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Human Primordial Germ Cell-Like Cell Induction from Pluripotent Stem Cells by SOX17 and PRDM1 Expression

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Germ Cell Development

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2770))

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Abstract

Human primordial germ cell (PGC) development initiates about 2 weeks after fertilization during embryogenesis. Unique molecular events follow, including epigenetic resetting, to establish functional gametes (egg and sperm). Due to the inaccessibility of human embryos, it is essential to have an amenable experimental platform to investigate the mechanisms and potential dysfunctions of the events. We previously established a PGC-like cell (PGCLC) differentiation method using human pluripotent stem cells (PSCs) via induction of precursor cells followed by stimulation with a cytokine cocktail including BMP. We also revealed that the expression of PGC specifiers, SOX17 and PRDM1, can robustly induce PGCLCs from PSCs without the cytokines. The balance of SOX17 and PRDM1 is critical for germ cell fate since the two factors also regulate endoderm differentiation. Here we describe a detailed procedure for PGCLC differentiation with the balanced induction of SOX17 and PRDM1. The protocol can be used for PGC induction in other mammalian species exhibiting PGCs with SOX17 expression. Together, these studies will advance the understanding of germ cell biology and its applications in reproductive technology and medicine.

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References

  1. Felici MD (2012) Origin, migration, and proliferation. Oogenesis:19–37. https://doi.org/10.1007/978-0-85729-826-3_2

  2. Tang WWC, Kobayashi T, Irie N et al (2016) Specification and epigenetic programming of the human germ line. Nat Rev Genet 17:585–600. https://doi.org/10.1038/nrg.2016.88

    Article  CAS  PubMed  Google Scholar 

  3. Saitou M, Hayashi K (2021) Mammalian in vitro gametogenesis. Science 374:eaaz6830. https://doi.org/10.1126/science.aaz6830

    Article  CAS  PubMed  Google Scholar 

  4. Fujimoto T, Miyayama Y, Fuyuta M (1977) The origin, migration and fine morphology of human primordial germ cells. Anat Rec 188:315–329. https://doi.org/10.1002/ar.1091880305

    Article  CAS  PubMed  Google Scholar 

  5. Tang WWC, Dietmann S, Irie N et al (2015) A unique gene regulatory network resets the human germline epigenome for development. Cell 161:1453–1467. https://doi.org/10.1016/j.cell.2015.04.053

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Gkountela S, Zhang KX, Shafiq TA et al (2015) DNA demethylation dynamics in the human prenatal germline. Cell 161:1425–1436. https://doi.org/10.1016/j.cell.2015.05.012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Guo F, Yan L, Guo H et al (2015) The transcriptome and DNA methylome landscapes of human primordial germ cells. Cell 161:1437–1452. https://doi.org/10.1016/j.cell.2015.05.015

    Article  CAS  PubMed  Google Scholar 

  8. Guo H, Hu B, Yan L et al (2017) DNA methylation and chromatin accessibility profiling of mouse and human fetal germ cells. Cell Res 27:165–183. https://doi.org/10.1038/cr.2016.128

    Article  CAS  PubMed  Google Scholar 

  9. Gruhn WH, Tang WWC, Dietmann S et al (2023) Epigenetic resetting in the human germ line entails histone modification remodeling. Sci Adv 9. https://doi.org/10.1126/sciadv.ade1257

  10. Irie N, Weinberger L, Tang WWC et al (2015) SOX17 is a critical specifier of human primordial germ cell fate. Cell 160:253–268. https://doi.org/10.1016/j.cell.2014.12.013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kobayashi T, Zhang H, Tang WWC et al (2017) Principles of early human development and germ cell program from conserved model systems. Nature 546:416–420. https://doi.org/10.1038/nature22812

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Sasaki K, Yokobayashi S, Nakamura T et al (2015) Robust in vitro induction of human germ cell fate from pluripotent stem cells. Cell Stem Cell 17:178–194. https://doi.org/10.1016/j.stem.2015.06.014

    Article  CAS  PubMed  Google Scholar 

  13. Kobayashi T, Castillo-Venzor A, Penfold CA et al (2021) Tracing the emergence of primordial germ cells from bilaminar disc rabbit embryos and pluripotent stem cells. Cell Rep 37:109812. https://doi.org/10.1016/j.celrep.2021.109812

    Article  CAS  PubMed  Google Scholar 

  14. Sasaki K, Nakamura T, Okamoto I et al (2016) The germ cell fate of cynomolgus monkeys is specified in the nascent amnion. Dev Cell 39:169–185. https://doi.org/10.1016/j.devcel.2016.09.007

    Article  CAS  PubMed  Google Scholar 

  15. Seita Y, Cheng K, McCarrey JR et al (2023) Efficient generation of marmoset primordial germ cell-like cells using induced pluripotent stem cells. elife 12:e82263. https://doi.org/10.7554/elife.82263

    Article  PubMed  PubMed Central  Google Scholar 

  16. Hayashi M, Zywitza V, Naitou Y et al (2022) Robust induction of primordial germ cells of white rhinoceros on the brink of extinction. Sci Adv 8:eabp9683. https://doi.org/10.1126/sciadv.abp9683

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Hara K, Kanai-Azuma M, Uemura M et al (2009) Evidence for crucial role of hindgut expansion in directing proper migration of primordial germ cells in mouse early embryogenesis. Dev Biol 330:427–439. https://doi.org/10.1016/j.ydbio.2009.04.012

    Article  CAS  PubMed  Google Scholar 

  18. Kanai-Azuma M, Kanai Y, Gad JM et al (2002) Depletion of definitive gut endoderm in Sox17 -null mutant mice. Development 129:2367–2379. https://doi.org/10.1242/dev.129.10.2367

    Article  CAS  PubMed  Google Scholar 

  19. Kobayashi T, Kobayashi H, Goto T et al (2020) Germline development in rat revealed by visualization and deletion of Prdm14. Development 147:dev.183798. https://doi.org/10.1242/dev.183798

    Article  CAS  Google Scholar 

  20. Oikawa M, Kobayashi H, Sanbo M et al (2022) Functional primordial germ cell–like cells from pluripotent stem cells in rats. Science 376:176–179. https://doi.org/10.1126/science.abl4412

    Article  CAS  PubMed  Google Scholar 

  21. Alberio R, Kobayashi T, Surani MA (2021) Conserved features of non-primate bilaminar disc embryos and the germline. Stem Cell Reports 16:1078–1092. https://doi.org/10.1016/j.stemcr.2021.03.011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kojima Y, Yamashiro C, Murase Y et al (2021) GATA transcription factors, SOX17 and TFAP2C, drive the human germ-cell specification program. Life Sci Alliance 4:e202000974. https://doi.org/10.26508/lsa.202000974

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Tang WWC, Castillo-Venzor A, Gruhn WH et al (2022) Sequential enhancer state remodelling defines human germline competence and specification. Nat Cell Biol:1–13. https://doi.org/10.1038/s41556-022-00878-z

  24. Shono M, Kishimoto K, Hikabe O et al (2023) Induction of primordial germ cell-like cells from common marmoset embryonic stem cells by inhibition of WNT and retinoic acid signaling. Sci Rep 13:3186. https://doi.org/10.1038/s41598-023-29850-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Irie N, Surani MA (2016) Methods molecular biology, pp 217–226. https://doi.org/10.1007/978-1-4939-4017-2_16

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Acknowledgments

The work was supported by an M.R.C. research grant (RG85305) and Rosetrees Trust to M.A.S. and N.I., and by a Wellcome Investigator Awards in Science (209475/Z/17/Z and 096738/Z/11/Z) and a BBSRC research grant (G103986) to M.A.S., and by a core grant to the Gurdon Institute by Wellcome and Cancer Research U.K. T.K. is supported by AMED (JP22bm1123008). We thank Dr. Carlos le Sage for the critical reading of the manuscript.

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Authors and Affiliations

  1. Wellcome Trust/Cancer Research U.K. Gurdon Institute, Henry Wellcome Building of Cancer and Developmental Biology, Cambridge, UK

    Naoko Irie & M. Azim Surani

  2. Metabolic Systems Laboratory, Live Imaging Center, Central Institute for Experimental Animals, Kawasaki-ku, Kanagawa, Japan

    Naoko Irie

  3. Division of Mammalian Embryology, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan

    Toshihiro Kobayashi

  4. Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Okazaki, Aichi, Japan

    Toshihiro Kobayashi

  5. Physiology, Development and Neuroscience Department, University of Cambridge, Cambridge, UK

    M. Azim Surani

Authors
  1. Naoko Irie
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  2. Toshihiro Kobayashi
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  3. M. Azim Surani
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Corresponding authors

Correspondence to Naoko Irie or M. Azim Surani .

Editor information

Editors and Affiliations

  1. Department of Biomedicine and Prevention, Section of Human Anatomy, Faculty of Medicine and Surgery, “Tor Vergata“ University of Rome, Rome, Italy

    Marco Barchi

  2. Department of Biomedicine and Prevention, Section of Histology and Embryology, Faculty of Medicine and Surgery, “Tor Vergata” University of Rome, Rome, Italy

    Massimo De Felici

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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Irie, N., Kobayashi, T., Azim Surani, M. (2024). Human Primordial Germ Cell-Like Cell Induction from Pluripotent Stem Cells by SOX17 and PRDM1 Expression. In: Barchi, M., De Felici, M. (eds) Germ Cell Development. Methods in Molecular Biology, vol 2770. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3698-5_7

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  • DOI: https://doi.org/10.1007/978-1-0716-3698-5_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3697-8

  • Online ISBN: 978-1-0716-3698-5

  • eBook Packages: Springer Protocols

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