ITGA6+ human testicular cell populations acquire a mesenchymal rather than germ cell transcriptional signature during long-term culture

Abstract: Autologous spermatogonial stem cell transplantation is an experimental technique aimed at restoring fertility in infertile men. Although effective in animal models, in vitro propagation of human spermatogonia prior to transplantation has proven to be difficult. A major limiting factor is endogenous somatic testicular cell overgrowth during long-term culture. This makes the culture both inefficient and necessitates highly specific cell sorting strategies in order to enrich cultured germ cell fractions prior to transplantation. Here, we employed RNAseq using differential gene expression and cell deconvolution analyses, to determine cell type composition in sorted integrin alpha-6 (ITGA6+) primary human testicular cells (n = 4 donors) cultured for up to two months. Our data and analyses reveal that long-term cultured ITGA6+ testicular cells are composed mainly of cells expressing markers of peritubular myoid cells, Leydig cell progenitors, fibroblasts and mesenchymal stromal cells and only a limited number of spermatogonial cells as compared to their uncultured counterparts. These findings provide valuable insights into the cell type composition of cultured human ITGA6+ testicular cells during in vitro propagation and may serve as a basis for optimizing future cell sorting strategies as well as optimizing the current human testicular cell culture system for clinical use. RNAseq analysis of ITGA6+ primary human testicular cells during in vitro culture. Cultures were initiated from 4 testicular tissue donors and analyzed at 6 time points in culture, totalling 24 samples.