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Defining the Specification of Human Primordial Germ Cells: SOX17 Surprises!

Review of “SOX17 Is a Critical Specifier of Human Primordial Germ Cell Fate” from Cell by Stuart P. Atkinson

While we understand a great deal about the mechanisms of specification of mouse primordial germ cells (PGCs), the cells that give rise to sperm and eggs, this is unfortunately not the case for humans. Inherent differences between mouse and human pluripotent cells [1, 2] and their early post-implantation development, and the scarcity of human PGC-like cells (hPGCLCs) generated from hESCs [3], have confounded their study. Now, the combined might of researchers from the laboratories of Jacob Hanna (Weizmann Institute of Science, Israel) and Azim Surani (Gurdon Institute, University of Cambridge, UK) have developed a method to specify hPGCLCs from germ cell competent or naïve hESCs/hiPSCs [4], and report on this and the subsequent analysis of these cells [5].

Growth of hESCs in “4i” medium reverts cells to a naïve state in which they resemble mouse ESCs, and it was in this pluripotent state from which the researchers attempted to induce hPGCLCs. The group first pre-induced both male and female naïve hESCs with bFGF, TGFβ, and 1% knockout serum replacement medium for 2 days, before using a previously established differentiation medium (BMP2 or BMP4, LIF, stem cell factor (SCF), epidermal growth factor (EGF), and Rho-kinase (ROCK) inhibitor) with cells cultured in low-attachment wells [6]. This generated embryoid bodies which strongly upregulated the expression of a PGC-specific reporter gene (NANOS3-mCherry) and TNAP (tissue non-specific alkaline phosphatase), another PGC-specific gene, within 3 days. At 4-5 days, where the hPGCLCs stopped proliferating, NANOS3/TNAP double-positive cells made up 27% of total cells, and these cells expressed key PGC genes (NANOS3, BLIMP1, TFAP2C, STELLA, TNAP, KIT, OCT4, NANOG, and PRDM14). Surprisingly, the researchers also found high levels of SOX17 expression.

In depth transcriptional analysis suggested that hPGCLCs represented a very early stage of the human germ cell lineage towards becoming PGCs, as while they expressed core germ cell genes, the lacked the expression of late germ cell markers, such as DAZL, VASA, and MAEL. Therefore, these cells represent an important chance to model mechanisms of PGC specification in humans, previously unmapped due to exclusion of early embryos from any investigation. The group assessed this using single cell analysis of temporal gene expression by immunofluorescence on day 1–8 embryoids after hPGCLC differentiation. This demonstrated that at early stages, a few randomly distributed few SOX17/BLIMP1 double-positive cells were present, and over time these cells expanded as a cluster which expressed PGC-specific genes, making SOX17-BLIMP1 expression a key determinant of PGC specification and maintenance. The researchers strengthened this hypothesis using BLIMP1 and SOX17 null cells, finding that BLIMP1 functions to suppress the somatic program during human germline development, while SOX17 works upstream to induce hPGCLC-linked gene expression to initiate the human germ cell transcriptional network.

Finally, and of great importance to patient specific therapeutic strategies which may follow from this research, various human induced pluripotent stem cell (hiPSC) lines could also undergo hPGCLC specification in an efficient manner after being maintained in 4i medium and differentiation in a similar manner.

This study will hopefully represent a launching pad for further studies towards understanding the establishment of the human germline, and towards the development of relevant therapies. The importance of studying this system in humans is clear given the surprising role of SOX17 in hPGCLC specification, a protein with no known germ cell-related role in mouse. More surprises may await us!

References

  1. Hackett JA and Surani MA Regulatory principles of pluripotency: from the ground state up. Cell Stem Cell 2014;15:416-430.
  2. Nichols J and Smith A Naive and primed pluripotent states. Cell Stem Cell 2009;4:487-492.
  3. Gkountela S, Li Z, Vincent JJ, 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 2013;15:113-122.
  4. Gafni O, Weinberger L, Mansour AA, et al. Derivation of novel human ground state naive pluripotent stem cells. Nature 2013;504:282-286.
  5. Irie N, Weinberger L, Tang WW, et al. SOX17 Is a Critical Specifier of Human Primordial Germ Cell Fate. Cell 2015;160:253-268.
  6. Hayashi K, Ohta H, Kurimoto K, et al. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 2011;146:519-532.