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.
Highlights
Published: 16 May 2021In vitro gametogenesis (IVG) is a promising avenue offering potential benefits both towards understanding germline development and factors contributing to infertility
Our data suggests that the combination of PDPN and TNAP is adequate to purify human primordial germ cell-like cells (hPGCLCs) in the context of embryoid body (EB) [19], but we demonstrate that the two main types of female fetal germ cells (FGCs) (‘mitotic’ and ‘RA-responsive’ FGCs) expressed different levels of PDPN and TNAP and could be efficiently separated or purified using this combination of antibodies in fluorescence-activated cell sorting (FACS)
This study addresses the challenges of the sex-specific characterization of early hFGCs and hPGCLCs and identifies the markers TNAP and PDPN as a suitable combination to purify this population from premeiotic hFGCs in both sexes
Summary
In vitro gametogenesis (IVG) is a promising avenue offering potential benefits both towards understanding germline development and factors contributing to infertility. IVG may one day be used in human medically assisted reproduction, technical advancement is majorly dependent on understanding the markers and genes governing germline development. Primordial germ cells (PGCs) are known to be the earliest lineage-specific diploid progenitors of the germline in animals. The origin of human PGCs (hPGCs) is unclear, but they are likely specified around four to five weeks of gestation (WG) (equivalent to two to three weeks of development) in the posterior epiblast of post-implantation embryos around the onset of gastrulation [1,2]. Gonadal fetal germ cells (FGCs) respond to environmental stimuli to undergo proliferation and sex-determination, prompting sex-specific development. While human FGCs are known to be relatively homogenous until about 10 WG in females and males, progress to the second and third trimesters is pronouncedly asynchronous [4,5,6,7]
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