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Isolation, characterization and propagation of mitotically active germ cells from adult mouse and human ovaries

Abstract

Accruing evidence indicates that production of new oocytes (oogenesis) and their enclosure by somatic cells (folliculogenesis) are processes not limited to the perinatal period in mammals. Endpoints ranging from oocyte counts to genetic lineage tracing and transplantation experiments support a paradigm shift in reproductive biology involving active renewal of oocyte-containing follicles during postnatal life. The recent purification of mitotically active oocyte progenitor cells, termed female germline stem cells (fGSCs) or oogonial stem cells (OSCs), from mouse and human ovaries opens up new avenues for research into the biology and clinical utility of these cells. Here we detail methods for the isolation of mouse and human OSCs from adult ovarian tissue, cultivation of the cells after purification, and characterization of the cells before and after ex vivo expansion. The latter methods include analysis of germ cell–specific markers and in vitro oogenesis, as well as the use of intraovarian transplantation to test the oocyte-forming potential of OSCs in vivo.

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Figure 1: Schematic overview of the OSC isolation protocol.
Figure 2: Oogenesis in OSC cultures.
Figure 3: In vivo differentiation of OSCs into oocytes.
Figure 4: Testing the functionality of OSC-derived oocytes.
Figure 5: Morphology of freshly isolated and ex vivo–expanded OSCs.
Figure 6: Expanded OSCs maintain a primitive germline gene expression profile.

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Acknowledgements

This work conducted by the authors and discussed herein was supported by a Method to Extend Research in Time (MERIT) Award from the National Institute on Aging (National Institutes of Health (NIH) R37-AG012279 to J.L.T.), a Ruth L. Kirschstein National Research Service Award (NIH F32-AG034809 to D.C.W.), a Glenn Foundation Award for Research in the Biological Mechanisms of Aging (J.L.T.) and the Henry and Vivian Rosenberg Philanthropic Fund (J.L.T.). We thank L. Prickett-Rice and K. Folz-Donahue of the Harvard Stem Cell Institute for outstanding technical assistance with flow cytometry, and M. Cooper (Cooper Graphics) for expert assistance with preparation of Figure 1.

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D.C.W. and J.L.T. contributed equally to the development and validation of the protocols described, and the preparation of this article for publication.

Corresponding author

Correspondence to Jonathan L Tilly.

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D.C.W. has no interests to declare; J.L.T. declares interest in intellectual property described in US Patent 7,195,775, US Patent 7,850,984 and US Patent 7,955,846, and he is a cofounder of OvaScience (Cambridge, Massachusetts, USA).

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Woods, D., Tilly, J. Isolation, characterization and propagation of mitotically active germ cells from adult mouse and human ovaries. Nat Protoc 8, 966–988 (2013). https://doi.org/10.1038/nprot.2013.047

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