Smad1 expands the hemangioblast population within a limited developmental window

Abstract Bone morphogenetic protein (BMP) signaling is an important regulator of hematovascular development. However, the progenitor population that responds to BMP signaling is undefined, and the relative role of downstream mediators including Smad1 is unclear. We find that Smad1 shows a distinctive expression profile as embryonic stem (ES) cells undergo differentiation in the embryoid body (EB) system, with peak levels in cell populations enriched for the hemangioblast. To test the functional relevance of this observation, we generated an ES cell line that allows temporal control of ectopic Smad1 expression. Continuous expression of Smad1 from day 2 of EB culture does not disturb hematopoiesis, according to colony assays. In contrast, a pulse of Smad1 expression exclusively between day 2 and day 2.25 expands the population of progenitors for primitive erythroblasts and other hematopoietic lineages. This effect correlates with increased levels of transcripts encoding markers for the hemangioblast, including Runx1, Scl, and Gata2. Indeed, the pulse of Smad1 induction also expands the blast colony-forming cell (BL-CFC) population at a level that is fully sufficient to explain subsequent increases in hematopoiesis. Our data demonstrate that Smad1 expression is sufficient to expand the number of cells that commit to hemangioblast fate.

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Inhibition of Cardiomyocytes Differentiation of Mouse Embryonic Stem Cells by CD38/cADPR/Ca2+ Signaling Pathway

Journal of Biological Chemistry ◽

10.1074/jbc.m112.392530 ◽

2012 ◽

Vol 287 (42) ◽

pp. 35599-35611 ◽

Cited By ~ 20

Author(s):

Wen-Jie Wei ◽

Hai-Ying Sun ◽

Kai Yiu Ting ◽

Li-He Zhang ◽

Hon-Cheung Lee ◽

...

Keyword(s):

Signaling Pathway ◽

Embryoid Body ◽

Troponin T ◽

Es Cells ◽

Embryonic Stem ◽

Es Cell ◽

Mouse Es Cells ◽

Adp Ribosyl Cyclase

Cyclic adenosine diphosphoribose (cADPR) is an endogenous Ca2+ mobilizing messenger that is formed by ADP-ribosyl cyclases from nicotinamide adenine dinucleotide (NAD). The main ADP-ribosyl cyclase in mammals is CD38, a multi-functional enzyme and a type II membrane protein. Here we explored the role of CD38-cADPR-Ca2+ in the cardiomyogenesis of mouse embryonic stem (ES) cells. We found that the mouse ES cells are responsive to cADPR and possess the key components of the cADPR signaling pathway. In vitro cardiomyocyte (CM) differentiation of mouse ES cells was initiated by embryoid body (EB) formation. Interestingly, beating cells appeared earlier and were more abundant in CD38 knockdown EBs than in control EBs. Real-time RT-PCR and Western blot analyses further showed that the expression of several cardiac markers, including GATA4, MEF2C, NKX2.5, and α-MLC, were increased markedly in CD38 knockdown EBs than those in control EBs. Similarly, FACS analysis showed that more cardiac Troponin T-positive CMs existed in CD38 knockdown or 8-Br-cADPR, a cADPR antagonist, treated EBs compared with that in control EBs. On the other hand, overexpression of CD38 in mouse ES cells significantly inhibited CM differentiation. Moreover, CD38 knockdown ES cell-derived CMs possess the functional properties characteristic of normal ES cell-derived CMs. Last, we showed that the CD38-cADPR pathway negatively modulated the FGF4-Erks1/2 cascade during CM differentiation of ES cells, and transiently inhibition of Erk1/2 blocked the enhanced effects of CD38 knockdown on the differentiation of CM from ES cells. Taken together, our data indicate that the CD38-cADPR-Ca2+ signaling pathway antagonizes the CM differentiation of mouse ES cells.

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Bone morphogenetic protein-4 promotes induction of cardiomyocytes from human embryonic stem cells in serum-based embryoid body development

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01288.2008 ◽

2009 ◽

Vol 296 (6) ◽

pp. H1793-H1803 ◽

Cited By ~ 62

Author(s):

Shunsuke Takei ◽

Hinako Ichikawa ◽

Kohei Johkura ◽

Akimi Mogi ◽

Heesung No ◽

...

Keyword(s):

Bone Morphogenetic Protein ◽

Embryoid Body ◽

Heart Diseases ◽

Es Cells ◽

Embryonic Stem ◽

Bmp Signaling ◽

Cardiac Differentiation ◽

Pcr Analysis ◽

Cell Based Therapy ◽

Morphogenetic Protein

Cardiomyocytes derived from human embryonic stem (ES) cells are a potential source for cell-based therapy for heart diseases. We studied the effect of bone morphogenetic protein (BMP)-4 in the presence of fetal bovine serum (FBS) on cardiac induction from human H1 ES cells during embryoid body (EB) development. Suspension culture for 4 days with 20% FBS produced the best results for the differentiation of early mesoderm and cardiomyocytes. The addition of Noggin reduced the incidence of beating EBs from 23.6% to 5.3%, which indicated the involvement of BMP signaling in the spontaneous cardiac differentiation. In this condition, treatment with 12.5–25 ng/ml BMP-4 during the 4-day suspension optimally promoted the cardiomyocyte differentiation. The incidence of beating EBs at 25 ng/ml BMP-4 reached 95.8% on day 6 of expansion and then plateaued until day 20. In real-time PCR analysis, the cardiac development-related genes MESP1 and Nkx2.5 were upregulated in the EB outgrowths by 25 ng/ml BMP-4. The activation of BMP signaling in EBs was confirmed by the increase in the phosphorylation of Smad1/5/8 and by the nuclear localization of phospho-Smad1/5/8 and Smad4. The addition of 150 ng/ml Noggin considerably decreased the incidence of beating EBs and Nkx2.5 expression, and Noggin alone increased Nestin expression and neural differentiation in EB outgrowths. The cardiomyocytes induced by 25 ng/ml BMP-4 showed proper cell biological characteristics and a course of differentiation as judged from isoproterenol administration, gene expression, protein assay, immunoreactivity, and subcellular structures. No remarkable change in the extent of apoptosis and proliferation in the cardiomyocytes was observed by BMP-4 treatment. These findings showed that BMP-4 in combination with FBS at the appropriate time and concentrations significantly promotes cardiomyocyte induction from human ES cells.

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398 DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS INTO CARDIOMYOCYTES BY USING SLOW TURNING LATERAL VESSEL (STLV/BIOREACTOR)

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab398 ◽

2010 ◽

Vol 22 (1) ◽

pp. 355

Author(s):

S. Rungarunlert ◽

K. Tar ◽

S. Muenthaisong ◽

M. Techakumphu ◽

M. Pirity ◽

...

Keyword(s):

Large Scale ◽

Embryoid Body ◽

Troponin T ◽

Es Cells ◽

Statistical Significance ◽

Embryonic Stem ◽

Industrial Applications ◽

Cardiac Differentiation ◽

Seeding Density ◽

Es Cell

Cardiomyocytes derived from embryonic stem (ES) cells are anticipated to be valuable for cardiovascular drug testing and disease therapies. The overall efficiency and quantity of cardiomyocytes obtained by differentiation of ES cells is still low. To enable a large-scale culture of ES-derived cells, we have tested a scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled, bioreactor/STLV (slow turning lateral vessel, Synthecon, Inc., Houston, TX, USA) following inoculation with a single cell suspension of mouse ES cells. Technical parameters for optimal cell expansion and efficient ES cell differentiation were compared, such as ES cell seeding density (3 × 105 and 5 × 105 cells mL-1) into the bioreactor and day of transfer and plating of EB on gelatinated petri dishes (Day 2, Day 3, Day 4, and Day 5). The quantity and quality of EB production including the yield and size of EB, as well as viability and apoptosis of cells, were analyzed. Furthermore, after cultivation, well-developed contracting EB with functional cardiac muscle were obtained in which the percentage of EB beating/well and several specific cardiac genes [cardiac Troponin T (cTnT) and α-actinin] expression were also determined. Data are expressed as mean ± SEM of at least 3 independent experiments. Statistical analyses included one-way ANOVA and Student’s t-test Statistical significance was set at P < 0.05. The results showed that 5 × 105 ES cells mL-1 seeded into the STLV significantly improved the homogeneity of size of EB formed compared with 3 × 105 ES cells mL-1. The EB derived from Days 2 or 3 culturing in STLV had less necrotic cells than Days 4 and 5 groups. Furthermore, plating these EB on Days 2 and 3 resulted in significantly more EB beating/well than that of Days 4 and 5 groups. For cardiac differentiation, the group with 5 × 105 ES cells mL-1 seeded into STLV and transferred and plated on Day 3 expressed more cardiac markers than other groups. In conclusion, the optimized rotary suspension culture method can produce a highly uniform population of efficiently differentiating EB in large quantities in a manner that can be easily implemented by basic research laboratories. This method provides a technological platform for the controlled large-scale generation of ES cell-derived cells for clinical and industrial applications. This work was financed by The Thailand Commission on Higher Education (CHE-PhD-SW-2005-100), EUFP6 CLONET (MRTN-CT-2006-035468), NKFP_07_1-ES2HEART-HU (OM-00202-2007), and EUFP7 (PartnErS, PIAP-GA-2008-218205).

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BMP4 and TGFbeta Differentially Regulate CD34+ Progenitor Development in Human Embryonic Stem Cells through SMAD-Dependent Pathway

Blood ◽

10.1182/blood.v112.11.889.889 ◽

2008 ◽

Vol 112 (11) ◽

pp. 889-889

Author(s):

ZacK Z. Wang ◽

Hao Bai ◽

Melanie Arzigian ◽

Yong-Xing Gao ◽

Wen-Shu Wu

Keyword(s):

Stem Cells ◽

Smooth Muscle ◽

Growth Factors ◽

Progenitor Cells ◽

Embryoid Body ◽

Es Cells ◽

Embryonic Stem ◽

Ips Cells ◽

Bmp Signaling ◽

Cd34 Cells

Abstract Pluripotent stem cells derived from patients, including embryonic stem (ES) cells and “induced pluripotent stem” (iPS) cells, are a promising area of regenerative medical research. A major roadblock toward human clinical therapies using ES cells or iPS cells is to define the factors that direct ES cell differentiation into lineage specific cells. We previously established a simple and efficient human embryonic stem cell (hESC) differentiation system to generate CD34+/CD31+ progenitor cells that gave rise to hematopoietic and endothelial cells (Nat Biotech.25:317, 2007). To advance potential clinical application and to define the effects of growth factors on hematopoietic and vascular differentiation, we assessed hESC differentiation on human feeder cells in serum-free condition without intermediate embryoid body (EB) formation. We investigated the roles of BMPs, TGFbeta, VEGF, and FGF2 in directing hESC differentiation. Growth factors were added into culture at different time points to test their stage specific roles. Our study demonstrated that BMP proteins, including BMP2, BMP4, and BMP7, but not BMP9, had synergic effects to VEGF and FGF-2 on hESC differentiation to CD34+/CD31+ progenitor cells. BMP4 was essential to initial CD34+/CD31+ cell development, whereas VEGF and FGF2 promoted the differentiation in later stage, suggesting the sequential roles of BMP4, VEGF and FGF2 in directing hESC differentiation to CD34+/CD31+ progenitor cells. TGFbeta or activin promoted hESC differentiation into CD34+/CD31− cells that were unable to give rise to hematopoietic, endothelial, and smooth muscle cells. Furthermore, TGFbeta or activin activated Smad2/3 signaling, and suppressed BMP4-induced CD34+/CD31+ cells. Microarray analysis revealed that BMP4-induced CD34+ cells expressed hematopoietic, endothelial and smooth muscle genes, including GATA2, gamma globins, VE-Cad, KDR, CD31, Tie2, and aortic smooth muscle actin, whereas TGFbeta-induced CD34+ cells expressed pluripotent markers and endoderm markers, including Oct3/4, Sox2, and Nanog, HHEX, GATA6, and FoxA2. Both canonical BMP signaling (Smad1/5/8-dependent) and non-canonical BMP signaling (p38 MAPK and p42 ERK pathway) were activated by BMP4 in hESCs. Dorsomorphin specifically inhibited BMP4-mediated phosphorylation of Smad1/5/8, and blocked hESC differentiation into CD34+/CD31+ cells. In summary, BMPs and TGFbeta regulate distinct populations of CD34+ cells in hESCs. BMP-Smad1/5/8 pathway is critical for hematopoietic and vascular progenitor development.

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UTX regulates mesoderm differentiation of embryonic stem cells independent of H3K27 demethylase activity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1204166109 ◽

2012 ◽

Vol 109 (38) ◽

pp. 15324-15329 ◽

Cited By ~ 111

Author(s):

Chaochen Wang ◽

Ji-Eun Lee ◽

Young-Wook Cho ◽

Ying Xiao ◽

Qihuang Jin ◽

...

Keyword(s):

Cell Differentiation ◽

Embryonic Development ◽

Es Cells ◽

Embryonic Stem ◽

Es Cell ◽

Mesoderm Development ◽

H3k27me3 Level ◽

Histone H3k27 ◽

Demethylase Activity

To investigate the role of histone H3K27 demethylase UTX in embryonic stem (ES) cell differentiation, we have generated UTX knockout (KO) and enzyme-dead knock-in male ES cells. Deletion of the X-chromosome-encoded UTX gene in male ES cells markedly decreases expression of the paralogous UTY gene encoded by Y chromosome, but has no effect on global H3K27me3 level, Hox gene expression, or ES cell self-renewal. However, UTX KO cells show severe defects in mesoderm differentiation and induction of Brachyury, a transcription factor essential for mesoderm development. Surprisingly, UTX regulates mesoderm differentiation and Brachyury expression independent of its enzymatic activity. UTY, which lacks detectable demethylase activity, compensates for the loss of UTX in regulating Brachyury expression. UTX and UTY bind directly to Brachyury promoter and are required for Wnt/β-catenin signaling-induced Brachyury expression in ES cells. Interestingly, male UTX KO embryos express normal levels of UTY and survive until birth. In contrast, female UTX KO mice, which lack the UTY gene, show embryonic lethality before embryonic day 11.5. Female UTX KO embryos show severe defects in both Brachyury expression and embryonic development of mesoderm-derived posterior notochord, cardiac, and hematopoietic tissues. These results indicate that UTX controls mesoderm differentiation and Brachyury expression independent of H3K27 demethylase activity, and suggest that UTX and UTY are functionally redundant in ES cell differentiation and early embryonic development.

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Murine embryonic stem cell differentiation is promoted by SOCS-3 and inhibited by the zinc finger transcription factor Klf4

Blood ◽

10.1182/blood-2004-07-2681 ◽

2005 ◽

Vol 105 (2) ◽

pp. 635-637 ◽

Cited By ~ 167

Author(s):

Yanjun Li ◽

Jeanette McClintick ◽

Li Zhong ◽

Howard J. Edenberg ◽

Mervin C. Yoder ◽

...

Keyword(s):

Cell Function ◽

Embryoid Body ◽

Es Cells ◽

Embryonic Stem ◽

Stem Cell Differentiation ◽

Northern Blotting ◽

Cytokine Signaling ◽

Embryonic Stem Cell Differentiation ◽

Es Cell ◽

Self Renewal

Abstract Embryonic stem (ES) cells homozygous for a Shp-2 mutation (Shp-2Δ46-110) demonstrate leukemia inhibitory factor (LIF) hypersensitivity and increased LIF-stimulated phosphorylation of signal transducer and activator of transcription (STAT3). We hypothesized that LIF-responsive genes in Shp-2Δ46-110 cells would represent potential candidates for molecules vital for ES cell self-renewal. Using microarray analysis, we detected 41 genes whose expression was modified by LIF in Shp-2Δ46-110 ES cells. Induction of 2 significantly up-regulated genes, suppressor of cytokine signaling–3 (SOCS-3) and Krüppel-like factor 4 (Klf4), was verified using Northern blotting. ES cells overexpressing SOCS-3 had an increased capacity to differentiate to hematopoietic progenitors, rather than to self-renew. In contrast, ES cells overexpressing Klf4 had a greater capacity to self-renew based on secondary embryoid body (EB) formation. Klf4-transduced d6 EBs expressed higher levels of Oct-4, consistent with the notion that Klf4 promotes ES cell self-renewal. These findings verify the negative role of SOCS-3 on LIF signaling and provide a novel role for Klf4 in ES cell function.

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Smad1 signaling restricts hematopoietic potential after promoting hemangioblast commitment

Blood ◽

10.1182/blood-2010-10-312389 ◽

2011 ◽

Vol 117 (24) ◽

pp. 6489-6497 ◽

Cited By ~ 11

Author(s):

Brandoch D. Cook ◽

Susanna Liu ◽

Todd Evans

Keyword(s):

Embryoid Body ◽

Embryonic Stem ◽

Indirect Evidence ◽

Bmp Signaling ◽

Hematopoietic Progenitors ◽

Mesoderm Development ◽

Morphogenetic Protein ◽

Smad Proteins ◽

Enhanced Expression ◽

Developmental Window

Abstract Bone morphogenetic protein (BMP) signaling regulates embryonic hematopoiesis via receptor-mediated activation of downstream SMAD proteins, including SMAD1. In previous work, we showed that Smad1 expression is sufficient to enhance commitment of mesoderm to hemangioblast fate. We also found indirect evidence to support a subsequent repressive function for Smad1 in hematopoiesis. To test this hypothesis directly, we developed a novel system allowing temporal control of Smad1 levels by conditional knockdown in embryonic stem cell derivatives. Depletion of Smad1 in embryoid body cultures before hemangioblast commitment limits hematopoietic potential because of a block in mesoderm development. Conversely, when Smad1 is depleted in FlK1+ mesoderm, at a stage after hemangioblast commitment, the pool of hematopoietic progenitors is expanded. This involves enhanced expression levels for genes specific to hematopoiesis, including Gata1, Runx1 and Eklf, rather than factors required for earlier specification of the hemangioblast. The phenotype correlates with increased nuclear SMAD2 activity, indicating molecular cross-regulation between the BMP and TGF-β signaling pathways. Consistent with this mechanism, hematopoiesis was enhanced when Smad2 was directly expressed during this same developmental window. Therefore, this study reveals a temporally defined function for Smad1 in restricting the expansion of early hematopoietic progenitors.

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A definitive role of Shp-2 tyrosine phosphatase in mediating embryonic stem cell differentiation and hematopoiesis

Blood ◽

10.1182/blood-2003-04-1171 ◽

2003 ◽

Vol 102 (6) ◽

pp. 2074-2080 ◽

Cited By ~ 65

Author(s):

Rebecca J. Chan ◽

Scott A. Johnson ◽

Yanjun Li ◽

Mervin C. Yoder ◽

Gen-Sheng Feng

Keyword(s):

Cell Differentiation ◽

Embryoid Body ◽

Es Cells ◽

Tyrosine Phosphatase ◽

Embryonic Stem ◽

Stem Cell Differentiation ◽

Initial Step ◽

Embryonic Stem Cell Differentiation ◽

Es Cell

Abstract Homozygous mutant (Shp-2Δ46-110) embryonic stem (ES) cells exhibit decreased hematopoiesis; however, the point at which Shp-2 is critical for ES cell differentiation to hematopoietic cells is unknown. We characterized the differentiation defect of Shp-2Δ46-110 ES cells by examining early points of differentiation, conducting leukemia inhibitory factor (LIF)–stimulated biochemical analysis, and performing in vitro reconstitution studies with wild-type (WT) Shp-2. ES cell in vitro differentiation assays were used to compare the differentiation of WT, Shp-2Δ46-110, and reconstituted ES cells to mesoderm, by measuring brachyury expression, to hemangioblasts, by measuring blast colony-forming cell (BL-CFC) formation and flk-1 expression, and to hematopoietic progenitor colony-forming cells, by performing secondary plating assays. LIF-stimulated phospho-Stat3 (known to be critical for ES cell self-renewal and maintenance of an undifferentiated state) and phospho-Erk levels were examined by immunoblotting. ES cell survival, using annexin V staining, and secondary embryoid body (EB) formation were also evaluated. Differentiation to both mesoderm and hemangioblasts was lower in Shp-2Δ46-110 cells compared to WT cells. On reconstitution with WT Shp-2, expression of brachyury and flk-1 and differentiation to hemangioblasts and primitive and definitive hematopoietic progenitors were restored. LIF-stimulated phospho-Stat3 levels were higher, whereas phospho-Erk levels were lower in Shp-2Δ46-110 ES cells than in WT and reconstituted cells. The increased phospho-Stat3 levels correlated with increased Shp-2Δ46-110 ES cell secondary EB formation and survival. We conclude that normal Shp-2 function is critical for the initial step of ES cell differentiation to mesoderm and to hemangioblasts and acts within the LIF-gp130-Stat3 pathway to maintain a proper balance of ES cell differentiation, pluripotency, and apoptosis.

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Antisense Transcription through theXist Locus Mediates Tsix Function in Embryonic Stem Cells

Molecular and Cellular Biology ◽

10.1128/mcb.21.24.8512-8520.2001 ◽

2001 ◽

Vol 21 (24) ◽

pp. 8512-8520 ◽

Cited By ~ 120

Author(s):

Sandra Luikenhuis ◽

Anton Wutz ◽

Rudolf Jaenisch

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Antisense Transcription ◽

Model System ◽

X Inactivation ◽

The Other ◽

Es Cell ◽

Targeted Deletion ◽

First Time

ABSTRACT Expression of the Xist gene, a key player in mammalian X inactivation, has been proposed to be controlled by the antisense Tsix transcript. Targeted deletion of theTsix promoter encompassing the DPXas34 locus leads to nonrandom inactivation of the mutant X, but it remains unresolved whether this phenotype is caused by loss of Tsixtranscription or by deletion of a crucial DNA element. In this study we determined the role of Tsix transcription in random X inactivation by using mouse embryonic stem (ES) cells as a model system. Two approaches were chosen to modulate Tsixtranscription with minimal disturbance of genomic sequences. First,Tsix transcription was functionally inhibited by introducing a transcriptional stop signal into the transcribed region of Tsix. In the second approach, an inducible system forTsix expression was created. We found that the truncation of the Tsix transcript led to complete nonrandom inactivation of the targeted X chromosome. Induction of Tsix transcription during ES cell differentiation, on the other hand, caused the targeted chromosome always to be chosen as the active chromosome. These results for the first time establish a function for antisense transcription in the regulation of X inactivation.

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Steroidogenic Factor-1 (SF-1)-Driven Differentiation of Murine Embryonic Stem (ES) Cells into a Gonadal Lineage

Endocrinology ◽

10.1210/en.2011-0219 ◽

2011 ◽

Vol 152 (7) ◽

pp. 2870-2882 ◽

Cited By ~ 24

Author(s):

Unmesh Jadhav ◽

J. Larry Jameson

Keyword(s):

Target Genes ◽

De Novo ◽

Embryoid Body ◽

Es Cells ◽

Embryonic Stem ◽

Cell Lineage ◽

Culture Conditions ◽

Steroidogenic Factor 1 ◽

Lineage Differentiation ◽

And Function

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells (SF-1-ES cells) has been shown to prime the cells for steroidogenesis. When provided with exogenous cholesterol substrate, and after treatment with retinoic acid and cAMP, SF-1-ES cells produce progesterone but do not produce other steroids such as cortisol, estradiol, or testosterone. In this study, we explored culture conditions that optimize SF-1-mediated differentiation of ES cells into defined steroidogenic lineages. When embryoid body formation was used to facilitate cell lineage differentiation, SF-1-ES cells were found to be restricted in their differentiation, with fewer cells entering neuronal pathways and a larger fraction entering the steroidogenic lineage. Among the differentiation protocols tested, leukemia inhibitory factor (LIF) removal, followed by prolonged cAMP treatment was most efficacious for inducing steroidogenesis in SF-1-ES cells. In this protocol, a subset of SF-1-ES cells survives after LIF withdrawal, undergoes morphologic differentiation, and recovers proliferative capacity. These cells are characterized by induction of steroidogenic enzyme genes, use of de novo cholesterol, and production of multiple steroids including estradiol and testosterone. Microarray studies identified additional pathways associated with SF-1 mediated differentiation. Using biotinylated SF-1 in chromatin immunoprecipitation assays, SF-1 was shown to bind directly to multiple target genes, with induction of binding to some targets after steroidogenic treatment. These studies indicate that SF-1 expression, followed by LIF removal and treatment with cAMP drives ES cells into a steroidogenic pathway characteristic of gonadal steroid-producing cells.

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