Bone morphogenetic protein-4 affects both trophoblast and non-trophoblast lineage-associated gene expression in human embryonic stem cells

Human embryonic stem cells (hESC) can be induced to differentiate to trophoblast by bone morphogenetic proteins (BMPs) and by aggregation to form embryoid bodies (EB), but there are many differences and controversies regarding the nature of the differentiated cells. Our goals herein were to determine if BG02 cells form trophoblast-like cells (a) in the presence of BMP4-plus-basic fibroblast growth factor (FGF-2) and (b) upon EB formation, and (c) whether the BMP4 antagonist noggin elicits direct effects on gene expression and hormone production in the cells. Transcriptome profiling of hESC incubated with BMP4/FGF-2 showed a down-regulation of pluripotency-associated genes, an up-regulation of trophoblast-associated genes, and either a down-regulation or no change in gene expression for many markers of the three embryonic germ layers. Yet, there was up-regulation of several genes associated with mesoderm, ectoderm, and endoderm, strongly suggesting that differentiation to trophoblast-like cells under the conditions used does not yield a homogeneous cell type. Several genes, heretofore unreported, were identified that are altered in hESC in response to BMP4-mediated differentiation. The production of human chorionic gonadotropin (hCG), progesterone, and estradiol in the differentiated cells confirmed that trophoblast-like cells were obtained. Gene expression by EB was characterized by an up-regulation of a number of genes associated with trophoblast, ectoderm, endoderm, and mesoderm, and the production of hCG and progesterone confirmed that trophoblast-like cells were formed. These results suggest that, in the presence of FGF-2, BG02 cells respond to BMP4 to yield trophoblast-like cells, which are also obtained upon EB formation. Thus, BMP4-mediated differentiation of hESC represents a viable cell system for studying early developmental events post-implantation; however, up-regulation of non-trophoblast genes suggests a somewhat diverse response to BMP4/FGF-2. Noggin altered the transcription of a limited number of genes but, not surprisingly, did not lead to secretion of hormones.


INTRODUCTION
In the human blastocyst, the first step of differentiation from the morula, composed of totipotent cells, yields the inner cell mass and trophoblast.The former differentiates into the hypoblast, leading to the extraembryonic endoderm and the epiblast, that differentiates to give the amniotic ectoderm and the primitive ectoderm.The primitive ectoderm, in turn, differentiates into the embryonic ectoderm and the primitive streak, the latter giving rise to extraembryonic mesoderm, primitive mesoderm, and embryonic endoderm.The trophectoderm is composed of epithelial cells and differentiates into several lineages.The trophoblast precursor cells begin rapid proliferation into cytotrophoblasts, with some fusing to form multinucleate syncytiotrophoblasts, a terminally differentiated trophoblast cell; in addition, villous and extravillous cytotrophoblasts are formed [1][2][3].One of the hallmarks of trophoblast differentiation is the production of the heterodimeric glycoprotein hormone, human chorionic gonadotropin (hCG), that is secreted throughout gestation, being essential for progesterone production by the corpus luteum in the first trimester of human pregnancy [4,5].There is a paucity of adequate cell models for studying early trophoblast development, and a reliable system will greatly facilitate progress in the area of human reproduction.
These cellular models for trophectoderm differentiation begin to fill a long-awaited need for new systems to study early development, particularly in view of the major differences between human and mouse trophoblast [35][36][37].Yet, the characteristics of the human trophoblast cells obtained by BMP-mediated differentiation vary, sometimes quite significantly, depending upon the cell type, culture conditions (including the presence or absence of growth factors, particularly FGF2), and mode of differentiation [10,18,38].Moreover, in a number of instances there are considerable differences and controversies regarding the product(s) of differentiation obtained with BMP.For example, BMP4-mediated differentiation was found to yield little evidence of trophoblast-like cells under standard culture conditions [38].Another study found that 14 genes were highly up-regulated as determined by microarrays, in addition to many others [21], while another report did not identify six of these particular genes [17].Also, differences in cell morphology and gene markers have been noted in BMP-treated hESCs [18,21].Explanations for many of these controversial reports were recently provided by two groups.Yu et al. [22] reported that bFGF (FGF-2), which acts via the MEK-ERK pathway, redirects BMP4-mediated differentiation from trophectoderm to mesendoderm as judged by the expression of brachyury.The activated MEK-ERK pathway sustains NANOG expression leading to a bFGF-independent induction of mesendoderm by BMP4.Another recent paper concluded that in the cooperative presence of FGF2, BMP4 (that acts through brachyury and CDX2) leads to the induction of mesoderm, not trophoblast [39].
The present study was undertaken with the goals of: 1) extending the previous reports that were based on a variety of cell lines, different culture conditions, and at times conflicting results, and 2) to critically examine the trophoblast and non-trophoblast BMP-regulated genes, particularly since a number of the non-trophoblast BMPregulated genes have been noted in the earlier studies.We chose to use the hESC line, BG02 (karyotype 46, XY), that has not been thoroughly studied in hESC differentiation to trophoblast.This cell line was established from a 6-day embryo with an embryo grade of 3CC [7].Herein, BMP4, in the presence of FGF2, was incubated with adherent colonies of BG02 cells to initiate trophoblast differentiation.Identical studies were also done in the presence of the BMP4 antagonist noggin, since it has been shown that noggin alters hESC gene expression and morphology, perhaps leading to differentiation toward early neuroectoderm [18].In addition, trophoblast differentiation was initiated by formation of EBs.Particular emphasis was placed on quantitative profiling of a variety of genes via qRT-PCR, and measurements were made to determine hCG and steroid hormone production by the cells and EBs.Our results have many similarities with the findings of others, including for example the down-regulation of pluripotency genes, the up-regulation of trophoblast genes, and placental hormone production, but there are also notable differences, particularly with the up-regulation of certain genes associated with the three primary germ layers.

BMP4-Mediated Differentiation
The cells were passaged by gentle enzymatic digestion using cell dissociation buffer (Gibco) into 10 cm Matrigel-coated dishes (BD Bioscience, Boca Raton, FL).The BG02 cells were cultured in 50% DMEM/F12 medium that was conditioned by MEF layers [21] and then supplemented with 4 ng/mL FGF2.Experimental groups included incubation with 100 ng/mL BMP-4 (Quest Diagnostics, Lyndberg, NJ) and with 250 ng/mL noggin (Quest Diagnostics), with untreated hESC serving as controls.The medium was collected each day for analysis of secreted hormones.On day 7 the cells were harvested and quick-frozen for RNA extraction

Formation of Embryoid Bodies
Colonies were sliced into small pieces, then removed gently from the MEF layer where they were allowed to aggregate randomly in suspension on agarose plates.EBs were grown on agarose dishes in 12 mL of the hESC medium described above.Each culture began with ~50 EBs and ended with ~12 EBs of varying sizes.The loss of EB was attributed to aggregation of the individual units and/or to atresia/necrosis.On alternate days the culture plates were swirled to aggregate the EB with 6 mL of medium being removed and frozen.The same volume of fresh medium was added with the EBs then dispersed to prevent clumping.

Hormone Assays
Media collected from cell cultures and EBs were analyzed for secretion of three placental hormones namely hCG (the assay recognizes both heterodimer and hCGβ), progesterone, and estradiol using immunofluorescencebased assays.The hormones were measured with an Immulite 1000 with a tri-level internal control in human serum, Con6, being used to standardize all kits (Diagnostic Product Corporation, Los Angeles, CA).

qRT-PCR
Total RNA was isolated from BG02 (day 0, i.e. control, and incubated for 7 days) and from EBs at days 5, 22, and 50 of culture.hESCs were resuspended in 1 mL Trizol (Invitrogen, Carlsbad, CA) and triturated until homogenized.The integrity of isolated RNA isolated from the homogenates (Trizol, Molecular Research Corporation, Albany, NY) was verified and quantified using a RNA 600 Nano Assay (Agilent Technologies, Foster City, CA) and the Agilent 2100 Bioanalyzer.The cDNA Archive Kit (Applied Biosystems, Inc., Foster City, CA) was used to reverse transcribe 5 μg total RNA with the MultiScribe Reverse Transcriptase.Initially, reactions were incubated at 25˚C for 10 min and subsequently at 37˚C for 120 min.Quantitative PCR (Taqman) assays were selected for the transcripts to be evaluated from Assays-On-Demand (Applied Biosystems, Inc.) and incorporated into 384-well Micro-Fluidic Cards.The cDNA samples and 50 μl of GeneAmp Fast PCR master mix (2×) (Applied Biosystems, Inc.) were loaded in duplicate into respective channels on each microfluidic card and briefly centrifuged.
qRT-PCR and relative quantification were performed with the ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Inc.), with the expression levels of all genes analyzed given relative to 18S rRNA expression.The results for differential expression between the treated and control samples were expressed as means and data with a C t value greater than 35 were not analyzed.The qRT-PCR data on gene expression of BMP4-treated and noggin-treated hESCs on day 7 of culture were analyzed as ddC t relative to day 0, i.e. undifferentiated hESCs.Data were collected for EBs at days 5, 22, and 50 of culture, in all cases using 18S rRNA as the internal reference gene.The values of ddC t on days 22 and 50 are expressed relative to day 5.In the equations below, std = internal gene standard (18S rRNA), goi = gene of interest under experimental conditions, and con = gene of interest under control conditions.It is assumed that the amplifycation efficiency is identical under all conditions to give the normalized fold-change of the mRNA of interest in treated cells (experimental) relative to that of control cells [40,41].
As defined, ddC t is negative if the gene of interest under experimental conditions, i.e.BG02 cells plus BMP4 or plus noggin on day 7, is expressed at a higher level than the same gene under control conditions, i.e. untreated cells at day 0.

Data Analysis
Most of the PCR experiments were performed in trip-licate, although in a few cases n was 2, 4, 5, or 6.Hormone measurements were done in triplicate, and the results are given as mean ± SEM.Data exceeding a foldchange of 2.0 (up-regulated) or -2.0 (down-regulated) were analyzed using a one-way analysis of variance (ANOVA) with the GraphPad Prism software.Results are given for changes with P < 0.05 and P < 0.10.Although the mean fold-change was large in some cases, significance was at times not reached, often due to n = 2 or to one value in three being particularly different from the others.Outliers were identified using the Grubbs' test (on-line GraphPad software).From over 400 hormone measurements, only about 1% was deemed outliers, and from over 700 qRT-PCR runs, only six and seven outliers were identified in the cell and EB data, respectively.

Effects of BMP4 and Noggin on Gene Expression and Hormone Production in hESC
Gene expression: The selection of 177 genes to investigate via qRT-PCR was based on several criteria.For example, it was important to include many that were established markers of pluripotency, trophoblast, ectoderm, endoderm, and mesoderm.In addition to these standard marker genes, others were also screened.Included in this list were genes encoding certain steroidogenic enzymes even if not specific for trophoblast and placenta, e.g.cytochrome P450 side-chain cleavage enzyme (CYP11A1) and aromatase (CYP19A1).Other genes encoding proteins for extracellular matrix and various aspects of cell function were monitored.
Genes highlighted above that were altered similarly by both BMP4 and noggin, albeit in some cases to a greater or lesser degree, include, up-regulation: AKT1, CDH5, CEBPG, IL6, TBX5, VCAM1; and down-regulation: GREM1 and SOX3.These results may imply non-specific action of BMP4 and/or direct effects of noggin.
Of the genes surveyed, 31 were not expressed sufficiently to be accurately determined and hence could not be included in Figure 1.These genes are listed in the Supplement (Figure S1).
Phase-contrast microscopic images of the hESCs are given in the Supplement (Figure S1) showing control cells and cells incubated with BMP4 and with noggin.Consistent with the transcriptome profiling results, heterogeneity is apparent, but the BMP4-treated cells give more the appearance of syncytiotrophoblast while the noggin-treated cells are quite distinct.
Hormone production: The media concentrations of hCG, progesterone, and estradiol were measured daily, up to seven days, for cells incubated with BMP4 and with noggin (Figure 2).Following incubation with BMP4, the three hormones increased on days 6 and 7 relative to control cells.As expected, noggin itself had no effect on hormone production.The concentrations reached on day 7 with BMP4 are about 15, 10, and 0.6 ng/mL for hCG, progesterone, and estradiol, respectively.

Gene Expression and Hormone
Production in EB Gene expression: Expression of 74 genes by EBs is presented as a heatmap (derived from mean values of ) in Figure 3 at 22 and 50 days, each relative to day 5.Some of the more prominently expressed genes up-regulated at day 22 and/or day 50 include AFP, CGB, CHRD, DAB2, HSPG2, INHBA, PGF, PTGS2, RUNX1, TIMP1, and VEGF, only a few of which are trophoblast-associated.Most of the genes shown in Figure 3 are, however, either down-regulated or exhibit no appreciable change over time.Of the genes surveyed, an arbitrary selection of the 15 most highly expressed at day 5, as assessed by dC t values, are (in decreasing order):  Considerable variability was noted in the hormone concentrations, attributed to the different sizes and numbers of EBs in each agarose dish.Moreover, viability may be decreasing after extended culture.hCG begins increasing on about day 18 and reaches a maximum on days 32 -36 days.Under the conditions of the assay, where 50% of the EB medium is replaced every two days to ensure EB viability, the concentrations measured reflect new synthesis to a large extent, and, to a lesser degree, accumulation.Assuming that hCG is stable in the medium, it is possible to correct the concentrations for the total accumulated values at each two days of measurement.Doing so shows that hCG is continually synthesized between days 20 -40 and then reaches a plateau of about 660 ng/mL between days 40 -50.Media concentrations of progesterone follow a similar pattern to that of hCG Figure 4(b) and, when concentrations were corrected as described above, reach a plateau of about 1 ng/mL between days 40 -50.The concentrations of estradiol were also measured (data not shown) and, when corrected as per hCG and progesterone, a maximal concentration of only about 0.04 ± 0.01 ng/mL was achieved.In view of the dynamic nature of EB size and number, quantification of hormone data is precluded.

BG02 Cells
This study has shown that, in the presence of FGF2, BMP4 leads to differentiation of the hESC line, BG02, to trophoblast-like cells, as has been reported by several groups using different cell lines, e.g.H1, H7, H9, H14, HES-2, HES-3 and various culture conditions (cf.[17,19,21,27]).We have also identified BMP4-mediated and specific up-regulation of the genes AKT1, CEBPB, RUNX2, SOX9, and VEGF.Of these, Xu et al. [21] reported only minimal and non-significant changes in AKT1 and CEBPB.These genes most likely reflect BMP4 signaling and are not specific to trophoblast, although VEGF is expected to become important in placental formation and development.AKT1 encodes an isoform of serine/threonine kinase B (PKBα) involved in cell survival, proliferation, metabolism, and angiogenesis.Its regulation by BMP4 and noggin may imply that the BMP4-regulated component may be via a non-canonical pathway.The intronless gene CEBPB encodes the bZIP transcriptional factor, CCAAT/enhancer binding pro-  days.The variability at each day is attributable to the dynamic nature of EB, in particular the changing size, number, and possibly atresia and necrosis.As discussed in the text, the conditions are such that new hormone synthesis and secretion is the primary contributor with accumulated prior secretion contributing some as well.A correction for the total accumulated hCG and progesterone, assuming complete stability in the medium, shows that the majority of synthesis occurs between days 20 -40, after which plateaus of about 660 ng/mL and 1 ng/mL, respectively, are reached.The concentrations of estradiol were also measured and found to be quite low, about 0.04 ± 0.01 ng/mL (data not shown).
tein-b, that forms homodimers or heterodimers with other members of the CEBP family, a, d, and g.RUNX2 is regulated by the BMP pathway and also encodes a transcriptional factor involved in osteogenesis.Another transcriptional factor, (sex determining region Y)-box 9, is encoded by SOX9 and participates in chondrogenesis.Vascular endothelial growth factor, a member of the cystine-knot growth factor family and encoded by VEGF, stimulates vasculogenesis and angiogenesis and is expected to function in placental development.The results reported herein demonstrate an effect of noggin on the expression of a number of genes.In some cases BMP4 and noggin alter gene expression similarly, suggesting either that the regulation by BMP4 may not be via a canonical signaling pathway or that BMP4 and noggin may alter transcription similarly.At this time it is not possible to relate the observed changes accompanying treatment with noggin with the early signs of neural differentiation noted by Pera et al. [18].Xu et al. [21] first reported that, in the presence of FGF2, BMP2, BMP4, and BMP7 promoted differentiation of H1, H7, H9, and H14 cells, cultured in mouse embryonic fibroblast conditioned medium, into trophoblast cells.Later, others found that the induction of trophoblast by BMP4 required an inhibition of the Activin/Nodal signaling [43].The seminal study by Xu et al.  [21] was followed by other reports confirming BMP-me- diated hESC differentiation to trophoblast-like cells.Das et al. [44], for example, also showed that BMP4 directed H1 and H9 cells to trophoblast, while FGF2 slowed this differentiation, with oxygen accelerating it and promoting formation of syncytiotrophoblast.Interestingly, it was found that FGF2, acting to maintain NANOG levels via the MEK-ERK pathway, is capable of switching BMP4- mediated differentiation of hESC to mesendoderm as documented by the expression of brachyury and other primitive streak markers [22].A similar conclusion was reached by Bernardo et al. [39] who showed that differentiation of hESCs by BMP4 in the presence of FGF2 led to formation of mesoderm and inhibition of endoderm.They also reported that this differentiation was via the ERK pathway and mediated by brachyury and CDX2.
In a recent comprehensive study of BMP-mediated differentiation of hESC to trophoblast, Marchand et al.  [17] performed microarray analysis using the Affymetrix Human Gene version 1.0 ST array, along with qRT-PCR on selected genes.H7 and H9 cells were incubated with BMP4 for various times, 0, 2, 4, 6, 8, and 10 days, following the removal of FGF2.They found that after 2 days POU5F1 and NANOG were dramatically downregulated while trophoblast markers were up-regulated.Many new genes were identified and suggested to be involved in trophoblast formation, and pathway analysis provided considerable insight into the myriad signaling systems operative in the differentiation of hESCs to trophoblast.This study was augmented by another report [42] in which the transcriptome of trophectoderm cells obtained from 13 human blastocysts were compared with those of BMP4-mediated differentiation of hESCs [17].Their results documented that BMP4-induced differentiation of hESCs offers a good model for studying trophoblasts and contributed significantly to a better delineation of the associated transcriptome.
Our results with BMP4 are, by and large, in agreement with the findings from the two major combined microarray and PCR investigations on BMP-induced differentiation of hESCs [17,21].This is somewhat surprising since we and Xu et al. [21] maintained FGF2 along with BMP4, while Marchand et al. [17] removed FGF2 from the medium when BMP4 was added.A summary of many of the genes found to be altered in the present study compared to other reports is given in the Supplement (S.2).A major difference between our results and those of Marchand et al. [17] is in the expression of the mesodermal markers, BMP4 and T. We observed increased expression of these two genes, while they reported a decrease or no change, attributable to their removal of FGF2 during BMP-mediated differentiation.Of interest, they found increased expression of KDR, while we noted a minimal decrease, albeit not significant.Increased expression of MMP9 was found herein, consistent with the findings of Xu et al. [21] and Schultz et al. [19], but Marchand et al. [17] reported down-regulation.With the BG02 cells, we found reduced expression of the pluripotent marker, FOXH1, whereas others did not [17,21].These discrepancies may reflect cell-specific differences, culture differences, e.g.±FGF2, or other factors.
While it has been convincingly documented that members of the BMP family lead to differentiation of hESC to trophoblast, there is also considerable evidence that experimental conditions have a profound effect on the type of differentiation obtained.For example, an earlier report on BMP4-mediated differentiation of BG02 cells identified the formation and outgrowth of an immature vascular system when the cells are grown in a 3D Matrigel substrate in an endothelial cell growth medium [13].Further, Pera et al. [18] found that, in response to BMP2, BMP4, or BMP2/7, HES-2 and HES-3 hESC differentiated to extra-embryonic endoderm, with only a few percent of the cells having the appearance of the trophoblast precursors described by Xu et al. [21].They also found that the BMP antagonist noggin blocks the differentiation to extra-embryonic endoderm and directs differentiation into neural precursors, differentiation that may be mimicked by secretion of gremlin by mouse embryo fibroblast feeder layers.Our results with noggin reflect some type(s) of differentiation, but there is no clear indication for preference of one major pathway.As judged by increased expression of T, MIXL1, and WNT3, others have found that short-term treatment of H1, H7, and H9 hESC with BMP4 resulted in the induction of mesoderm progenitor cells that can differentiate into hematopoietic and cardiac lineages [24].Working with H7 cells, it was reported that BMP4 treatment failed to yield trophectoderm using mouse embryonic fibroblasts as a feeder layer; this, however, was overcome by using feeder-free cells on Geltrex-coated plates in StemPro [38].Lastly, West et al. [45] have demonstrated that, in the presence of BMP4, the KIT ligand enhances differentiation to germ-like cells.Hence, additional work is needed to clarify the many experimental parameters associated with BMP-induced differentiation of hESC.

EB Derived from BG02 Cells
EB formation by these cells also yielded some degree of differentiation to trophoblast as evidenced by the up-regulation of CGB and the production of hCG and progesterone.The most highly expressed genes in EB on day 5 are: GJA1, CREBBP, CDH2, ID3, CDH1, DLX5, SOX2, CREB1, HRAS, AR, and AFP.Most of these were surveyed in the studies on cells receiving BMP4, and none were up-regulated.The results suggest that trophoblast-like cells are not forming to any significant extent by day 5, findings consistent with the absence of hCG and progesterone production until days 18 -20.These data are consistent with those by Gerami-Naini et al. [8] on H1 cell-derived EB growing in Matrigel.They could not detect measureable hormone until about day 20 of culture, and, depending upon the conditions used, maximal production of hCG was reached on days 35 -40, followed by a decline.In contrast to the results with the Matrigel-embedded EB, they found that in suspension culture EBs were producing hCG, progesterone, and estradiol by 48 h.
In our studies, a comparison of gene expression by EB on days 22 and 50, relative to day 5, with that of hESC receiving BMP4 for 7 days, provides additional evidence that differentiation to trophoblast is occurring by day 22, as evidenced by the up-regulation of CGB, PGF, PTGS2, RUNX1, and VEGF.These results are consistent with the hormone secretion data for hCG and progesterone.In addition to the above genes, there is also up-regulation of AFP, BMP6, CHD11, CHRD, DAB2, HSPG2, INHBA, PTGS2, and TIMP1.Not surprisingly, the EBs are apparently more heterogeneous in terms of constituent cell types than the BMP4-treated hESCs.For example, the early appearance (day 5) of GJA1, CREBBP, CDH2, ID3, CDH1, DLX5, SOX2, CREB1, HRAS, AR, AFP, CDH11, NES, GATA3, and GREM1, followed by the later up-regulation of AFP, BMP6, CHD11, CHRD, DAB2, HSPG2, INHBA, PTGS2, and TIMP1, indicates formation of trophoblast, endoderm, ectoderm, and mesoderm.Consistent with this finding was the observation some years ago that markers for the three embryonic germ layers were expressed in EBs prepared from H9 cells [46].

CONCLUSION
Overall, the results presented herein strongly support other reports concluding that, under certain conditions, BMP4 directs differentiation of hESCs to trophoblastlike cells.This conclusion notwithstanding, it is clear that experimental conditions, particularly the inclusion or exclusion of FGF2 during BMB-mediated differentiation, have a profound effect on the type of differentiation achieved, and, moreover, based on our transcriptome profiling, it is highly likely that other cell types may be forming in response to BMP4.On the other hand, it may emerge that some of the other genes we found to have been up-regulated by BMP4 function in differentiation to trophectoderm and then to cytotrophoblasts (villous and extravillous) and syncytiotrophoblasts, or be involved in placental formation and function.The heterogeneity of the differentiated cells needs to be carefully established, but the BMP-mediated differentiation of hESCs to trophoblast, particularly in the absence of FGF2, certainly provides an attractive in vitro system for studying early differentiation events and gives a more homogeneous system than that of embryoid bodies.Lastly, our observation that the BMP4 antagonist noggin alters gene transcription of a subset of genes investigated may correlate with the morphological changes reported by others; however, more studies are required to map the transcriptional changes to neural differentiation.

Figure 1 .
Figure 1.Heatmap showing the alterations in gene expression in hESC after seven days of incubation with either BMP4 (B) or noggin (N) relative to control.The data reflect qRT-PCR results given as mean values of .The gray bars denote sufficiently low expression that could not be accurately determined, i.e.C t greater than 35.t ddc 2 −

Figure 2 .
Figure 2. Medium concentrations of hCG (a), progesterone (b), and estradiol (c) of hESC.The cells were incubated in medium alone (CON), in medium containing BMP4 (BMP), or in medium with noggin (NOG).

Figure 3 . 2 −
Figure 3. Heatmap showing changes in gene expression of EB at 22 and 50 days, relative to day 5 (mean values of ).The most prominently altered genes concomitant with time in culture are discussed in the text.t ddc 2 −

Figure 4 .
Figure 4. Medium concentration of hCG (a) and progesterone (b) production by EB cultured up to 50days.The variability at each day is attributable to the dynamic nature of EB, in particular the changing size, number, and possibly atresia and necrosis.As discussed in the text, the conditions are such that new hormone synthesis and secretion is the primary contributor with accumulated prior secretion contributing some as well.A correction for the total accumulated hCG and progesterone, assuming complete stability in the medium, shows that the majority of synthesis occurs between days 20 -40, after which plateaus of about 660 ng/mL and 1 ng/mL, respectively, are reached.The concentrations of estradiol were also measured and found to be quite low, about 0.04 ± 0.01 ng/mL (data not shown).

Table 1 .
BMP4-mediated changes in gene expression of hESCs a .