An Extended Culture System that Supports Human Primordial Germ Cell-like Cell Survival and Initiation of DNA Methylation Erasure

Abstract

The development of an in vitro system in which human primordial germ cell-like cells (hPGCLCs) are generated from human pluripotent stem cells (hPSCs) has been invaluable to further our understanding of human primordial germ cell (hPGC) specification. However, the means to evaluate the next fundamental steps in germ cell development have not been well established. In this study we describe a two dimensional extended culture system that promotes proliferation of specified hPGCLCs, without reversion to a pluripotent state. We demonstrate that hPGCLCs in extended culture undergo partial epigenetic reprogramming, mirroring events described in hPGCs in vivo, including a genome-wide reduction in DNA methylation and maintenance of depleted H3K9me2. This extended culture system provides a new approach for expanding the number of hPGCLCs for downstream technologies, including transplantation, molecular screening, or possibly the differentiation of hPGCLCs into gametes by in vitro gametogenesis.

Divergent roles for KLF4 and TFCP2L1 in Naive Ground State Pluripotency and Human Primordial Germ Cell Development

“…Our results show that upon extended culture, TFCP2L1 protein becomes detectable in hPGCLCs, and that TFCP2L1 but not KLF4 regulates proliferation and survival of the hPGCLC population. Success of the hPGCLC extended culture system depends on addition of cAMP agonists including Forskolin and Rolipram 27 , which increase the intracellular content of cAMP. This is important because in placental cell lines, the expression of TFCP2L1 protein is associated with cAMP signaling 30 .…”

“…In addition to cAMP signaling, hPGCLCs in extended culture initiate DNA demethylation 27 . Using naive mouse iPSCs as a model, KLF4 and TFCP2L1 are both capable of binding ten eleven translocation 2 (TET2) at enhancers, and this is associated with targeted enhancer DNA demethylated during reprogramming 31 .…”

“…Since critical aspects of naive-like ground state pluripotency such as genome-wide DNA demethylation do not occur until D20 in early nonhuman primate PGCs 15 . Therefore, we next evaluated a potential role for KLF4 and TFCP2L1 with extended culture of hPGCLCs to D25, when hPGCLCs initiate DNA demethylation 27 (Fig. 4a and Supplementary Fig.…”

“…3a). To achieve this, we used FACS to isolate D4 hPGCLCs with ITGA6/EPCAM, and cultured the hPGCLCs for an additional 10 and 21 days, corresponding to D14 and D25 after hPGCLC specification 27 .…”

“…D4 hPGCLCs were cultured (C) for an additional 10 or 21 days as described previously 27 . Sorted hPGCLCs were plated at densities ranging 200-3000 cells in either a chamber well (D4C10) or 24-well (D4C21) in FR10 media.…”