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Human primordial germ cell-like cells specified from resetting precursors develop in human hindgut organoids

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Abstract

Human primordial germ cells (hPGCs), the precursors of eggs and sperm, start their complex development shortly after specification and during their migration to the primitive gonads. Here, we describe protocols for specifying hPGC-like cells (hPGCLCs) from resetting precursors and progressing them with the support of human hindgut organoids. Resetting hPGCLCs (rhPGCLCs) are specified from human embryonic stem cells (hESCs) transitioning from the primed into the naive state of pluripotency. Hindgut organoids are also derived from hESCs after a sequential differentiation into a posterior endoderm/hindgut fate. Both rhPGCLCs and hindgut organoids are combined and co-cultured for 25 d. The entire procedure takes ~1.5 months and can be successfully implemented by a doctoral or graduate student with basic skills and experience in hESC cultures. The co-culture system supports the progression of rhPGCLCs at a developmental timing analogous to that observed in vivo. Compared with previously developed hPGCLC progression protocols, which depend on co-cultures with mouse embryonic gonadal tissue, our co-culture system represents a developmentally relevant model closer to the environment that hPGCs first encounter after specification. Together with the potential for investigations of events during hPGC specification and early development, these protocols provide a practical approach to designing efficient models for in vitro gametogenesis. Notably, the rhPGCLC-hindgut co-culture system can also be adapted to study failings in hPGC migration, which are associated with the etiology of some forms of infertility and germ cell tumors.

Key points

  • This protocol describes the specification of rhPGCLCs with enhanced progression capabilities and introduces a co-culture system with human hindgut organoids, which enables rhPGCLC progression at a pace that closely mirrors in vivo hPGC development.

  • By reconstituting early gametogenesis from hPSCs, this method will facilitate biochemical and molecular analysis of hPGC specification and help to clarify the complex crosstalk between hPGCs and the hindgut, critical for understanding various reproductive disorders.

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Fig. 1: Overview of the protocol.
Fig. 2: Primed and resetting hESC morphology.
Fig. 3: EB morphology.
Fig. 4: FACS strategy for rhPGCLC isolation from day 4 EBs.
Fig. 5: Human hindgut organoid differentiation.
Fig. 6: rhPGCLC-human hindgut organoid co-culture.
Fig. 7: Progression analysis and FACS isolation strategy for rhPGCLC co-cultures with human hindgut organoids.
Fig. 8: Final co-culture ratios between rhPGCLCs and human hindgut organoids (HGs).

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Data availability

The data generated or analyzed for the elaboration of the protocol are included in this publication.

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Acknowledgements

We thank all members of the M.A.S. laboratory for their discussions and suggestions. J.P.A.-L. was supported by a Marie Skłodowska-Curie Actions Individual Fellowship (836291: GERMINOID) from the European Commission, by an International Postdoc Fellowship (2019-06316) from the Swedish Research Council, and by a Starting Grant in Medicine and Health (2022-01467) from the Swedish Research Council. M.A.S. was supported by Wellcome Investigator Awards in Science (209475/Z/17/Z and 096738/Z/11/Z), an MRC research grant (RG85305) and a BBSRC research grant (G103986). Some of the schematic diagrams were created with Biorender.com.

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Authors and Affiliations

  1. Wellcome/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK

    João Pedro Alves-Lopes, Frederick C. K. Wong & M. Azim Surani

  2. Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK

    João Pedro Alves-Lopes, Frederick C. K. Wong & M. Azim Surani

  3. NORDFERTIL Research Lab Stockholm, Childhood Cancer Research Unit, J9:30, Department of Women’s and Children’s Health, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden

    João Pedro Alves-Lopes

  4. Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge, UK

    Frederick C. K. Wong

Authors
  1. João Pedro Alves-Lopes
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Contributions

J.P.A.-L. and F.C.K.W. conceived and designed the experiments and performed cell culture and FACS analysis. J.P.A.-L. conceived the hypothesis and executed organoid co-culture experiments and immunostainings. J.P.A.-L. wrote the manuscript with contributions from F.C.K.W. and M.A.S. M.A.S. supervised the project and gave intellectual input.

Corresponding authors

Correspondence to João Pedro Alves-Lopes or M. Azim Surani.

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The authors declare no competing interests.

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Nature Protocols thanks Ernesto Rojas, Toshishiro Shioda, Patrick Western 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

Alves-Lopes, J. P. et al. Cell Rep. 42, 111907 (2023): https://doi.org/10.1016/j.celrep.2022.111907

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Alves-Lopes, J.P., Wong, F.C.K. & Surani, M.A. Human primordial germ cell-like cells specified from resetting precursors develop in human hindgut organoids. Nat Protoc 19, 1149–1182 (2024). https://doi.org/10.1038/s41596-023-00945-1

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  • DOI: https://doi.org/10.1038/s41596-023-00945-1

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