Abstract
Human pluripotent stem cells (hPSCs) form an ideal system to study the formation of placental cells, from an undifferentiated human embryonic stem cell state. The conventional human in vitro model systems to study the human placenta cannot be employed for understanding placental dysfunctions or the development of specialized placental cell types. Hence, human PSCs make an ideal model system to study human placental development and disorders. Here, we describe an efficient and validated protocol to reproducibly study the formation of human cytotrophoblasts (CTBs) and syncytiotrophoblast (STBs) from undifferentiated hPSCs. CTBs are the trophoblast stem cells that can differentiate into specialized placental cell types such as STBs. The multinucleated STB plays vital role in the exchange of nutrients and gases across the placenta and secretes several hormones during pregnancy, such as human chorionic gonadotropin β (hCGβ). Here we describe two methods of seeding the hPSCs: chemical (clumps method) and enzymatic methods (single cells) to differentiate them to CTB and STB, activating BMP (B) signaling and inhibiting ACTIVIN/NODAL and FGF signaling pathways (2i), thus naming our protocol as “B2i” (Sudheer et al., Stem Cells Dev 21:2987–3000, 2012). This protocol forms the perfect model system for understanding in vitro placentation, modeling diseases arising from abnormal placentation that cause complications such as miscarriage, preeclampsia or intrauterine growth restriction (IUGR), and drug discovery for placental disorders.
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Acknowledgments
We are very much grateful to Dr. R. V Shaji, CSCR, Vellore, India, for his support and the generous gift of the hiPSCs (D14C2). We thank Dr. Raghu Bhushan, YRC, Yenepoya (Deemed to be) University, Mangalore, India, for reading the chapter and helpful comments. SM.S. acknowledges the financial support from the Department of Biotechnology (DBT) (BT/PR29717/PFN/20/1361/2018), Indian Council of Medical Research (ICMR) (2019-3008/SCR/ADHOC/BMS) and Council of Scientific and Industrial Research (CSIR) (09/1108(13739)/2022-EMR-1), New Delhi, India.
Authors’ Contributions
A.S.A. and D.A.S. conducted the experiments and analysis. A.S.A., D.A.S., and SM.S. contributed to the analysis, validation, and writing of the original draft. SM.S. reviewed and edited the chapter and contributed to the conceptualization, methodology, supervision, project administration, and funding acquisition.
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Fig. S1
B2i treatment of hiPSCs seeded using the Chemical (clumps) method differentiates them to trophoblast. (a) Temporal changes observed in the cell morphology of hiPSCs before (0 HR) and during the B2i treatment (after 2 and 8 days). Quantitative RT PCR analysis of the expression of the CTB markers (b), Pan-trophoblast marker (c) and STB markers (d) in the undifferentiated hiPSC and the hiPSCs-derived CTB and STB-like cells. The data is normalized against the housekeeping gene, GAPDH. Error bars: Mean ± SD (n = 2) (DOCX 1218 kb)
Fig. S2
B2i treatment of hiPSCs seeded using the enzymatic (single cells) method differentiates them to trophoblast. (a) Temporal changes observed in cell morphology of hiPSCs before (0 HR) and during the B2i treatment (after 2 and 8 days). Quantitative RT PCR analysis of the expression of the CTB markers (b) and STB markers (c, d) in the undifferentiated hiPSC and the hiPSCs-derived CTB and STB-like cells. The data is normalized against the housekeeping gene, ACTB. Error bars: Mean ± SD (n = 2) (DOCX 1207 kb)
Table S1
List of primers and their sequences (5′- 3′) used for qRT PCR (DOCX 13 kb)
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Antony, A.S., Devika, A.S., Sudheer, S. (2023). Directed Differentiation of Human Pluripotent Stem Cells to Cytotrophoblast and Syncytiotrophoblast. In: Zernicka-Goetz, M., Turksen, K. (eds) Embryo Models In Vitro. Methods in Molecular Biology, vol 2767. Humana, New York, NY. https://doi.org/10.1007/7651_2022_469
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DOI: https://doi.org/10.1007/7651_2022_469
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