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

Abstract

The second trimester of human development is marked by asynchronous gonadal development hampering the isolation of homogenous populations of early and late fetal germ cells (FGCs). We evaluated the feasibility of using surface markers TNAP, PDPN, EPCAM and ITGA6 to isolate FGCs as well as human primordial germ cell-like cells (hPGCLCs) derived from embryonic stem cells (hESCs) from both sexes by fluorescence-activated cell sorting (FACS). Our results suggest that a combination of TNAP and PDPN was sufficient to separate populations of premeiotic FGCs and hPGCLCs in both sexes. This combination of antibodies also proved efficient in separating ‘mitotic’ from ‘retinoic-acid responsive’ female FGCs. Furthermore, we report that the differentiation efficiency of TNAP+PDPN+ hPGCLCs from hESCs was sex-independent, but the ability to propagate differed considerably between the sexes. In contrast to male, female hPGCLCs retained their characteristics and exhibited robust colony-forming ability when cultured for five days in medium containing LIF, forskolin and FGF2. We conclude that marked sex differences exist in the isolation and propagation of human FGCs and hPGCLCs. Our study provides novel insights relevant for the optimization of in vitro gametogenesis in humans.

Tissue of Origin, but Not XCI State, Influences Germ Cell Differentiation from Human Pluripotent Stem Cells

“…During early mammalian development, primordial germ cells (PGCs) are the first embryonic lineage to be fate restricted, marking the definitive separation between germline and soma in the embryo. One of the key events that takes place during human PGC (hPGC) specification is the upregulation of specific PGC-markers, such as TFAP2C and SOX17 , but also of surface makers such as ALPL , PDPN , EPCAM and ITGA6 [ 22 , 23 , 24 ]. In addition, PGCs undergo significant epigenetic reprogramming, including genome-wide DNA demethylation and remodelling of histone marks/variants, leading to the erasure of genomic imprints [ 25 , 26 ] and, in female hPGCs, the reactivation of the Xi, a process known as X chromosome reactivation (XCR) [ 26 , 27 ].…”

Improving In Vitro Culture of Human Male Fetal Germ Cells

“…In order to quantify the enrichment of male hFGCs obtained in the non-adherent fraction, we determined the percentage of hFGCs after the differential adhesion procedure by using surface markers THY1 and ITGA6, which are both established markers for identifying hFGCs and Sertoli cells [48][49][50][51], in the dissociated male gonads (before plating) and in the adherent fraction and non-adherent fraction by flow cytometry analysis. Cells were washed with FACS-buffer consisting of 10% fetal bovine serum (FBS) in phosphate-buffered saline buffer (PBS) and incubated for 30 min at 4 • C with conjugated antibodies diluted in FACS-buffer (Table S1).…”

“…As the human fetal testes have comparable developmental age and hFGCs exhibit characteristics in common with pluripotent stem cells, we reasoned that preplating for 30 min would be sufficient for the somatic cells in the male testes to adhere to the gelatin-coated plate. By using FACS, we compared the percentage of male hFGCs known to express the surface markers THY1 (also known as CD90) and ITGA6 [48][49][50][51] before and after enrichment and observed an 3 fold enrichment (average of 7% in the gonad compared with 23% in the non-adherent fraction) (Figure 1D; Figure S1B). Note that after 30 min, the percentage of hFGCs that remained in the adherent fraction was on average 3%, suggesting that longer incubation time, such as overnight incubation as used by Kanatsu-Shinohara and colleagues [53], may not be necessary.…”

“…Note that after 30 min, the percentage of hFGCs that remained in the adherent fraction was on average 3%, suggesting that longer incubation time, such as overnight incubation as used by Kanatsu-Shinohara and colleagues [53], may not be necessary. After 30 min, the adherent fraction consisted of a 4-5 fold enrichment in THY1-ITGA6+ Sertoli cells [49,51] (average of 10% in the gonad compared with 45% in the adherent fraction) (Figure 1D).…”

FGFR3 is expressed by human primordial germ cells and is repressed after meiotic initiation to form primordial oocytes

“…These include cKIT, TNAP, PDPN, CD38, ITGA6, and EPCAM ( Gkountela et al., 2012 ; MacGregor et al. 1995 ; Sasaki et al., 2015 ; Irie et al., 2015 ; Fernandes et al., 2018 ; Mishra et al., 2021 ). In most cases, combinations of antibodies that recognize two or more of these cell-surface proteins are used in FACS strategies to isolate highly enriched PGC populations for further analysis.…”