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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.

Data availability

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

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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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