The Proliferation Potential of Differentiated and Undifferentiated Spermatogonial Stem Cells on Diverse Feeder Layers

Spermatogonial stem cells (SSCs) play an essential role in the transfer of genetic information through generations, making studying their cellular and molecular mechanisms critical. However, since SSCs are few in mice, directly studying them is limited, requiring specialized in vitro cultivation. Feeder layers such as mouse embryonic fibroblasts (MEFs), SNL, neonate, and adult mouse testicular stromal feeder cells (TSCs) support in vitro survival and growth. To understand the effectiveness of these feeder layers on SSC proliferation, we compared MEF, SNL, neonatal, and adult TSCs. Furthermore, we identified hub genes and potential pathways in spermatogenesis. Two populations of differentiated and undifferentiated SSCs were compared for mouse SSC colony formation and proliferation effectiveness. Additionally, Cytoscape and STRING databases were employed for protein-protein interaction networks and functional gene enrichment. The expression of three hub genes, including Dazl, Zbtb16, and Stra8, was analyzed using dynamic array chips (Fluidigm) followed by statistical analysis. Our results indicated that undifferentiated SSCs favored MEF feeders, while differentiated SSCs thrived on SNL and primary TSC feeders for long-term culture. Functional enrichment results demonstrated hub genes involvement in cell differentiation, meiosis, regulation of meiotic nuclear division, cell development, and spermatogenesis. Furthermore, mRNA expression levels of Stra8, Zbtb16, and Dazl genes show different patterns among feeder layers and SSC differentiation phases.