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Establishment of human induced trophoblast stem cells via reprogramming of fibroblasts

Nature Protocols volume 17, pages 2739–2759 (2022)Cite this article

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Abstract

During early mammalian embryonic development, trophoblast cells play an essential role in establishing cell–cell interactions at the maternal–fetal interface to ensure a successful pregnancy. In a recent study, we showed that human fibroblasts can be reprogrammed into induced trophoblast stem (iTS) cells by transcription factor-mediated nuclear reprogramming using the Yamanaka factors OCT4, KLF4, SOX2 and c-MYC (OKSM) and a selection of TS cell culture conditions. The derivation of TS cells from human blastocysts or first-trimester placenta can be limited by difficulties in obtaining adequate material as well as ethical implications. By contrast, the described approach allows the generation of iTS cells from the adult cells of individuals with diverse genetic backgrounds, which are readily accessible to many laboratories around the world. Here we describe a step-by-step protocol for the generation and establishment of human iTS cells directly from dermal fibroblasts using a non-integrative reprogramming method. The protocol consists of four main sections: (1) recovery of cryopreserved human dermal fibroblasts, (2) somatic cell reprogramming, (3) passaging of reprogramming intermediates and (4) derivation of iTS cell cultures followed by routine maintenance of iTS cells. These iTS cell lines can be established in 2–3 weeks and cultured long term over 50 passages. We also discuss several characterization methods that can be performed to validate the iTS cells derived using this approach. Our protocol allows researchers to generate patient-specific iTS cells to interrogate the trophoblast and placenta biology as well as their interactions with embryonic cells in health and diseases.

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Fig. 1: Schematic diagram of the iTS cell derivation workflow.
Fig. 2: Sendai virus transduction for iTS cell derivation.
Fig. 3: Routine maintenance of iTS cells.
Fig. 4: Molecular characterization of iTS cells.
Fig. 5: iTS cell differentiation into STs and EVTs.
Fig. 6: In vivo engraftment assay of iTS cells into NOD/SCID IL-2R-gamma knockout mice.

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Data availability

The main data discussed in this protocol were generated as part of the studies published in the supporting primary research paper15.

References

  1. Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    Article  CAS  PubMed  Google Scholar 

  2. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

    Article  CAS  PubMed  Google Scholar 

  4. Orendi, K. et al. Placental and trophoblastic in vitro models to study preventive and therapeutic agents for preeclampsia. Placenta 32 (Suppl.), S49–S54 (2011).

    Article  CAS  PubMed  Google Scholar 

  5. Sooranna, S. R., Oteng-Ntim, E., Meah, R., Ryder, T. A. & Bajoria, R. Characterization of human placental explants: morphological, biochemical and physiological studies using first and third trimester placenta. Hum. Reprod. 14, 536–541 (1999).

    Article  CAS  PubMed  Google Scholar 

  6. Turco, M. Y. & Moffett, A. Development of the human placenta. Development 146, dev163428 (2019).

    Article  CAS  PubMed  Google Scholar 

  7. Heazlewood, C. F. et al. High incidence of contaminating maternal cell overgrowth in human placental mesenchymal stem/stromal cell cultures: a systematic review. Stem Cells Transl. Med. 3, 1305–1311 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Apps, R. et al. Genome-wide expression profile of first trimester villous and extravillous human trophoblast cells. Placenta 32, 33–43 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Pattillo, R. A. & Gey, G. O. The establishment of a cell line of human hormone-synthesizing trophoblastic cells in vitro. Cancer Res. 28, 1231–1236 (1968).

    CAS  PubMed  Google Scholar 

  10. Kohler, P. O. & Bridson, W. E. Isolation of hormone-producing clonal lines of human choriocarcinoma. J. Clin. Endocrinol. Metab. 32, 683–687 (1971).

    Article  CAS  PubMed  Google Scholar 

  11. Hertz, R. Choriocarcinoma of women maintained in serial passage in hamster and rat. Proc. Soc. Exp. Biol. Med. 102, 77–81 (1959).

    Article  CAS  PubMed  Google Scholar 

  12. Amita, M. et al. Complete and unidirectional conversion of human embryonic stem cells to trophoblast by BMP4. Proc. Natl Acad. Sci. Us. A 110, E1212–E1221 (2013).

    Article  CAS  Google Scholar 

  13. Xu, R.-H. et al. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat. Biotechnol. 20, 1261–1264 (2002).

    Article  CAS  PubMed  Google Scholar 

  14. Okae, H. et al. Derivation of human trophoblast stem cells. Cell Stem Cell 22, 50–63.e6 (2018).

    Article  CAS  PubMed  Google Scholar 

  15. Liu, X. et al. Reprogramming roadmap reveals route to human induced trophoblast stem cells. Nature 586, 101–107 (2020).

    Article  CAS  PubMed  Google Scholar 

  16. Mischler, A., Karakis, V., Mahinthakumar, J. & Carberry, C. Two distinct trophectoderm lineage stem cells from human pluripotent stem cells. J. Biol. Chem. 296, 100386 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Wei, Y. et al. Efficient derivation of human trophoblast stem cells from primed pluripotent stem cells. Sci Adv 7, (2021).

  18. Li, Z., Kurosawa, O. & Iwata, H. Establishment of human trophoblast stem cells from human induced pluripotent stem cell-derived cystic cells under micromesh culture. Stem Cell Res. Ther. 10, 245 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Dong, C. et al. Derivation of trophoblast stem cells from naïve human pluripotent stem cells. eLife 9, e52504 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Cinkornpumin, J. K. et al. Naive human embryonic stem cells can give rise to cells with a trophoblast-like transcriptome and methylome. Stem Cell Rep. 15, 198–213 (2020).

    Article  CAS  Google Scholar 

  21. Guo, G. et al. Human naive epiblast cells possess unrestricted lineage potential. Cell. Stem Cell. 28, 1040–1056.e6 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Io, S. et al. Capturing human trophoblast development with naive pluripotent stem cells in vitro. Cell Stem Cell 28, 1023–1039.e13 (2021).

    Article  CAS  PubMed  Google Scholar 

  23. Castel, G. et al. Induction of human trophoblast stem cells from somatic cells and pluripotent stem cells. Cell Rep. 33, 108419 (2020).

    Article  CAS  PubMed  Google Scholar 

  24. Bayerl, J. et al. Principles of signaling pathway modulation for enhancing human naive pluripotency induction. Cell Stem Cell 28, 1549–1565.e12 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Castel, G. & David, L. Induction of human trophoblast stem cells. Nat. Protoc. https://doi.org/10.1038/s41596-022-00744-0 (2022).

  26. Chang, C.-W., Wakeland, A. K. & Parast, M. M. Trophoblast lineage specification, differentiation and their regulation by oxygen tension. J. Endocrinol. 236, R43–R56 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Scifres, C. M. & Nelson, D. M. Intrauterine growth restriction, human placental development and trophoblast cell death. J. Physiol. 587, 3453–3458 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Sheridan, M. A. et al. Early onset preeclampsia in a model for human placental trophoblast. Proc. Natl Acad. Sci. USA 116, 4336–4345 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Alici-Garipcan, A., Özçimen, B., Suder, I. & Ülker, V. NLRP7 plays a functional role in regulating BMP4 signaling during differentiation of patient-derived trophoblasts. bioRxiv (2019).

  30. Horii, M. et al. Human pluripotent stem cells as a model of trophoblast differentiation in both normal development and disease. Proc. Natl Acad. Sci. USA 113, E3882–E3891 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Takahashi, S. et al. Loss of p57KIP2 expression confers resistance to contact inhibition in human androgenetic trophoblast stem cells. Proc. Natl Acad. Sci. USA https://doi.org/10.1073/pnas.1916019116 (2019).

  32. Yu, J. et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797–801 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Warren, L. et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7, 618–630 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Turco, M. Y. et al. Trophoblast organoids as a model for maternal-fetal interactions during human placentation. Nature 564, 263–267 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Haider, S. et al. Self-renewing trophoblast organoids recapitulate the developmental program of the early human placenta. Stem Cell Rep. 11, 537–551 (2018).

    Article  CAS  Google Scholar 

  36. Paynter, J. M., Chen, J., Liu, X. & Nefzger, C. M. Propagation and maintenance of mouse embryonic stem cells. Methods Mol. Biol. 1940, 33–45 (2019).

    Article  CAS  PubMed  Google Scholar 

  37. Liu, X. et al. Generation of mouse-induced pluripotent stem cells by lentiviral transduction. Methods Mol. Biol. 1940, 63–76 (2019).

    Article  CAS  PubMed  Google Scholar 

  38. CytoTuneTM-iPS 2.0 Sendai Reprogramming Kit. https://www.thermofisher.com/order/catalog/product/A16517 (2022).

  39. Muir, A., Lever, A. M. L. & Moffett, A. Human endogenous retrovirus-W envelope (syncytin) is expressed in both villous and extravillous trophoblast populations. J. Gen. Virol. 87, 2067–2071 (2006).

    Article  CAS  PubMed  Google Scholar 

  40. Lee, C. Q. E. et al. What is trophoblast? A combination of criteria define human first-trimester trophoblast. Stem Cell Rep. 6, 257–272 (2016).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank the staff at Monash Flowcore Facility for providing high-quality cell sorting service and technical input. The authors acknowledge the use of the services and facilities of Micromon, Monash Micro Imaging and Monash Histology Platforms at Monash University. We also thank J. Hatwell-Humble and S. Nilsson for assistance with the mouse work. The schematics were created with BioRender.com. This work was supported by National Health and Medical Research Council (NHMRC) project grants APP1104560 and APP2004774 to J.M.P.; a Silvia and Charles Viertel Senior Medical Research Fellowship; and an ARC Future Fellowship FT180100674. J.P.T. was supported by a Monash International Tuition Scholarship and Research Training Program Scholarship. X.L. was supported by the Monash International Postgraduate Research Scholarship, a Monash Graduate Scholarship and the Carmela and Carmelo Ridolfo Prize in Stem Cell Research. The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government.

Author information

Authors and Affiliations

  1. Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia

    Jia Ping Tan, Xiaodong Liu & Jose M. Polo

  2. Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Clayton, Victoria, Australia

    Jia Ping Tan, Xiaodong Liu & Jose M. Polo

  3. Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria, Australia

    Jia Ping Tan, Xiaodong Liu & Jose M. Polo

  4. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China

    Xiaodong Liu

  5. School of Life Sciences, Westlake University, Hangzhou, China

    Xiaodong Liu

  6. Westlake Institute for Advanced Study, Hangzhou, China

    Xiaodong Liu

  7. Adelaide Centre for Epigenetics, Faculty of Medicine Nursing and Medical Sciences, The University of Adelaide, Adelaide, Australia

    Jose M. Polo

  8. The South Australian Immunogenomics Cancer Institute, Faculty of Medicine Nursing and Medical Sciences, The University of Adelaide, Adelaide, Australia

    Jose M. Polo

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Contributions

J.P.T. and X.L. drafted the protocol. J.P.T., X.L. and J.M.P. wrote the protocol. Most experiments presented in this protocol were performed by J.P.T. with supervision from X.L. and J.M.P.

Corresponding authors

Correspondence to Xiaodong Liu or Jose M. Polo.

Ethics declarations

Competing interests

Although not directly related to this paper, J.M.P. is a co-founder and shareholder of Mogrify Ltd., a cell therapy company. X.L. and J.M.P. are co-inventors on a PCT patent application (application number 2019904283) filed by Monash University, National University of Singapore and Université de Nantes related to work on derivation of iTS cells. The other authors declare no competing interests.

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Nature Protocols thanks Robert Morey, Kaela Varberg and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key reference using this protocol

Liu, X et al. Nature 586, 101–107 (2020): https://doi.org/10.1038/s41586-020-2734-6

Supplementary information

Supplementary Information

Flow cytometry gating strategy for live cells

Source data

Source Data Fig. 4

Values of the relative expression for each gene in Fig. 4b.

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Tan, J.P., Liu, X. & Polo, J.M. Establishment of human induced trophoblast stem cells via reprogramming of fibroblasts. Nat Protoc 17, 2739–2759 (2022). https://doi.org/10.1038/s41596-022-00742-2

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