Long‐term expansion with germline potential of human primordial germ cell‐like cells in vitro

Abstract

Human germ cells perpetuate human genetic and epigenetic information. However, the underlying mechanism remains elusive, due to a lack of appropriate experimental systems. Here, we show that human primordial germ cell-like cells (hPGCLCs) derived from human-induced pluripotent stem cells (hiPSCs) can be propagated to at least~10 6-fold over a period of 4 months under a defined condition in vitro. During expansion, hPGCLCs maintain an early hPGC-like transcriptome and preserve their genome-wide DNA methylation profiles, most likely due to retention of maintenance DNA methyltransferase activity. These characteristics contrast starkly with those of mouse PGCLCs, which, under an analogous condition, show a limited propagation (up to~50-fold) and persist only around 1 week, yet undergo cell-autonomous genome-wide DNA demethylation. Importantly, upon aggregation culture with mouse embryonic ovarian somatic cells in xenogeneic-reconstituted ovaries, expanded hPGCLCs initiate genome-wide DNA demethylation and differentiate into oogonia/gonocyte-like cells, demonstrating their germline potential. By creating a paradigm for hPGCLC expansion, our study uncovers critical divergences in expansion potential and the mechanism for epigenetic reprogramming between the human and mouse germ cell lineage.

GATA transcription factors, SOX17 and TFAP2C, drive the human germ-cell specification program

“…To examine whether the GATA3/SOX17/TFAP2C clone-derived BT + AG + cells at ag77 undergo epigenetic reprogramming, we determined their genome-wide DNA methylation profile by whole-genome bisulfite sequence (WGBS) analysis. Importantly, we found that the genome-wide DNA methylation properties of the GATA3/SOX17/TFAP2C clone-derived cells at ag77 were similar to those of oogonia/gonocytes reported previously ( 10 , 47 , 50 ) with regard to both their distribution profiles ( Fig 7E ) and total levels (reduced to as low as ∼17.5%) ( Fig 7F ). Accordingly, the GATA3/SOX17/TFAP2C clone-derived cells erased their DNA methylation throughout their genomic regions, including promoters, exons, introns, intergenic regions, and non-promoter CpG islands (CGIs) ( Fig 7G ), as well as parental imprint control regions (ICRs) ( Fig 7H ).…”

“…Crucially, we showed that the TF-induced BT + AG + cells, when cultured in xrOvaries, underwent epigenetic reprogramming and differentiated into oogonia/gonocytes ( Fig 7 ), demonstrating that the TF-induced BT + AG + cells bear one of the key functions of bona fide hPGCs. Unlike mPGCLC specification, which is directly coupled with epigenetic reprogramming ( 65 , 66 , 67 , 68 ), hPGCLC specification itself does not appear to be sufficient to elicit the epigenetic reprogramming: further signaling/environmental cues, including those provided by xrOvaries, are necessary to activate such key processes ( 10 , 50 ). Upon mPGCLC specification, Blimp1, Prdm14, and Tfap2c repress the expression of genes such as Dnmt3a/b and Uhrf1 , and create a cellular state with little, if any, de novo and maintenance DNA methyltransferase (DNMT) activities ( 50 , 65 , 66 , 67 , 68 ), and this leads to a replication-coupled passive genome-wide DNA demethylation upon mPGCLC proliferation ( 67 , 68 ).…”

“…In contrast, the mechanism of epigenetic reprogramming, including genome-wide DNA demethylation, in humans is unclear, and may involve a divergence from that in mice. The identification of the TFs sufficient to create the hPGCLC state (this study), coupled with the development of a method for hPGCLC expansion ( 50 ), will be instrumental in clarifying the mechanism of epigenetic reprogramming in human germ cells.…”

Sex-Specific Isolation and Propagation of Human Premeiotic Fetal Germ Cells and Germ Cell-Like Cells

“…This has led to the hypothesis that it might be possible to propagate hPGCLCs under pluripotent culture conditions. Recently, a few studies have demonstrated the extended culture of hPGCLCs, that retained their identity for up to 21 days [ 22 ] as well as proliferative capacity for over four months [ 20 ]. Previous studies have tested culture conditions for the derivation of pluripotent human embryonic germ cells (hEGCs) from hPGCs in medium containing leukemia inhibitory factor (LIF), forskolin and fibroblast growth factor 2 (FGF2) (LFF medium) [ 23 , 24 ].…”

Mammalian Germ Cell Development: From Mechanism to In Vitro Reconstitution

“…Importantly, the hPGCLC induction system revealed that there are critical differences between the transcriptional regulation of hPGC(LC) specification and that of mPGC specification: SOX17 , which is dispensable for mPGC specification, functions upstream of BLIMP1 for hPGC(LC) specification, and both the transcription factors themselves and their hierarchy of actions are different between human and mouse germ cell specification ( Chen et al., 2018 ; Irie et al., 2015 ; Kojima et al., 2017 ; Pierson Smela et al., 2019 ; Sasaki et al., 2015 ; Sybirna et al., 2020 ). More recently, an hPGCLC expansion system was developed, and experiments using this system revealed that, in stark contrast to mPGCLCs, hPGCLCs propagate ∼10 6 -fold over a period of 4 months and maintain not only their transcriptome, but also their epigenome during the expansion ( Murase et al., 2020 ). Thus, unlike the specification and propagation of mPGC(LC)s, hPGC(LC) specification and propagation are genetically dissociable from epigenetic reprogramming.…”