A Fundamental Research in In Vitro Spermatogonial Stem Cell Culturing: What Are Clump Cells?

Spermatogonial stem cells (SSCs) are a small group of testicular cells located in the basement membrane of seminiferous tubules and can balance self-renewal and differentiation during spermatogenesis. Our in vitro culture experiments of mouse SSCs indicated heterogeneity of cultured cells. Highly compact colonies were observed next to SSC colonies, which we call clump cells. We used immunocytochemical staining to identify SSCs and somatic cells with VASA and Vimentin antibodies. Subsequently, we compared mRNA expression levels of VASA, DAZL, PLZF, GFRA1, Lin28, Kit, Myc and Vimentin genes using Fluidigm real-time RT-polymerase chain reaction in clump cells, SSCs, and testicular stromal cells. To better understand the functions of selected genes, we created a protein-protein interaction network and performed an enrichment analysis using different databases. Based on the data collected, we state that clump cells do not express the molecular markers of SSCs, so we cannot consider them as SSCs; however, we claim that these cells are altered SSCs. The molecular mechanism of this conversion is still obscure. Therefore, this study can support the analysis of germ cell development both in vitro and in vivo. In addition, it can be effective in finding new and more efficient treatments for male infertility.