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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

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.

References

  1. Ohinata, Y. et al. A signaling principle for the specification of the germ cell lineage in mice. Cell 137, 571–584 (2009).

    CAS  Article  Google Scholar 

  2. Hayashi, K. et al. Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice. Science 16, 971–975 (2012).

    Article  Google Scholar 

  3. Reijo, R. et al. Mouse autosomal homolog of DAZ, a candidate male sterility gene in humans, is expressed in male germ cells before and after puberty. Genomics 35, 346–352 (1996).

    CAS  Article  Google Scholar 

  4. Park, T. S. et al. Derivation of primordial germ cells from human embryonic and induced pluripotent stem cells is significantly improved by co-culture with human fetal gonadal cells. Stem Cells 27, 783–795 (2009).

    CAS  Article  Google Scholar 

  5. Gaskell, T. L., Esnal, A., Robinson, L. L. L., Anderson, R. A. & Saunders, P. T. K. Immunohistochemical profiling of germ cells within the human fetal testis: identification of three subpopulations. Biol. Reprod. 71, 2012–2021 (2004).

    CAS  Article  Google Scholar 

  6. Robinson, L., Gaskell, T., Saunders, P. & Anderson, R. Germ cell specific expression of c-kit in the human fetal gonad. Mol. Human Reprod. 7, 845 (2001).

    CAS  Article  Google Scholar 

  7. Høyer, P. E., Byskov, A. G. & Møllgård, K. Stem cell factor and c-Kit in human primordial germ cells and fetal ovaries. Mol. Cell. Endocrinol. 234, 1–10 (2005).

    Article  Google Scholar 

  8. Pauls, K. Spatial expression of germ cell markers during maturation of human fetal male gonads: an immunohistochemical study. Hum. Reprod. 21, 397–404 (2005).

    Article  Google Scholar 

  9. Kerr, C., Hill, C., Blumenthal, P. & Gearhart, J. Expression of pluripotent stem cell markers in the human fetal ovary. Hum. Reprod. 23, 589–599 (2008).

    CAS  Article  Google Scholar 

  10. Kerr, C. L., Hill, C. M., Blumenthal, P. D. & Gearhart, J. D. Expression of pluripotent stem cell markers in the human fetal testis. Stem Cells 26, 412–421 (2008).

    Article  Google Scholar 

  11. Seki, Y. et al. Extensive and orderly reprogramming of genome-wide chromatin modifications associated with specification and early development of germ cells in mice. Dev. Biol. 278, 440–458 (2005).

    CAS  Article  Google Scholar 

  12. Hajkova, P. et al. Chromatin dynamics during epigenetic reprogramming in the mouse germ line. Nature 452, 877–881 (2008).

    CAS  Article  Google Scholar 

  13. Hajkova, P. et al. Genome-wide reprogramming in the mouse germ line entails the base excision repair pathway. Science 329, 78–82 (2010).

    CAS  Article  Google Scholar 

  14. Popp, C. et al. Genome-wide erasure of DNA methylation in mouse primordial germ cells is affected by AID deficiency. Nature 463, 1101–1105 (2010).

    CAS  Article  Google Scholar 

  15. Sinclair, A. H. et al. A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 346, 240–244 (1990).

    CAS  Article  Google Scholar 

  16. Atlasi, Y., Mowla, S. J., Ziaee, S. A. M., Gokhale, P. J. & Andrews, P. W. OCT4 spliced variants are differentially expressed in human pluripotent and nonpluripotent cells. Stem Cells 26, 3068–3074 (2008).

    CAS  Article  Google Scholar 

  17. Warthemann, R., Eildermann, K., Debowski, K. & Behr, R. False-positive antibody signals for the pluripotency factor OCT4A (POU5F1) in testis-derived cells may lead to erroneous data and misinterpretations. Mol. Hum. Reprod. 18, 605–612 (2012).

    CAS  Article  Google Scholar 

  18. Ruzov, A. et al. Lineage-specific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development. Cell Res. 21, 1332–1342 (2011).

    CAS  Article  Google Scholar 

  19. Ito, S. et al. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature 466, 1129–1133 (2010).

    CAS  Article  Google Scholar 

  20. Ko, M. et al. Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2. Nature 468, 839–843 (2010).

    CAS  Article  Google Scholar 

  21. Koh, K. P. et al. Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells. Stem Cell 8, 200–213 (2011).

    CAS  Google Scholar 

  22. Thomson, J. A. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    CAS  Article  Google Scholar 

  23. Diaz Perez, S. V. et al. Derivation of new human embryonic stem cell lines reveals rapid epigenetic progression in vitro that can be prevented by chemical modification of chromatin. Hum. Mol. Genet. 21, 751–764 (2012).

    CAS  Article  Google Scholar 

  24. Clark, A. T. Spontaneous differentiation of germ cells from human embryonic stem cells in vitro. Hum. Mol. Genet. 13, 727–739 (2004).

    CAS  Article  Google Scholar 

  25. Zwaka, T. P. A germ cell origin of embryonic stem cells? Development 132, 227–233 (2005).

    CAS  Article  Google Scholar 

  26. Bao, S. et al. The germ cell determinant blimp1 is not required for derivation of pluripotent stem cells. Cell Stem Cell 11, 110–117 (2012).

    CAS  Article  Google Scholar 

  27. Kamei, K. et al. Microfluidic image cytometry for quantitative single-cell profiling of human pluripotent stem cells in chemically defined conditions. Lab. Chip 10, 1113–1119 (2010).

    CAS  Article  Google Scholar 

  28. Briddell, R. et al. Further phenotypic characterization and isolation of human hematopoietic progenitor cells using a monoclonal-antibody to the c-kit receptor. Blood 79, 3159–3167 (1992).

    CAS  PubMed  Google Scholar 

  29. Anderson, R., Fulton, N., Cowan, G., Coutts, S. & Saunders, P. Conserved and divergent patterns of expression of DAZL, VASA and OCT 4 in the germ cells of the human fetal ovary and testis. BMC Dev. Biol. 7, 136 (2007).

    Article  Google Scholar 

  30. Bhutani, N. et al. Reprogramming towards pluripotency requires AID-dependent DNA demethylation. Nature 463, 1042–1047 (2010).

    CAS  Article  Google Scholar 

  31. Maiti, A. & Drohat, A. C. Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: potential implications for active demethylation of CpG sites. J. Biol. Chem. 286, 35334–35338 (2011).

    CAS  Article  Google Scholar 

  32. Hajkova, P. et al. Epigenetic reprogramming in mouse primordial germ cells. Mech. Dev. 117, 15–23 (2002).

    CAS  Article  Google Scholar 

  33. Ohinata, Y., Sano, M., Shigeta, M., Yamanaka, K. & Saitou, M. A comprehensive, non-invasive visualization of primordial germ cell development in mice by the Prdm1-mVenus and Dppa3-ECFP double transgenic reporter. Reproduction 136, 503–514 (2008).

    CAS  Article  Google Scholar 

  34. Kurimoto, K. et al. Complex genome-wide transcription dynamics orchestrated by Blimp1 for the specification of the germ cell lineage in mice. Genes Dev. 22, 1617–1635 (2008).

    CAS  Article  Google Scholar 

  35. Kee, K., Gonsalves, J. M., Clark, A. T. & Pera, R. A. R. Bone morphogenetic proteins induce germ cell differentiation from human embryonic stem cells. Stem. Cells Dev. 15, 831–837 (2006).

    CAS  Article  Google Scholar 

  36. Kee, K., Angeles, V. T., Flores, M., Nguyen, H. N. & Pera, R. A. R. Human DAZL, DAZ and BOULE genes modulate primordial germ-cell and haploid gamete formation. Nature 462, 222–225 (2009).

    CAS  Article  Google Scholar 

  37. Aflatoonian, B. et al. In vitro post-meiotic germ cell development from human embryonic stem cells. Hum. Reprod. 24, 3150–3159 (2009).

    CAS  Article  Google Scholar 

  38. Tilgner, K. et al. Expression of GFP under the control of the RNA helicase VASA permits fluorescence-activated cell sorting isolation of human primordial germ cells. Stem Cells 28, 84–92 (2010).

    CAS  PubMed  Google Scholar 

  39. Panula, S. et al. Human germ cell differentiation from fetal-and adult-derived induced pluripotent stem cells. Hum. Mol. Genet. 20, 752–762 (2011).

    CAS  Article  Google Scholar 

  40. Medrano, J. V., Ramathal, C., Nguyen, H. N., Simon, C. & Reijo Pera, R. A. Divergent RNA-binding proteins, DAZL and VASA, induce meiotic progression in human germ cells derived in vitro. Stem Cells 30, 441–451 (2012).

    CAS  Article  Google Scholar 

  41. Chuang, C. Y. et al. Meiotic Competent human germ cell-like cells derived from human embryonic stem cells induced by BMP4/WNT3A signaling and OCT4/EpCAM (epithelial cell adhesion molecule) selection. J. Biol. Chem. 287, 14389–14401 (2012).

    CAS  Article  Google Scholar 

  42. Plath, K. Role of histone H3 Lysine 27 methylation in X inactivation. Science 300, 131–135 (2003).

    CAS  Article  Google Scholar 

  43. Ficz, G. et al. Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 473, 398–402 (2011).

    CAS  Article  Google Scholar 

  44. Vincent, J. J. et al. Single cell analysis facilitates staging of blimp1-dependent primordial germ cells derived from mouse embryonic stem cells. PLoS ONE 6, e28960 (2011).

    CAS  Article  Google Scholar 

  45. Boissonnas, C. et al. Specific epigenetic alterations of IGF2-H19 locus in spermatozoa from infertile men. Eue. J. Human Gene. 18, 73–80 (2009).

    Article  Google Scholar 

  46. Geuns, E., Hilven, P., Van Steirteghem, A., Liebaers, I. & De Rycke, M. Methylation analysis of KvDMR1 in human oocytes. J. Med. Genet. 44, 144–147 (2006).

    Article  Google Scholar 

  47. Kagami, M. et al. The IG-DMR and the MEG3-DMR at human chromosome 14q32.2: hierarchical interaction and distinct functional properties as imprinting control centers. PLoS Genet. 6, e1000992 (2010).

    Article  Google Scholar 

  48. Zechner, U. et al. Quantitative methylation analysis of developmentally important genes in human pregnancy losses after ART and spontaneous conception. Mol. Hum. Reprod. 16, 704–713 (2010).

    CAS  Article  Google Scholar 

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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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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.

Corresponding author

Correspondence to Amander T. Clark.

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The authors declare no competing financial interests.

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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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