Abstract
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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
The data generated or analyzed during this study are included in this published article.
References
Saitou, M. & Miyauchi, H. Gametogenesis from pluripotent stem cells. Cell Stem Cell 18, 721–735 (2016).
Tang, W. W., Kobayashi, T., Irie, N., Dietmann, S. & Surani, M. A. Specification and epigenetic programming of the human germ line. Nat. Rev. Genet. 17, 585–600 (2016).
Lee, H. J., Hore, T. A. & Reik, W. Reprogramming the methylome: erasing memory and creating diversity. Cell Stem Cell 14, 710–719 (2014).
Spiller, C., Koopman, P. & Bowles, J. Sex determination in the mammalian germline. Annu. Rev. Genet. 51, 265–285 (2017).
Edson, M. A., Nagaraja, A. K. & Matzuk, M. M. The mammalian ovary from genesis to revelation. Endocr. Rev. 30, 624–712 (2009).
McGee, E. A. & Hsueh, A. J. Initial and cyclic recruitment of ovarian follicles. Endocr. Rev. 21, 200–214 (2000).
Manku, G. & Culty, M. Mammalian gonocyte and spermatogonia differentiation: recent advances and remaining challenges. Reproduction 149, R139–R157 (2015).
Griswold, M. D. Spermatogenesis: the commitment to meiosis. Physiol. Rev. 96, 1–17 (2016).
Hermann, B. P., Sukhwani, M., Hansel, M. C. & Orwig, K. E. Spermatogonial stem cells in higher primates: are there differences from those in rodents? Reproduction 139, 479–493 (2010).
Li, L. et al. Single-cell RNA-seq analysis maps development of human germline cells and gonadal niche interactions. Cell Stem Cell 20, 858–873.e4 (2017).
Yamashiro, C. et al. Generation of human oogonia from induced pluripotent stem cells in vitro. Science 362, 356–360 (2018).
Irie, N. et al. SOX17 is a critical specifier of human primordial germ cell fate. Cell 160, 253–268 (2015).
Chen, D. et al. Germline competency of human embryonic stem cells depends on eomesodermin. Biol. Reprod. 97, 850–861 (2017).
Hayashi, K., Ohta, H., Kurimoto, K., Aramaki, S. & Saitou, M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 146, 519–532 (2011).
Hayashi, K. et al. Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice. Science 338, 971–975 (2012).
Ishikura, Y. et al. In vitro derivation and propagation of spermatogonial stem cell activity from mouse pluripotent stem cells. Cell Rep. 17, 2789–2804 (2016).
Hikabe, O. et al. Reconstitution in vitro of the entire cycle of the mouse female germ line. Nature 539, 299–303 (2016).
Miyauchi, H. et al. Bone morphogenetic protein and retinoic acid synergistically specify female germ-cell fate in mice. EMBO J. 36, 3100–3119 (2017).
Nichols, J. & Smith, A. Naive and primed pluripotent states. Cell Stem Cell 4, 487–492 (2009).
Sasaki, K. et al. Robust in vitro induction of human germ cell fate from pluripotent stem cells. Cell Stem Cell 17, 178–194 (2015).
Yokobayashi, S. et al. Clonal variation of human induced pluripotent stem cells for induction into the germ cell fate. Biol. Reprod. 96, 1154–1166 (2017).
Kojima, Y. et al. Evolutionarily distinctive transcriptional and signaling programs drive human germ cell lineage specification from pluripotent stem cells. Cell Stem Cell 21, 517–532.e5 (2017).
Hayashi, K. & Saitou, M. Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells. Nat. Protoc. 8, 1513–1524 (2013).
Hayashi, K., Hikabe, O., Obata, Y. & Hirao, Y. Reconstitution of mouse oogenesis in a dish from pluripotent stem cells. Nat. Protoc. 12, 1733–1744 (2017).
Gkountela, S. et al. DNA demethylation dynamics in the human prenatal germline. Cell 161, 1425–1436 (2015).
Guo, F. et al. The transcriptome and DNA methylome landscapes of human primordial germ cells. Cell 161, 1437–1452 (2015).
Tang, W. W. et al. A unique gene regulatory network resets the human germline epigenome for development. Cell 161, 1453–1467 (2015).
Aramaki, S. et al. A mesodermal factor, T, specifies mouse germ cell fate by directly activating germline determinants. Dev. Cell 27, 516–529 (2013).
Sasaki, K. et al. The germ cell fate of cynomolgus monkeys is specified in the nascent amnion. Dev. Cell 39, 169–185 (2016).
Kobayashi, T. et al. Principles of early human development and germ cell program from conserved model systems. Nature 546, 416–420 (2017).
Gafni, O. et al. Derivation of novel human ground state naive pluripotent stem cells. Nature 504, 282–286 (2013).
Ohta, H. et al. In vitro expansion of mouse primordial germ cell-like cells recapitulates an epigenetic blank slate. EMBO J. 36, 1888–1907 (2017).
Patel, S. et al. Human embryonic stem cells do not change their X Inactivation status during differentiation. Cell Rep. 18, 54–67 (2017).
Sahakyan, A. et al. Human naive pluripotent stem cells model X chromosome dampening and X inactivation. Cell Stem Cell 20, 87–101 (2017).
Acknowledgements
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.
Author information
Authors and Affiliations
Contributions
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.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Peer review information Nature Protocols thanks Diana Laird and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Related links
Key references using this protocol
Yamashiro, C. et al. Science 362, 356–360 (2018): https://doi.org/10.1126/science.aat1674
Kojima, Y. et al. Cell Stem Cell 21, 517–532 (2017): https://doi.org/10.1016/j.stem.2017.09.005
Sasaki, K. et al. Cell Stem Cell 17, 178–194 (2015): https://doi.org/10.1016/j.stem.2015.06.014
Supplementary information
Rights and permissions
About this article
Cite this article
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). https://doi.org/10.1038/s41596-020-0297-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41596-020-0297-5
This article is cited by
-
Versatile extracellular vesicle-mediated information transfer: intercellular synchronization of differentiation and of cellular phenotypes, and future perspectives
Inflammation and Regeneration (2024)
-
Human primordial germ cell-like cells specified from resetting precursors develop in human hindgut organoids
Nature Protocols (2024)
-
Immunological Underpinnings of Autoimmune Primary Ovarian Insufficiency
Current Obstetrics and Gynecology Reports (2024)
-
Artificial Gametes and Human Reproduction in the 21st Century: An Ethical Analysis
Reproductive Sciences (2024)
-
The stromal microenvironment and ovarian aging: mechanisms and therapeutic opportunities
Journal of Ovarian Research (2023)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.