2023 journal article
Laminin switches terminal differentiation fate of human trophoblast stem cells under chemically defined culture conditions
JOURNAL OF BIOLOGICAL CHEMISTRY, 299(5).
MeSH headings : Humans; Cell Differentiation / drug effects; Colforsin / pharmacology; Colforsin / metabolism; HLA-G Antigens / genetics; HLA-G Antigens / metabolism; Laminin / pharmacology; Stem Cells / cytology; Stem Cells / drug effects; Transforming Growth Factor beta / metabolism; Trophoblasts / cytology; Trophoblasts / metabolism; Culture Media / chemistry; Hypoxia-Inducible Factor 1, alpha Subunit / genetics; Gene Expression Regulation, Developmental / drug effects; Cytological Techniques / methods
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Human trophoblast stem cells (hTSCs) have emerged as a powerful tool to model early placental development in vitro. Analogous to the epithelial cytotrophoblast in the placenta, hTSCs can differentiate into cells of the extravillous trophoblast (EVT) lineage or the multinucleate syncytiotrophoblast (STB). Here we present a chemically defined culture system for STB and EVT differentiation of hTSCs. Notably, in contrast to current approaches, we neither utilize forskolin for STB formation nor transforming growth factor-beta (TGFβ) inhibitors or a passage step for EVT differentiation. Strikingly, the presence of a single additional extracellular cue–laminin-111–switched the terminal differentiation of hTSCs from STB to the EVT lineage under these conditions. In the absence of laminin-111, STB formation occurred, with cell fusion comparable to that obtained with differentiation mediated by forskolin; however, in the presence of laminin-111, hTSCs differentiated to the EVT lineage. Protein expression of nuclear hypoxia-inducible factors (HIF1α and HIF2α) was upregulated during EVT differentiation mediated by laminin-111 exposure. A heterogeneous mixture of Notch1+ EVTs in colonies and HLA-G+ single-cell EVTs were obtained without a passage step, reminiscent of heterogeneity in vivo. Further analysis showed that inhibition of TGFβ signaling affected both STB and EVT differentiation mediated by laminin-111 exposure. TGFβ inhibition during EVT differentiation resulted in decreased HLA-G expression and increased Notch1 expression. On the other hand, TGFβ inhibition prevented STB formation. The chemically defined culture system for hTSC differentiation established herein facilitates quantitative analysis of heterogeneity that arises during hTSC differentiation and will enable mechanistic studies in vitro. Human trophoblast stem cells (hTSCs) have emerged as a powerful tool to model early placental development in vitro. Analogous to the epithelial cytotrophoblast in the placenta, hTSCs can differentiate into cells of the extravillous trophoblast (EVT) lineage or the multinucleate syncytiotrophoblast (STB). Here we present a chemically defined culture system for STB and EVT differentiation of hTSCs. Notably, in contrast to current approaches, we neither utilize forskolin for STB formation nor transforming growth factor-beta (TGFβ) inhibitors or a passage step for EVT differentiation. Strikingly, the presence of a single additional extracellular cue–laminin-111–switched the terminal differentiation of hTSCs from STB to the EVT lineage under these conditions. In the absence of laminin-111, STB formation occurred, with cell fusion comparable to that obtained with differentiation mediated by forskolin; however, in the presence of laminin-111, hTSCs differentiated to the EVT lineage. Protein expression of nuclear hypoxia-inducible factors (HIF1α and HIF2α) was upregulated during EVT differentiation mediated by laminin-111 exposure. A heterogeneous mixture of Notch1+ EVTs in colonies and HLA-G+ single-cell EVTs were obtained without a passage step, reminiscent of heterogeneity in vivo. Further analysis showed that inhibition of TGFβ signaling affected both STB and EVT differentiation mediated by laminin-111 exposure. TGFβ inhibition during EVT differentiation resulted in decreased HLA-G expression and increased Notch1 expression. On the other hand, TGFβ inhibition prevented STB formation. The chemically defined culture system for hTSC differentiation established herein facilitates quantitative analysis of heterogeneity that arises during hTSC differentiation and will enable mechanistic studies in vitro. The placenta is a complex fetal organ with a vast network of villi that ensures efficient exchange of nutrients and waste across the maternal-fetal interface. Epithelial cytotrophoblasts (CTBs) of the early human placenta give rise to all trophoblast cell types in the placenta (1Aplin J.D. Developmental cell biology of human villous trophoblast: current research problems.Int. J. Dev. Biol. 2010; 54: 323-329Crossref PubMed Scopus (95) Google Scholar, 2Knöfler M. Vasicek R. Schreiber M. Key regulatory transcription factors involved in placental trophoblast development - a review.Placenta. 2001; 22: S83-S92Crossref PubMed Scopus (45) Google Scholar, 3Knöfler M. Haider S. Saleh L. Pollheimer J. Gamage T.K.J.B. James J. Human placenta and trophoblast development: key molecular mechanisms and model systems.Cell. Mol. Life Sci. 2019; 76: 3479-3496Crossref PubMed Scopus (311) Google Scholar, 4Loregger T. Pollheimer J. Knöfler M. Regulatory transcription factors controlling function and differentiation of human trophoblast - a review.Placenta. 2003; 24: S104-S110Crossref PubMed Scopus (26) Google Scholar). 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Inhibition of TGFβ during EVT differentiation precludes investigation into the role of TGFβ signaling during normal or pathological trophoblast differentiation. Similarly, STB differentiation in vitro predominantly relies on the use of forskolin to induce cell fusion. Forskolin raises cyclic AMP concentrations causing upregulation of GCM1, which controls expression of fusion genes, syncytin-1 and syncytin-2 (55Chang C.-W. Chang G.-D. Chen H. A novel cyclic AMP/Epac1/CaMKI signaling cascade promotes GCM1 desumoylation and placental cell fusion.Mol. Cell. Biol. 2011; 31: 3820-3831Crossref PubMed Scopus (39) Google Scholar). The use of forskolin impedes studies on extracellular cues regulating STB differentiation. Finally, it is important to note that heterogeneity of cell types is a central feature of EVT differentiation in vivo. Initially, epithelial CTBs form proliferative proximal cell columns that express column markers MYC, VE-cadherin, Notch1, as well as CTB markers EGFR and ITGA6 (3Knöfler M. Haider S. Saleh L. Pollheimer J. Gamage T.K.J.B. James J. Human placenta and trophoblast development: key molecular mechanisms and model systems.Cell. Mol. Life Sci. 2019; 76: 3479-3496Crossref PubMed Scopus (311) Google Scholar, 8Vićovac L. Aplin J.D. Epithelial-mesenchymal transition during trophoblast differentiation.Acta Anat. (Basel). 1996; 156: 202-216Crossref PubMed Scopus (209) Google Scholar, 11Benirschke K. Burton G.J. Baergen R.N. Pathology of the Human Placenta.6th Ed. Springer, Berlin Heidelberg2012Crossref Scopus (64) Google Scholar, 32Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. et al.Derivation of human trophoblast stem cells.Cell Stem Cell. 2018; 22: 50-63.e6Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 50Pollheimer J. Knöfler M. 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Under these culture conditions, we identified laminin-111-mediated upregulation of hypoxia-inducible factor-alpha (HIFα) as the critical input that switches differentiation hTSCs from STB to the EVT lineage. We also investigated the effect of inhibiting TGFβ signaling on EVT and STB differentiation. Placenta-derived CT29 and CT30 hTSCs and hiPSC-derived SC102A-1 hTSCs were cultured in trophoblast stem cell medium (TSCM) as described previously (32Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. et al.Derivation of human trophoblast stem cells.Cell Stem Cell. 2018; 22: 50-63.e6Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 38Mischler A. Karakis V. Mahinthakumar J. Carberry C.K. Miguel A.S. Rager J.E. et al.Two distinct trophectoderm lineage stem cells from human pluripotent stem cells.J. Biol. Chem. 2021; 296: 100386Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Differentiation was induced by passaging hTSCs into a defined trophoblast differentiation medium (DTDM) supplemented with epidermal growth factor (EGF) and the ROCK inhibitor, Y-27632, at passage for 2 days, and culturing them for an additional 4 days in DTDM (Fig. 1A). Upon passage, we initially observed an increase in cell number, but b