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Directed Differentiation of Human iPSCs to Functional Ovarian Granulosa-Like Cells via Transcription Factor Overexpression

Merrick Pierson Smela, Christian Kramme, Patrick Fortuna, Jessica Adams, Edward Dong, Mutsumi Kobayashi, Garyk Brixi, Emma Tysinger, Richie. E. Kohman, Toshi Shioda, View ORCID ProfilePranam Chatterjee, George M. Church
doi: https://doi.org/10.1101/2022.07.04.498717
Merrick Pierson Smela
1Wyss Institute, Harvard Medical School
2Department of Genetics, Harvard Medical School
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Christian Kramme
1Wyss Institute, Harvard Medical School
2Department of Genetics, Harvard Medical School
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Patrick Fortuna
1Wyss Institute, Harvard Medical School
2Department of Genetics, Harvard Medical School
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Jessica Adams
1Wyss Institute, Harvard Medical School
2Department of Genetics, Harvard Medical School
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Edward Dong
1Wyss Institute, Harvard Medical School
2Department of Genetics, Harvard Medical School
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Mutsumi Kobayashi
3Massachusetts General Hospital Center for Cancer Research, Harvard Medical School
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Garyk Brixi
4Department of Biomedical Engineering, Duke University
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Emma Tysinger
5Center for Bits and Atoms, Massachusetts Institute of Technology
6Media Lab, Massachusetts Institute of Technology
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Richie. E. Kohman
1Wyss Institute, Harvard Medical School
2Department of Genetics, Harvard Medical School
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Toshi Shioda
3Massachusetts General Hospital Center for Cancer Research, Harvard Medical School
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Pranam Chatterjee
1Wyss Institute, Harvard Medical School
2Department of Genetics, Harvard Medical School
4Department of Biomedical Engineering, Duke University
5Center for Bits and Atoms, Massachusetts Institute of Technology
6Media Lab, Massachusetts Institute of Technology
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  • ORCID record for Pranam Chatterjee
  • For correspondence: pranam.chatterjee@duke.edu
George M. Church
1Wyss Institute, Harvard Medical School
2Department of Genetics, Harvard Medical School
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Abstract

An in vitro model of human ovarian follicles would greatly benefit the study of female reproduction. Ovarian development requires the combination of germ cells and their supporting somatic cells, known as granulosa cells. Whereas efficient protocols exist for generating human primordial germ cell-like cells (hPGCLCs) from human iPSCs, a method of generating granulosa cells has been elusive. Here we report that simultaneous overexpression of two transcription factors (TFs) can direct the differentiation of human iPSCs to granulosa-like cells. We elucidate the regulatory effects of several granulosa-related TFs, and establish that overexpression of NR5A1 and either RUNX1 or RUNX2 is necessary and sufficient to generate granulosa-like cells. Our granulosa-like cells form ovary-like organoids (ovaroids) when aggregated with hPGCLCs, and recapitulate key ovarian phenotypes including support of germ cell maturation, follicle formation, and steroidogenesis. This model system will provide unique opportunities for studying human ovarian biology, and may enable the development of therapies for female reproductive health.

Competing Interest Statement

P.C., C.K., M.P.S., and G.M.C. are listed as inventors for U.S. Provisional Application No. 63/326,640, entitled “Methods and Compositions for Producing Granulosa-Like Cells.” P.C. is a co-founder and scientific advisor to Gameto, Inc. C.K. is the Chief Scientific Officer of Gameto, Inc. G.M.C. serves on the scientific advisory board of Gameto, Inc., Colossal Biosciences, and GCTx.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted July 04, 2022.
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Directed Differentiation of Human iPSCs to Functional Ovarian Granulosa-Like Cells via Transcription Factor Overexpression
Merrick Pierson Smela, Christian Kramme, Patrick Fortuna, Jessica Adams, Edward Dong, Mutsumi Kobayashi, Garyk Brixi, Emma Tysinger, Richie. E. Kohman, Toshi Shioda, Pranam Chatterjee, George M. Church
bioRxiv 2022.07.04.498717; doi: https://doi.org/10.1101/2022.07.04.498717
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Directed Differentiation of Human iPSCs to Functional Ovarian Granulosa-Like Cells via Transcription Factor Overexpression
Merrick Pierson Smela, Christian Kramme, Patrick Fortuna, Jessica Adams, Edward Dong, Mutsumi Kobayashi, Garyk Brixi, Emma Tysinger, Richie. E. Kohman, Toshi Shioda, Pranam Chatterjee, George M. Church
bioRxiv 2022.07.04.498717; doi: https://doi.org/10.1101/2022.07.04.498717

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