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The ontogeny of cKIT+ human primordial germ cells proves to be a resource for human germ line reprogramming, imprint erasure and in vitro differentiation

Nature Cell Biology volume 15, pages 113–122 (2013)Cite this article

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Abstract

The generation of research-quality, clinically relevant cell types in vitro from human pluripotent stem cells requires a detailed understanding of the equivalent human cell types. Here we analysed 134 human embryonic and fetal samples from 6 to 20 developmental weeks and identified the stages at which cKIT+ primordial germ cells (PGCs), the precursors of gametes, undergo whole-genome epigenetic reprogramming with global depletion of 5mC, H3K27me3 and H2A.Z, and the time at which imprint erasure is initiated and 5hmC is present. Using five alternative in vitro differentiation strategies combined with single-cell microfluidic analysis and a bona fide human cKIT+ PGC signature, we show the stage of cKIT+ PGC formation in the first 16 days of differentiation. Taken together, our study creates a resource of human germ line ontogeny that is essential for future studies aimed at in vitro differentiation and unveiling the mechanisms necessary to pass human DNA from one generation to the next.

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Figure 1: The dynamics of cKIT, OCT4A and VASA expression in the fetal gonad.
Figure 2: Molecular characterization of cKIT+ PGCs from 7 to 20 developmental weeks.
Figure 3: Global loss of 5mC precedes loss of 5hmC.
Figure 4: Epigenetic reprogramming of H3K27me3 and H2A.Z occurs in the common PGC progenitor.
Figure 5: RNA-Seq reveals the transcriptional identity of cKIT+ PGCs.
Figure 6: In vitro hESC differentiation generates rare germ line progenitors that are cKIT/TRA-1-81 positive.
Figure 7: In vitro PGC differentiation from hESCs using five alternative differentiation techniques.
Figure 8: Summarized roadmap of human germ line development.

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Acknowledgements

The authors would like to thank the UCLA Translational Pathology Core Laboratory and the UCLA Gene and Cellular Core Laboratory for some of the gonadal samples used in this study. We also thank J. Hargan-Calvopina, M. Oliveros-Etter and S. Diaz-Perez for critical reading of the manuscript, F. Codrea and J. Scholes for FACS and S. Peckman from the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research for critical assistance with human subject and embryonic stem cell review. This work was supported primarily by fund number 1R01HD058047 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD; ATC), as well as the Iris Cantor-UCLA Women’s Health Pilot Project (ATC) and 1P01GM081621 from NIGMS. The Laboratory of Developmental Biology, University of Washington, Seattle is supported by NIH Award Number 5R24HD000836 from the NICHD. Human fetal tissue requests can be made to: bdrl@u.washington.edu.

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Authors and Affiliations

  1. Department of Molecular Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California 90095, USA

    Sofia Gkountela, Ziwei Li, John J. Vincent, Matteo Pellegrini & Amander T. Clark

  2. Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California Los Angeles, Los Angeles, California 90095, USA

    Sofia Gkountela, Ziwei Li, John J. Vincent & Amander T. Clark

  3. Molecular Biology Institute, University of California Los Angeles, Los Angeles, California 90095, USA

    John J. Vincent & Amander T. Clark

  4. Department of Biological Chemistry, Howard Hughes Medical Institute, University of California Los Angeles, Los Angeles, California 90095, USA

    Kelvin X. Zhang

  5. Obstetrics & Gynecology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California 90095, USA

    Angela Chen

  6. Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, California 90095, USA

    Amander T. Clark

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Contributions

S.G. designed and performed the experiments, analysed data and wrote the manuscript; Z.L. performed flow analyses, RNA and DNA extraction for some gonadal samples, J.J.V. performed the single-cell analysis of female ovary at 14 weeks; K.X.Z. and M.P. performed the RNA-Seq data analysis; A.C. provided gonadal samples used in this study; A.T.C. designed the experiments, analysed data and wrote the manuscript.

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Correspondence to Amander T. Clark.

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Gkountela, S., Li, Z., Vincent, J. et al. The ontogeny of cKIT+ human primordial germ cells proves to be a resource for human germ line reprogramming, imprint erasure and in vitro differentiation. Nat Cell Biol 15, 113–122 (2013). https://doi.org/10.1038/ncb2638

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