2021 journal article

Two distinct trophectoderm lineage stem cells from human pluripotent stem cells

JOURNAL OF BIOLOGICAL CHEMISTRY, 296.

By: A. Mischler n, V. Karakis n, J. Mahinthakumar n, C. Carberry *, A. San Miguel n , J. Rager *, R. Fry *, B. Rao n

MeSH headings : CDX2 Transcription Factor / metabolism; Cell Culture Techniques; Cell Differentiation; Cell Lineage; Culture Media; Embryonic Stem Cells / cytology; Embryonic Stem Cells / metabolism; Female; Humans; Placenta / cytology; Placenta / metabolism; Pluripotent Stem Cells / cytology; Pluripotent Stem Cells / metabolism; Pregnancy; Trophoblasts / cytology; Trophoblasts / metabolism
TL;DR: Derivation of hTSCs and hTESCs from pluripotent stem cells significantly enables construction of models for normal and pathological placental development. (via Semantic Scholar)
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Source: Web Of Science
Added: September 7, 2021

The trophectoderm layer of the blastocyst-stage embryo is the precursor for all trophoblast cells in the placenta. Human trophoblast stem (TS) cells have emerged as an attractive tool for studies on early trophoblast development. However, the use of TS cell models is constrained by the limited genetic diversity of existing TS cell lines and restrictions on using human fetal tissue or embryos needed to generate additional lines. Here we report the derivation of two distinct stem cell types of the trophectoderm lineage from human pluripotent stem cells. Analogous to villous cytotrophoblasts in vivo, the first is a CDX2- stem cell comparable with placenta-derived TS cells—they both exhibit identical expression of key markers, are maintained in culture and differentiate under similar conditions, and share high transcriptome similarity. The second is a CDX2+ stem cell with distinct cell culture requirements, and differences in gene expression and differentiation, relative to CDX2- stem cells. Derivation of TS cells from pluripotent stem cells will significantly enable construction of in vitro models for normal and pathological placental development. The trophectoderm layer of the blastocyst-stage embryo is the precursor for all trophoblast cells in the placenta. Human trophoblast stem (TS) cells have emerged as an attractive tool for studies on early trophoblast development. However, the use of TS cell models is constrained by the limited genetic diversity of existing TS cell lines and restrictions on using human fetal tissue or embryos needed to generate additional lines. Here we report the derivation of two distinct stem cell types of the trophectoderm lineage from human pluripotent stem cells. Analogous to villous cytotrophoblasts in vivo, the first is a CDX2- stem cell comparable with placenta-derived TS cells—they both exhibit identical expression of key markers, are maintained in culture and differentiate under similar conditions, and share high transcriptome similarity. The second is a CDX2+ stem cell with distinct cell culture requirements, and differences in gene expression and differentiation, relative to CDX2- stem cells. Derivation of TS cells from pluripotent stem cells will significantly enable construction of in vitro models for normal and pathological placental development. Specification of the trophectoderm and the inner cell mass is the first differentiation event during human embryonic development. The trophectoderm mediates blastocyst implantation in the uterus and is the precursor to all trophoblast cells in the placenta. Upon embryo implantation, the trophectoderm forms the cytotrophoblast (CTB), a putative stem cell that can differentiate to form the two major cell types in the placenta, the extravillous trophoblast (EVT) and the syncytiotrophoblast (STB) (1Bischof P. Irminger-Finger I. The human cytotrophoblastic cell, a mononuclear chameleon.Int. J. Biochem. Cel. Biol. 2005; 37: 1-16Crossref PubMed Scopus (125) Google Scholar, 2Benirschke Kurt. Baergen R.N. Burton G. Graham J. Pathology of the Human Placenta [electronic Resource]. Springer, Heidelberg2012Crossref Scopus (48) Google Scholar). The EVTs are involved in remodeling of uterine arteries, which is critical to ensure adequate perfusion of the placenta with maternal blood, whereas the multinucleated STB mediates the nutrient and gas exchange at the maternal–fetal interface (3Yabe S. Alexenko A.P. Amita M. Yang Y. Schust D.J. Sadovsky Y. Ezashi T. Roberts R.M. Comparison of syncytiotrophoblast generated from human embryonic stem cells and from term placentas.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E2598-E2607Crossref PubMed Scopus (68) Google Scholar, 4Moser G. Orendi K. Gauster M. Siwetz M. Helige C. Huppertz B. The art of identification of extravillous trophoblast.Placenta. 2011; 32: 197-199Crossref PubMed Scopus (29) Google Scholar). Abnormalities in trophoblast development are associated with pregnancy-related pathologies such as miscarriage, preeclampsia, and placenta accreta. Yet, despite its relevance to maternal and fetal health, constraints on research with human embryos and early fetal tissue impede mechanistic insight into early trophoblast development. Trophoblast stem (TS) cells derived from first-trimester human placental samples and blastocyst-stage embryos have emerged as an attractive in vitro model system for early human trophoblast (5Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of human trophoblast stem cells.Cell stem cell. 2018; 22: 50-63.e6Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). However, restricted accessibility of embryos and placental samples from early gestation and low genetic diversity of existing cell lines limit the use of this model. In contrast, human pluripotent stem cells (hPSCs) are a more accessible source for generating in vitro models of human trophoblast. Of more importance, unlike early gestation primary samples where the projected pregnancy outcome is uncertain, human induced pluripotent stem cells (hiPSCs) can potentially provide models of validated normal and pathological trophoblast development (6Sheridan M.A. Yang Y. Jain A. Lyons A.S. Yang P. Brahmasani S.R. Dai A. Tian Y. Ellersieck M.R. Tuteja G. Schust D.J. Schulz L.C. Ezashi T. Roberts R.M. Early onset preeclampsia in a model for human placental trophoblast.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 4336-4345Crossref PubMed Scopus (20) Google Scholar). However, whether bona fide trophoblast can be obtained from hPSCs has been a subject of intense debate (7Roberts R.M. Loh K.M. Amita M. Bernardo A.S. Adachi K. Alexenko A.P. Schust D.J. Schulz L.C. Telugu B.P.V.L. Ezashi T. Pedersen R.A. Differentiation of trophoblast cells from human embryonic stem cells: To be or not to be?.Reproduction (Cambridge, England). 2014; 147: D1-D12Crossref PubMed Scopus (45) Google Scholar). A rigorous head-to-head comparison between trophoblast derived from hPSCs and their in vivo counterparts has proven difficult owing to multiple reasons. Previous studies have used varying experimental protocols (8Roberts R.M. Ezashi T. Sheridan M.A. Yang Y. Specification of trophoblast from embryonic stem cells exposed to BMP4†.Biol. Reprod. 2018; 99: 212-224Crossref PubMed Scopus (22) Google Scholar); both primary placental samples and cultures of terminally differentiated trophoblast obtained from hPSCs exhibit heterogeneity and contain many cell types, and until recently self-renewing TS-like cells had not been derived from hPSCs (9Dong C. Beltcheva M. Gontarz P. Zhang B. Popli P. Fischer L.A. Khan S.A. Park K.-M. Yoon E.-J. Xing X. Kommagani R. Wang T. Solnica-Krezel L. Theunissen T.W. Derivation of trophoblast stem cells from naïve human pluripotent stem cells.eLife. 2020; 9: e52504Crossref PubMed Scopus (57) Google Scholar, 10Cinkornpumin J.K. Kwon S.Y. Guo Y. Hossain I. Sirois J. Russett C.S. Tseng H.W. Okae H. Arima T. Duchaine T.F. Liu W. Pastor W.A. Naive human embryonic stem cells can give rise to cells with a trophoblast-like transcriptome and Methylome.Stem Cell Rep. 2020; 15: 198-213Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 11Li Z. Kurosawa O. Iwata H. Development of trophoblast cystic structures from human induced pluripotent stem cells in limited-area cell culture.Biochem. Biophysical Res. Commun. 2018; 505: 671-676Crossref PubMed Scopus (5) Google Scholar, 12Gao X. Nowak-Imialek M. Chen X. Chen D. Herrmann D. Ruan D. Chen A.C.H. Eckersley-Maslin M.A. Ahmad S. Lee Y.L. Kobayashi T. Ryan D. Zhong J. Zhu J. Wu J. et al.Establishment of porcine and human expanded potential stem cells.Nat. Cell Biol. 2019; 21: 687-699Crossref PubMed Scopus (120) Google Scholar). In this study, we report the derivation and maintenance of two distinct trophectoderm lineage stem cell types from hPSCs, specifically human embryonic stem cells (hESCs) and hiPSCs, in chemically defined culture conditions. The first is a CDX2- stem cell that is comparable with TS cells derived from early-gestation placental samples and similar to the villous CTB. The second is a CDX2+ cell type with distinct cell culture requirements, and differences in gene expression and differentiation, relative to CDX2- stem cells. Critically, the isolation of self-renewing stem cell populations allowed a direct comparison of placenta-derived TS cells with TS cells from hPSCs; genome-wide transcriptomic analysis and functional differentiation assays demonstrate very high similarity between placenta- and hPSC-derived CDX2- TS cells. The routine derivation of TS cells from hPSCs will provide powerful tools for mechanistic studies on normal and pathological early trophoblast development. Media formulations in previous studies on trophoblast differentiation of hESCs included components such as knockout serum replacement (KSR) or bovine serum albumin (BSA) that act as carriers for lipids. Albumin-associated lipids have been implicated in activation of G-protein–coupled receptor–mediated signaling (13Yu F.-X. Zhao B. Panupinthu N. Jewell J.L. Lian I. Wang L.H. Zhao J. Yuan H. Tumaneng K. Li H. Fu X.-D. Mills G.B. Guan K.-L. Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling.Cell. 2012; 150: 780-791Abstract Full Text Full Text PDF PubMed Scopus (974) Google Scholar, 14Mendelson K. Evans T. Hla T. Sphingosine 1-phosphate signalling.Development (Cambridge, England). 2014; 141: 5-9Crossref PubMed Scopus (165) Google Scholar). For instance, the phospholipid sphingosine-1 phosphate (S1P) present in KSR can activate YAP signaling. YAP plays a critical role in specification of the trophectoderm in mouse (15Yagi R. Kohn M.J. Karavanova I. Kaneko K.J. Vullhorst D. DePamphilis M.L. Buonanno A. Transcription factor TEAD4 specifies the trophectoderm lineage at the beginning of mammalian development.Development (Cambridge, England). 2007; 134: 3827-3836Crossref PubMed Scopus (353) Google Scholar, 16Knott J.G. Paul S. Transcriptional regulators of the trophoblast lineage in mammals with hemochorial placentation.Reproduction (Cambridge, England). 2014; 148: R121-R136Crossref PubMed Scopus (40) Google Scholar, 17Nishioka N. Yamamoto S. Kiyonari H. Sato H. Sawada A. Ota M. Nakao K. Sasaki H. Tead4 is required for specification of trophectoderm in pre-implantation mouse embryos.Mech. Dev. 2008; 125: 270-283Crossref PubMed Scopus (331) Google Scholar), as well as human trophoblast development (18Saha B. Ganguly A. Home P. Bhattacharya B. Ray S. Ghosh A. Rumi M.A.K. Marsh C. French V. Gunewardena S. Paul S. TEAD4 ensures postimplantation development by promoting trophoblast self-renewal: An implication in early human pregnancy loss.Proc. Natl. Acad. Sci. 2020; 117: 202002449Crossref Scopus (20) Google Scholar, 19Meinhardt G. Haider S. Kunihs V. Saleh L. Pollheimer J. Fiala C. Hetey S. Feher Z. Szilagyi A. Than N.G. Knöfler M. Pivotal role of the transcriptional co-activator YAP in trophoblast stemness of the developing human placenta.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 13562-13570Crossref PubMed Scopus (25) Google Scholar). We investigated the use of S1P in the context of trophoblast differentiation of hESCs under chemically defined culture conditions, by modifying our previous protocol that utilized KSR (20Sarkar P. Randall S.M. Collier T.S. Nero A. Russell T.A. Muddiman D.C. Rao B.M. Activin/nodal signaling Switches the terminal fate of human embryonic stem cell-derived trophoblasts.J. Biol. Chem. 2015; 290: 8834-8848Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 21Sarkar P. Mischler A. Randall S.M. Collier T.S. Dorman K.F. Boggess K.A. Muddiman D.C. Rao B.M. Identification of epigenetic factor proteins expressed in human embryonic stem cell-derived trophoblasts and in human placental trophoblasts.J. Proteome Res. 2016; 15: 2433-2444Crossref PubMed Scopus (6) Google Scholar). H1 and H9 hESCs cultured in E8 medium were differentiated for 6 days in E7 medium (E8 without transforming growth factor-beta1 [TGFβ1]) supplemented with S1P, by treatment with BMP4 and the activin/nodal inhibitor SB431542 (Fig. 1A). Under these conditions, we observed upregulation of the trophectoderm marker CDX2 and the CTB marker ELF5 (Fig. S1, A and B). Upregulation of TBX4 was observed after 6 days. However, overall there were no significant changes in markers associated with neural or mesodermal differentiation after 6 days suggesting that differentiation to these lineages did not occur (Fig. S1, A and B). Immunofluorescence analysis at day 6 confirmed expression of the pan-trophoblast marker KRT7, and CTB markers P63 and GATA3; expression of CDX2 was not observed (Figs. 1B and S1C). The putative CTB cells obtained at day 6 were investigated for their ability to differentiate to EVTs and STB, using protocols similar to those previously employed (20Sarkar P. Randall S.M. Collier T.S. Nero A. Russell T.A. Muddiman D.C. Rao B.M. Activin/nodal signaling Switches the terminal fate of human embryonic stem cell-derived trophoblasts.J. Biol. Chem. 2015; 290: 8834-8848Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). We observed formation of mesenchymal cells from epithelial cells over a 6-day period when passaged into E8 medium supplemented with epidermal growth factor (EGF) and SB431542. Immunofluorescence analysis showed expression of KRT7 and the EVT markers VE-Cadherin and HLA-G (Figs. 1C, S1D). Alternatively, passaging CTB-like cells in E6 medium (E8 without TGFβ1 and fibroblast growth factor-2 [FGF2]) supplemented with activin and EGF resulted in the formation of KRT7+ multinucleate cells expressing the STB markers hCG and syncytin over an 8-day period (Figs. 1D, S1E). Removal of S1P from the medium during hESC differentiation to CTB-like cells abolished the formation of EVTs that express HLA-G and VE-Cadherin (Figs. 1E, S2A) under identical differentiation conditions (Fig. 1A). Differentiation to STB also did not occur in the absence of S1P, as evidenced by lack of expression of syncytin and KRT7 (Figs. 1F, S2B). Also, downregulation of the trophectoderm marker CDX2 and upregulation of transcripts of neural and mesoderm markers was observed in cells after 6 days of differentiation, upon removal of S1P (Fig. S2C). Taken together these results show that CTB-like cells, similar to those in previous studies utilizing more complex culture conditions (20Sarkar P. Randall S.M. Collier T.S. Nero A. Russell T.A. Muddiman D.C. Rao B.M. Activin/nodal signaling Switches the terminal fate of human embryonic stem cell-derived trophoblasts.J. Biol. Chem. 2015; 290: 8834-8848Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar), can be obtained by differentiation of hESCs in a chemically defined medium containing S1P. Furthermore, addition of exogenous S1P is necessary for hESC differentiation to trophoblast in our chemically defined culture medium. Rho GTPase signaling, downstream of G-protein–coupled receptors activated by S1P, has been implicated in nuclear localization of YAP (22Ohgushi M. Minaguchi M. Sasai Y. Rho-signaling-directed YAP/TAZ activity Underlies the long-term Survival and Expansion of human embryonic stem cells.Cell stem cell. 2015; 17: 448-461Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 23Mo J.-S. Yu F.-X. Gong R. Brown J.H. Guan K.-L. Regulation of the Hippo-YAP pathway by protease-activated receptors (PARs).Genes Dev. 2012; 26: 2138-2143Crossref PubMed Scopus (195) Google Scholar). Both Rho/RhoA associated kinase (ROCK) and nuclear YAP play a critical role in trophectoderm specification in the mouse (24Nishioka N. Inoue K. Adachi K. Kiyonari H. Ota M. Ralston A. Yabuta N. Hirahara S. Stephenson R.O. Ogonuki N. Makita R. Kurihara H. Morin-Kensicki E.M. Nojima H. Rossant J. et al.The Hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass.Dev. Cel. 2009; 16: 398-410Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar, 25Kono K. Tamashiro D.A.A. Alarcon V.B. Inhibition of RHO-ROCK signaling enhances ICM and suppresses TE characteristics through activation of Hippo signaling in the mouse blastocyst.Dev. Biol. 2014; 394: 142-155Crossref PubMed Scopus (79) Google Scholar). Therefore, we investigated the role of Rho/ROCK signaling and YAP in trophoblast differentiation of hESCs. The Rho/ROCK inhibitor Y-27632 was included during differentiation of hESCs to CTB-like cells and subsequent differentiation to EVT and STB to investigate the role of Rho/ROCK signaling. Under these conditions, HLA-G expression was observed in cells obtained from H9 hESCs; however, VE-Cadherin expression was weak and observed in only a few cells (Fig. S3A). On the other hand, expression of EVT markers was not observed in cells derived from H1 hESCs. In addition, presence of ROCK inhibition abolished STB formation, as shown by the lack of expression of syncytin and KRT7 (Fig. S3B). To investigate the role of YAP signaling in CTB formation from hESCs, we used an hESC cell line (H9) that expresses an inducible shRNA against YAP (H9-YAP-ishRNA) or a scrambled shRNA control (26Hsiao C. Lampe M. Nillasithanukroh S. Han W. Lian X. Palecek S.P. Human pluripotent stem cell culture density modulates YAP signaling.Biotechnol. J. 2016; 11: 662-675Crossref PubMed Scopus (26) Google Scholar). YAP knockdown abolished differentiation to EVT and STB, as evidenced by lack of expression of the relevant markers. It is notable that high cell death was observed (Fig. S3, A and B). Gene expression analysis revealed a significant reduction in ELF5 upon YAP knockdown, relative to the scrambled shRNA control (Fig. S3C). Significant downregulation of the mesodermal genes TBX4 and LMO2 was observed, whereas T was upregulated, in H9-YAP-ishRNA, relative to the scrambled control. Taken together, these results show that Rho/ROCK signaling and YAP are necessary for differentiation of hESCs to functional CTB that can give rise to both EVTs and STB, in our chemically defined culture medium. S1P acts through both receptor-mediated and receptor-independent pathways (14Mendelson K. Evans T. Hla T. Sphingosine 1-phosphate signalling.Development (Cambridge, England). 2014; 141: 5-9Crossref PubMed Scopus (165) Google Scholar, 27Maceyka M. Harikumar K.B. Milstien S. Spiegel S. Sphingosine-1-phosphate signaling and its role in disease.Trends Cell Biol. 2012; 22: 50-60Abstract Full Text Full Text PDF PubMed Scopus (695) Google Scholar). To investigate the specific mechanism of S1P action during hESC differentiation to trophoblast, we replaced S1P with D-erythro-dihydrospingosine-1-phosphate (dhS1P) in our protocol. dhS1P acts as an agonist for the S1P receptors (S1PRs) but does not mediate an intracellular effect (28Van Brocklyn J.R. Lee M.-J. Menzeleev R. Olivera A. Edsall L. Cuvillier O. Thomas D.M. Coopman P.J.P. Thangada S. Liu C.H. Hla T. Spiegel S. Dual actions of sphingosine-1-phosphate: Extracellular through the G i -coupled receptor Edg-1 and intracellular to regulate proliferation and Survival.J. Cell Biol. 1998; 142: 229-240Crossref PubMed Scopus (444) Google Scholar). Replacing S1P with dhS1P yielded similar results—CTB-like cells showed expression of CDX2, GATA3, P63, and TEAD4 (Figs. 2A and S4A). Upon further differentiation as previously described (Fig. 1A), STB expressing KRT7 and hCG, and EVT expressing HLA-G and VE-Cadherin were obtained (Fig. 2, B and C; Fig. S4, B and C). These results suggest that S1PR signaling mediates the effect of exogenous S1P during hESC differentiation to trophoblast in our chemically defined medium. S1P acts extracellularly through S1PR1-5 (14Mendelson K. Evans T. Hla T. Sphingosine 1-phosphate signalling.Development (Cambridge, England). 2014; 141: 5-9Crossref PubMed Scopus (165) Google Scholar, 27Maceyka M. Harikumar K.B. Milstien S. Spiegel S. Sphingosine-1-phosphate signaling and its role in disease.Trends Cell Biol. 2012; 22: 50-60Abstract Full Text Full Text PDF PubMed Scopus (695) Google Scholar); however, TBs have been shown to only express S1PR1-3 (29Johnstone E.D. Chan G. Sibley C.P. Davidge S.T. Lowen B. Guilbert L.J. Sphingosine-1-phosphate inhibition of placental trophoblast differentiation through a G(i)-coupled receptor response.J. lipid Res. 2005; 46: 1833-1839Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). We further used selective chemical agonists for S1PR1-3—CYM5442 hydrochloride, CYM5520, and CYM5541, respectively—to replace S1P in differentiation protocols previously discussed. Expression of CDX2, GATA3, P63, and TEAD4 was observed in CTB-like cells for all three agonists (Figs. 2A and S4A). Similarly, use of each agonist resulted in expression of the EVT markers HLA-G and VE-Cadherin and formation of multinucleate STB expressing KRT7 and hCG (Fig. 2, B and C; Fig. S4, B and C). However, we observed some variability between the agonists (Fig. S5). For instance, use of the S1PR2 agonist resulted in strong cytoplasmic expression of P63 and high heterogeneity in staining at day 6 relative to the other agonists. Formation of large multinucleated STB was more pronounced when the S1PR2 or S1PR3 agonists were used, as compared with the S1PR1 agonist. On the other hand, the S1PR1 and S1PR3 agonists enhanced the formation of mesenchymal EVTs, relative to the S1PR2 agonist. Taken together, our results further confirmed that S1PR signaling mediates effects of exogenous S1P during trophoblast differentiation of hESCs in our culture system. Since our qualitative observations showed that use of the S1PR3 agonist resulted in expression of CTB markers, and both multinucleate STB and mesenchymal EVTs could be obtained when the S1PR3 agonist was used, we chose the S1PR3 agonist for subsequent studies. We investigated whether CTB-like cells obtained by treatment of hESCs with BMP4 and SB431542 in E7 medium supplemented with the S1PR3 agonist CYM5541 for 6 days could be passaged and maintained under conditions used for culture of blastocyst- and placenta-derived primary TS cells (5Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of human trophoblast stem cells.Cell stem cell. 2018; 22: 50-63.e6Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Upon plating in trophoblast stem cell medium (TSCM) developed by Okae et al. (5Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of human trophoblast stem cells.Cell stem cell. 2018; 22: 50-63.e6Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar), hESC-derived CTB-like cells underwent differentiation and epithelial colonies could not be retained after a single passage. CDX2 expression is upregulated significantly in as little as 2 days after initiation of hESC differentiation but decreases by day 6 (Fig. S1, A and B). In addition, previous studies have reported differentiation of hESCs to CDX2+/p63+ cells upon treatment with BMP for 4 days (30Horii M. Li Y. Wakeland A.K. Pizzo D.P. Nelson K.K. Sabatini K. Laurent L.C. Liu Y. Parast M.M. Human pluripotent stem cells as a model of trophoblast differentiation in both normal development and disease.Proc. Natl. Acad. Sci. United States America. 2016; 113: E3882-E3891Crossref PubMed Scopus (66) Google Scholar). Therefore, we explored the use of a shorter differentiation step for obtaining CTB-like cells (Fig. 3A). After 3 days of differentiation, H9 and H1 hESCs expressed nuclear CDX2, P63, and TEAD4 uniformly (Fig. 3B). However, by day 6 most differentiated H1 and H9 hESCs lose expression of CDX2 (Fig. 3C). Quantitative image analysis showed that nearly all cells are CDX2+ at day 3, in contrast to CTB-like cells at day 6. Of note, use of a 6-day protocol resulted in a significantly reduced fraction of CDX2+ cells in the case of H1 hESCs in comparison with the 3-day protocol; on the other hand, a significant fraction of H9 cells retained CDX2+ at day 6 (Fig. 3D). Transcriptome analysis using RNA sequencing identified 291 genes with significantly higher expression levels and 330 genes with significantly lower expression levels in day 3 differentiated hESCs versus undifferentiated hESCs (Tables S1 and S2).Expression of other trophectoderm-associated markers such as HAND1, GATA3, and TFAP2A, in addition to CDX2, was upregulated in differentiated hESCs at day 3, whereas expression of pluripotency-associated NANOG was downregulated. Gene set enrichment analysis of differentially expressed genes identified 567 and 202 gene ontology (GO) categories (of 9996 queried categories) associated with higher and lower gene expression in day 3 differentiated cells versus undifferentiated hESCs, respectively (Tables S3 and S4). Consistent with differentiation to epithelial trophoblast, genes associated with the GO terms for epithelium development, epithelial cell proliferation, and epithelial cell differentiation were upregulated in day 3 differentiated hESCs. CDX2+ cells at day 3 were passaged into a chemically defined medium containing four major components (denoted TM4), the S1PR3 agonist CYM5541, the GSK3β inhibitor CHIR99021, the TGFβ inhibitor A83-01, and FGF10. CHIR99021 and A83-01 are components of TSCM used for culture of primary TS cells; FGF10 was included because FGFR2b signaling is active in blastocyst- and placenta-derived TS cells and the early placenta (5Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of human trophoblast stem cells.Cell stem cell. 2018; 22: 50-63.e6Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Cells in TM4 could be maintained as epithelial colonies for 30+ passages over the course of 5 months. In TM4 medium, cells derived from H9 and H1 hESCs retained expression of the trophoblast markers CDX2, TFAP2C, YAP, TEAD4, and GATA3 (Figs. 3E and S6) (15Yagi R. Kohn M.J. Karavanova I. Kaneko K.J. Vullhorst D. DePamphilis M.L. Buonanno A. Transcription factor TEAD4 specifies the trophectoderm lineage at the beginning of mammalian development.Development (Cambridge, England). 2007; 134: 3827-3836Crossref PubMed Scopus (353) Google Scholar, 17Nishioka N. Yamamoto S. Kiyonari H. Sato H. Sawada A. Ota M. Nakao K. Sasaki H. Tead4 is required for specification of trophectoderm in pre-implantation mouse embryos.Mech. Dev. 2008; 125: 270-283Crossref PubMed Scopus (331) Google Scholar, 31Choi I. Carey T.S. Wilson C.A. Knott J.G. Transcription factor AP-2γ is a core regulator of tight junction biogenesis and cavity formation during mouse early embryogenesis.Development (Cambridge, England). 2012; 139: 4623-4632Crossref PubMed Scopus (40) Google Scholar, 32Home P. Saha B. Ray S. Dutta D. Gunewardena S. Yoo B. Pal A. Vivian J.L. Larson M. Petroff M. Gallagher P.G. Schulz V.P. White K.L. Golos T.G. 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Upon passage, cells showed no HLA-G and minimal VE-Cadherin expression (Fig. 3G). Furthermore, cells maintained an epithelial flattened morp