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Efficient Induction and Isolation of Human Primordial Germ Cell-Like Cells from Competent Human Pluripotent Stem Cells

  • Naoko Irie
  • M. Azim SuraniEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1463)

Abstract

We recently reported a robust and defined culture system for the specification of human primordial germ cell-like cells (hPGCLCs) from human pluripotent stem cells (hPSCs), both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in vitro (Irie et al. Cell 160: 253–268, 2015). Similar attempts previously produced hPGCLCs from hPSCs at a very low efficiency, and the resulting cells were not fully characterized. A key step, which facilitated efficient hPGCLC specification from hPSCs, was the induction of a “competent” state for PGC fate via the medium containing a cocktail of four inhibitors. The competency of hPSCs can be maintained indefinitely and interchangeably with the conventional/low-competent hPSCs. Specification of hPGCLC occurs following sequential expression of key germ cell fate regulators, notably SOX17 and BLIMP1, as well as initiation of epigenetic resetting over 5 days. The hPGCLCs can be isolated using specific cell surface markers without the need for generating germ cell-specific reporter hPSC lines. This powerful method for the induction and isolation of hPGCLCs can be applied to both hESCs and iPSCs, which can be used for advances in human germ line biology.

Key words

Human primordial germ cells Human pluripotent stem cells Embryonic stem cells Induced pluripotent stem cells Primordial germ cell-like cells Germ cell specification Epiblasts Epigenetic resetting 

Notes

Acknowledgement

This work was supported by a Wellcome Trust Investigator Award, and by a BIRAX Grant. We thank Dr. Toshihiro Kobayashi, Dr. Roopsha Sengupta, and Dr. Carlos le Sage for critical reading of the manuscript. The institute is funded by a core grant from the Wellcome Trust (092096) and Cancer Research UK (C6946/A14492).

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Wellcome Trust Cancer Research UK Gurdon InstituteUniversity of CambridgeCambridgeUK
  2. 2.Department of Physiology, Development and NeuroscienceUniversity of CambridgeCambridgeUK

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