Improving In Vitro Culture of Human Male Fetal Germ Cells

Abstract

Male human fetal germ cells (hFGCs) give rise to spermatogonial stem cells (SSCs), which are the adult precursors of the male gametes. Human SSCs are a promising (autologous) source of cells for male fertility preservation; however, in contrast to mouse SSCs, we are still unable to culture them in the long term. Here, we investigated the effect of two different culture media and four substrates (laminin, gelatin, vitronectin and matrigel) in the culture of dissociated second trimester testes, enriched for hFGCs. After 6 days in culture, we quantified the presence of POU5F1 and DDX4 expressing hFGCs. We observed a pronounced difference in hFGC number in different substrates. The combination of gelatin-coated substrate and medium containing GDNF, LIF, FGF2 and EGF resulted in the highest percentage of hFGCs (10% of the total gonadal cells) after 6 days of culture. However, the vitronectin-coated substrate resulted in a comparable percentage of hFGCs regardless of the media used (3.3% of total cells in Zhou-medium and 4.8% of total cells in Shinohara-medium). We provide evidence that not only the choices of culture medium but also choices of the adequate substrate are crucial for optimizing culture protocols for male hFGCs. Optimizing culture conditions in order to improve the expansion of hFGCs will benefit the development of gametogenesis assays in vitro.

Application of single-cell RNA sequencing on human testicular samples: a comprehensive review

“…So, they chose scRNA-seq data (the data used here were previously reported by the same team) to see the expression of KASH5 and found it to be enriched in SPCs, especially leptotene to pachytene SPCs, and finally validated it by IF staining. This process of “finding target gene” and then “detecting it in human testicular scRNAs-seq data” (sometimes with a third step as “validating genes using experiments”) was quite common for studies with interested genes 136 - 141 . Meanwhile, the interested genes were not restricted to one gene or several genes, the researchers could detect a gene family (such as Interleukins) or a series of function-related genes (such as inflammatory cytokine receptors) in scRNA-seq data 142 , which could give researchers a view on macroscale.…”

Effects of clinical medications on male fertility and prospects for stem cell therapy

“…Further exploration is needed on the conditions for long-term stable cryopreservation of testicular tissue and the location of transplantation ( Moraveji et al, 2019 ; Sharma et al, 2019 ). The above technologies have not yet reached the clinical stage ( Diao et al, 2022 ; Martin-Inaraja et al, 2021 ; Sanou et al, 2022 ).…”

Roles of Spermatogonial Stem Cells in Spermatogenesis and Fertility Restoration

“…Extensive effort has been focused on optimization of culture conditions for long-term maintenance and propagation of human SSCs. Multiple culture substrates, including hydrogel, matrigel, and laminin, have been shown to promote the propagation of human SSCs under feeder-free conditions ( 82 ). Currently, several markers are used for the verification of human SSCs.…”