguyen with human ES cell culture. We thank Peter ten Dijke
for the antibody to phosphorylated Smad-1and Leon Tertoolen for
help with the confocal microscopy.
References
Arteaga-Solis, E., Gayraud, B., Lee, S. Y., Shum, L., Sakai, L. and
Ramirez, F. (2001). Regulation of limb patterning by extracellular
microfibrils. J. Cell Biol. 154, 275-281.
Bachiller, D., Klingensmith, J., Kemp, C., Belo, J. A., Anderson, R. M.,
May, S. R., McMahon, J. A., McMahon, A. P., Harland, R. M., Rossant,
J. et al. (2000). The organizer factors Chordin and Noggin are required for
mouse forebrain development. Nature 403, 658-661.
Beddington, R. S. and Robertson, E. J. (1999). Axis development and early
asymmetry in mammals. Cell 96, 195-209.
Biben, C., Stanley, E., Fabri, L., Kotecha, S., Rhinn, M., Drinkwater, C.,
Lah, M., Wang, C. C., Nash, A., Hilton, D. et al. (1998). Murine cerberus
homologue mCer-1: a candidate anterior patterning molecule. Dev. Biol.
194, 135-151.
Cooper, S., Bennett, W., Andrade, J., Reubinoff, B. E., Thomson, J. and
Pera, M. F. (2002). Biochemical properties of a keratan sulphate/
chondroitin sulphate proteoglycan expressed in primate pluripotent stem
cells. J. Anat. 200, 259-265.
Coucouvanis, E. and Martin, G. R. (1999). BMP signaling plays a role in
visceral endoderm differentiation and cavitation in the early mouse embryo.
Development 126, 535-546.
Enders, A. C., Lantz, K. C. and Schlafke, S. (1990). Differentiation of the
inner cell mass of the baboon blastocyst. Anat. Rec. 226, 237-248.
Enders, A. C. and Schlafke, S. (1981). Differentiation of the Blastocyst of
the rhesus monkey. Am. J. Anat. 162, 1-21.
Enders, A. C., Schlafke, S. and Hendrickx, A. G. (1986). Differentiation of
the embryonic disc, amnion, and yolk sac in the rhesus monkey. Am. J. Anat.
177, 161-185.
Hancock, W. W., Kraft, N. and Atkins, R. C. (1982). The
immunohistochemical demonstration of major histocompatibility
antigens in the human kidney using monoclonal antibodies. Pathology 14,
409-414.
Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A., Yanuka, O.,
Amit, M., Soreq, H. and Benvenisty, N. (2000). Differentiation of human
embryonic stem cells into embryoid bodies compromising the three
embryonic germ layers. Mol. Med. 6, 88-95.
Katagiri, T., Yamaguchi, A., Komaki, M., Abe, E., Takahashi, N., Ikeda,
T., Rosen, V., Wozney, J. M., Fujisawa-Sehara, A. and Suda, T.
(1994). Bone morphogenetic protein-2 converts the differentiation
pathway of C2C12 myoblasts into the osteoblast lineage. J. Cell Biol. 127,
1755-1766.
Lake, J., Rathjen, J., Remiszewski, J. and Rathjen, P. D. (2000). Reversible
programming of pluripotent cell differentiation. J. Cell Sci. 113, 555-566.
Levenberg, S., Golub, J. S., Amit, M., Itskovitz-Eldor, J. and Langer, R.
(2002). Endothelial cells derived from human embryonic stem cells. Proc.
Natl. Acad. Sci. USA 99, 4391-4396.
Luckett, W. P. (1978). Origin and differentiation of the yolk sac and
extraembryonic mesoderm in presomite human and rhesus monkey
embryos. Am. J. Anat. 152, 59-97.
Mizushima, S. and Nagata, S. (1990). pEF-BOS, a powerful mammalian
expression vector. Nucleic Acids Res. 18, 5322.
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.
Pera, M. F., Cooper, S., Mills, J. and Parrington, J. M. (1989). Isolation
and characterization of a multipotent clone of human embryonal carcinoma
cells. Differentiation 42, 10-23.
Pera, M. F. and Herszfeld, D. (1998). Differentiation of human pluripotent
teratocarcinoma stem cells induced by bone morphogenetic protein-2.
Reprod. Fertil. Dev. 10, 551-555.
Pera, M. F., Reubinoff, B. and Trounson, A. (2000). Human embryonic stem
cells. J. Cell Sci. 113, 5-10.
Persson, U., Izumi, H., Souchelnytskyi, S., Itoh, S., Grimsby, S., Engstrom,
U., Heldin, C. H., Funa, K. and ten Dijke, P. (1998). The L45 loop in type
I receptors for TGF-beta family members is a critical determinant in
specifying Smad isoform activation. FEBS Lett. 434, 83-87.
Reubinoff, B. E., Itsykson, P., Turetsky, T., Pera, M. F., Reinhartz, E., Itzik,
A. and Ben-Hur, T. (2001). Neural progenitors from human embryonic
stem cells. Nat. Biotechnol. 19, 1134-1140.
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.
Roach, S., Schmid, W. and Pera, M. F. (1994). Hepatocytic transcription
factor expression in human embryonal carcinoma and yolk sac carcinoma
cell lines: expression of HNF-3 alpha in models of early endodermal cell
differentiation. Exp. Cell Res. 215, 189-198.
Schuldiner, M., Yanuka, O., Itskovitz-Eldor, J., Melton, D. A. and
Benvenisty, N. (2000). From the cover: effects of eight growth factors on
the differentiation of cells derived from human embryonic stem cells. Proc.
Natl. Acad. Sci. USA 97, 11307-11312.
Smith, W. C. and Harland, R. M. (1992). Expression cloning of noggin, a
new dorsalizing factor localized to the Spemann organizer in Xenopus
embryos. Cell 70, 829-840.
Stanley, E., Biben, C., Kotecha, S., Fabri, L., Tajbakhsh, S., Wang, C. C.,
Hatzistavrou, T., Roberts, B., Drinkwater, C., Lah, M. et al. (1998). DAN
is a secreted glycoprotein related to Xenopus cerberus. Mech. Dev. 77, 173184.
Streit, A. and Stern, C. D. (1999). Neural induction. A bird’s eye view. Trends
Genet. 15, 20-24.
Suzawa, M., Takeuchi, Y., Fukumoto, S., Kato, S., Ueno, N., Miyazono,
K., Matsumoto, T. and Fujita, T. (1999). Extracellular matrix-associated
bone morphogenetic proteins are essential for differentiation of murine
osteoblastic cells in vitro. Endocrinology 140, 2125-2133.
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.
Topol, L. Z., Bardot, B., Zhang, Q., Resau, J., Huillard, E., Marx, M.,
Calothy, G. and Blair, D. G. (2000). Biosynthesis, post-translation
modification, and functional characterization of Drm/Gremlin. J. Biol.
Chem. 275, 8785-8793.
Xu, C., Police, S., Rao, N. and Carpenter, M. K. (2002a). Characterization
and enrichment of cardiomyocytes derived from human embryonic stem
cells. Circ. Res. 91, 501-508.
Xu, R. H., Chen, X., Li, D. S., Li, R., Addicks, G. C., Glennon, C., Zwaka,
T. P. and Thomson, J. A. (2002b). BMP4 initiates human embryonic stem
cell differentiation to trophoblast. Nat. Biotechnol. 20, 1261-1264.
Ying, Y. and Zhao, G. Q. (2001a). Cooperation of endoderm-derived bmp2
and extraembryonic ectoderm-derived bmp4 in primordial germ cell
generation in the mouse. Dev. Biol. 232, 484-492.
Ying, Y. and Zhao, G. Q. (2001b). Cooperation of endoderm-derived BMP2
and extraembryonic ectoderm-derived BMP4 in primordial germ cell
generation in the mouse. Dev. Biol. 232, 484-492.
Zhang, H. and Bradley, A. (1996). Mice deficient for BMP2 are nonviable
and have defects in amnion/chorion and cardiac development. Development
122, 2977-2986.