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

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Alves Lopes, Joao 
Wong, F 
Surani, Azim 

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 naïve state of pluripotency. Hindgut organoids are also derived from hESCs following a sequential differentiation into posterior endoderm/hindgut fate. Both rhPGCLCs and hindgut organoids are combined and co-cultured for 25 days. The entire procedure takes approximately 1.5 months and can be successfully implement 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 aetiology of some forms of infertility and germ cell tumours.

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Nature Protocols
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Nature Research
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Wellcome Trust (209475/Z/17/Z)
Wellcome Trust (096738/Z/11/Z)
Medical Research Council (MR/P009948/1)
BBSRC (BB/T01346X/1)
European Commission Horizon 2020 (H2020) Marie Sk?odowska-Curie actions (836291)
Swedish Research Council (2019-06316) Swedish Research Council (2022-01467)