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Generation of human oogonia from induced pluripotent stem cells in culture


The human germ-cell lineage originates as human primordial germ cells (hPGCs). hPGCs undergo genome-wide epigenetic reprogramming and differentiate into oogonia or gonocytes, precursors for oocytes or spermatogonia, respectively. Here, we describe a protocol to differentiate human induced pluripotent stem cells (hiPSCs) into oogonia in vitro. hiPSCs are induced into incipient mesoderm-like cells (iMeLCs) using activin A and a WNT pathway agonist. iMeLCs, or, alternatively, hPSCs cultured with divergent signaling inhibitors, are induced into hPGC-like cells (hPGCLCs) in floating aggregates by cytokines including bone morphogenic protein 4. hPGCLCs are aggregated with mouse embryonic ovarian somatic cells to form xenogeneic reconstituted ovaries, which are cultured under an air–liquid interface condition for ~4 months for hPGCLCs to differentiate into oogonia and immediate precursory states for oocytes. To date, this is the only approach that generates oogonia from hPGCLCs. The protocol is suitable for investigating the mechanisms of hPGC specification and epigenetic reprogramming.

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Fig. 1: A scheme for human germ-cell development.
Fig. 2: Overview of the protocol.
Fig. 3: Phase-contrast images of hiPSCs and iMeLCs.
Fig. 4: A photomicrograph of a tungsten needle.
Fig. 5: A photomicrograph of a mouth pipette equipped with a glass capillary.
Fig. 6: Bright-field images of the iMeLC aggregates for hPGCLC induction and xrOvaries.
Fig. 7: The FACS gatings for sorting hPGCLCs or hPGCLC-derived cells.
Fig. 8: FACS plots of xrOvaries for detecting the differentiation of oogonia and RA-responsive FGCs from d6 hPGCLCs at ag77 and ag120.

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We thank the members of our laboratory for their helpful input on this study. We are grateful to Y. Nagai, N. Konishi, E. Tsutsusmi and M. Kawasaki for their technical assistance. This work was supported by a Grant-in-Aid for Specially Promoted Research from JSPS (17H06098), a JST-ERATO Grant (JPMJER1104), a grant from HFSP (RGP0057/2018) and grants from the Pythias Fund and Open Philanthropy Project to M.S.

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Authors and Affiliations



C.Y. and M.S. conceived the study and experimental design and co-wrote the manuscript. K.S., S.Y. and Y.K. contributed to the hPGCLC induction. C.Y. produced the images for the figures and the schematics. C.Y. analyzed the data and performed the experiments.

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Correspondence to Mitinori Saitou.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Diana Laird and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Yamashiro, C. et al. Science 362, 356–360 (2018):

Kojima, Y. et al. Cell Stem Cell 21, 517–532 (2017):

Sasaki, K. et al. Cell Stem Cell 17, 178–194 (2015):

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Yamashiro, C., Sasaki, K., Yokobayashi, S. et al. Generation of human oogonia from induced pluripotent stem cells in culture. Nat Protoc 15, 1560–1583 (2020).

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