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Efficient Human Germ Cell Specification from Stem Cells via Combinatorial Expression of Transcription Factors

Christian Kramme, Merrick Pierson Smela, Bennett Wolf, Patrick R. Fortuna, Garyk Brixi, Kalyan Palepu, Edward Dong, Jessica Adams, Suhaas Bhat, Sabrina Koseki, Emma Tysinger, Teodora Stan, Richie E. Kohman, Songlei Liu, Mutsumi Kobayashi, Toshi Shioda, George M. Church, View ORCID ProfilePranam Chatterjee
doi: https://doi.org/10.1101/2022.07.11.499564
Christian Kramme
1Wyss Institute, Harvard Medical School
2Department of Genetics, Harvard Medical School
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Merrick Pierson Smela
1Wyss Institute, Harvard Medical School
2Department of Genetics, Harvard Medical School
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Bennett Wolf
1Wyss Institute, Harvard Medical School
2Department of Genetics, Harvard Medical School
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Patrick R. Fortuna
1Wyss Institute, Harvard Medical School
2Department of Genetics, Harvard Medical School
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Garyk Brixi
1Wyss Institute, Harvard Medical School
2Department of Genetics, Harvard Medical School
3Department of Biomedical Engineering, Duke University
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Kalyan Palepu
1Wyss Institute, Harvard Medical School
2Department of Genetics, Harvard Medical School
3Department of Biomedical Engineering, Duke University
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Edward Dong
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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Suhaas Bhat
1Wyss Institute, Harvard Medical School
2Department of Genetics, Harvard Medical School
3Department of Biomedical Engineering, Duke University
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Sabrina Koseki
4Center for Bits and Atoms, Massachusetts Institute of Technology
5Media Lab, Massachusetts Institute of Technology
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Emma Tysinger
4Center for Bits and Atoms, Massachusetts Institute of Technology
5Media Lab, Massachusetts Institute of Technology
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Teodora Stan
4Center for Bits and Atoms, Massachusetts Institute of Technology
5Media 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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Songlei Liu
1Wyss Institute, Harvard Medical School
2Department of Genetics, Harvard Medical School
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Mutsumi Kobayashi
6Massachusetts General Hospital Center for Cancer Research, Harvard Medical School
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Toshi Shioda
6Massachusetts General Hospital Center for Cancer Research, Harvard Medical School
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George M. Church
1Wyss Institute, Harvard Medical School
2Department of Genetics, Harvard Medical School
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Pranam Chatterjee
1Wyss Institute, Harvard Medical School
2Department of Genetics, Harvard Medical School
3Department of Biomedical Engineering, Duke University
4Center for Bits and Atoms, Massachusetts Institute of Technology
5Media Lab, Massachusetts Institute of Technology
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  • ORCID record for Pranam Chatterjee
  • For correspondence: pranam.chatterjee@duke.edu
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Abstract

Germ cells are the vehicle of human reproduction, arising early in embryonic development and developing throughout adult life until menopause onset in women. Primordial germ cells are the common precursors of germline cells in both sexes, undergoing sexual specification into oogonia or gonocytes which further develop into oocytes or spermatocytes during development. Methods for recapitulation of primordial germ cell and oogonia formation have been developed extensively in recent decades, but fundamental technical limitations in their methodologies, throughput, and yield limit their utilization. Recently, transcription factor (TF)-based methods for human primordial germ cell-like cell (hPGCLC) formation, mouse meiotic entry, and mouse oocyte maturation have demonstrated the feasibility of gene overexpression screening in identifying potent regulators of germ cell development. Here we screened 47 folliculogenesis-regulating TFs for their role in hPGCLC and oogonia formation, identifying DLX5, HHEX, and FIGLA whose individual overexpression enhances hPGCLC formation from hiPSCs. Additionally, we identify a set of three TFs, ZNF281, LHX8, and SOHLH1, whose combinatorial overexpression drives direct oogonia-like formation from hiPSCs in a four-day, feeder-free monolayer culture condition with additional feeder-free culture capabilities post-isolation. We characterize these TF-based germ cells via gene and protein expression analyses, and demonstrate their broad similarity to in vivo germ cells. Together, these results identify novel regulators of human germ cell development and establish new TF-based tools for human in vitro oogenesis research.

Competing Interest Statement

P.C., C.K., M.P.S., and G.C. are listed as inventors for U.S. Provisional Application No. 63/326,656, entitled: “Methods and Compositions for Producing Primordial Germ Cell-Like Cells,” and U.S. Provisional Application No. 63/326,607, entitled: “Methods and Compositions for Producing Oogonia-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.

Copyright 
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 4.0 International license.
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Posted July 12, 2022.
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Efficient Human Germ Cell Specification from Stem Cells via Combinatorial Expression of Transcription Factors
Christian Kramme, Merrick Pierson Smela, Bennett Wolf, Patrick R. Fortuna, Garyk Brixi, Kalyan Palepu, Edward Dong, Jessica Adams, Suhaas Bhat, Sabrina Koseki, Emma Tysinger, Teodora Stan, Richie E. Kohman, Songlei Liu, Mutsumi Kobayashi, Toshi Shioda, George M. Church, Pranam Chatterjee
bioRxiv 2022.07.11.499564; doi: https://doi.org/10.1101/2022.07.11.499564
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Efficient Human Germ Cell Specification from Stem Cells via Combinatorial Expression of Transcription Factors
Christian Kramme, Merrick Pierson Smela, Bennett Wolf, Patrick R. Fortuna, Garyk Brixi, Kalyan Palepu, Edward Dong, Jessica Adams, Suhaas Bhat, Sabrina Koseki, Emma Tysinger, Teodora Stan, Richie E. Kohman, Songlei Liu, Mutsumi Kobayashi, Toshi Shioda, George M. Church, Pranam Chatterjee
bioRxiv 2022.07.11.499564; doi: https://doi.org/10.1101/2022.07.11.499564

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