In Vitro Testicular Niche Modelling: The Ratio of Spermatagonial Stem Cells (SSCs) to Supporting Somatic Cells is Critical for Optimal SSC Proliferation In Vitro

Introduction: SSCs, with somatic cell support, are responsible for sperm production. Differential plating (DP) is a germ cell enrichment technique that enables the separation of adherent testicular somatic cells from non-adherent (floating) germ cell populations containing rare SSCs. Our aim was to determine whether the proportion of germ / somatic cells co-cultured in vitro impacts SSC propagation in murine model.

Materials & Methods: Cell suspensions were obtained by mechanical and enzymatic digestion of 6-8 week old CD1 murine testes. DP was performed to separate floating and adherent cells, which were then co-cultured on laminin-coated plates at 5 floating to adherent cells ratios - 5:1, 10:1, 15:1, 20:1 and 25:1. A sixth culture included only floating cells as control. Total cell numbers and number of germ cell colonies were counted every 14-18 days during culture passages (P0, P1 and P2). After P2, cells from suitable conditions were characterised by qPCR, flow cytometry and immunocytochemistry.

Results: 15:1 and 20:1 ratio conditions showed significantly higher overall cell expansion and total number of germ cell-like colonies compared to lower ratios of 5:1 or 10:1 and highest ratio of 25:1, as well as control without somatic cell support (P<0.05). Among the optimal ratio of 20:1 after P2, 25.8% of cells were GFRα1-positive on flow cytometry accompanied by 2.3 fold change on qPCR fo GFRα1. Colonies stained positive for both GFRα1 and UTF1 with rare somatic marker Sox9 positive stainig (Figure 1).

Conclusions: Co-culture ratios of 15:1 and 20:1 resulted in significantly increased SSC proliferation and enrichment. Delicate balance between somatic support cells and SSCs is critical for optimal SSC expansion in vitro.