Post-meiotic male germ cell differentiation of mouse embryonic stem cells by exposure to conditioned medium

Abstract

To determine whether the exposure of mouse embryonic stem cells (mESCs) to medium conditioned by adult peritubular or neonatal germinal epithelial cells efficiently increases differentiation into primordial germ cell–like cells (PGCLCs) and induces meiosis. We tested a novel culture system utilizing a mesh interphase system to initiate meiotic differentiation of mESCs under media conditioned by adult peritubular cells consisting mainly of Leydig cells, or by neonatal germinal epithelial cells containing pre-Sertoli cells, which are adapted to paracrine signaling. mESCs without exposure of these cells served as a control. Over a 6-month period, mESCs were differentiated into epiblast-like cells (EpiLCs) and PGCLCs. Peritubular cells derived from adult mouse testes were isolated by differential plating; germline cells from neonatal mice were isolated by trypsinization of testes and placed on the mesh. After 7 passages, 1.2 x 106 mESCs were plated on each well with MEFs and cultured in the mesh interphase system subsidized with either adult or neonatal cells, or voided at 37oC and 5% CO2. Germ cell stage–specific biomarkers (Nanog, Oct4, DAZL, and VASA) were assessed at regular intervals of culture. At day 3 of differentiating mESCs in mesh interphase–conditioned medium containing Activin A, bFGF, and KSR, decreased positivity of Nanog (45%) indicated the successful differentiation of mESCs into EpiLCs. Continued expression of Oct4 (>90%) was detected in the cells at day 3, suggesting the retention of stemness. Cytoplasmic DAZL positivity at day 5 demonstrated early meiotic differentiation into spermatocyte lineage. On day 8, approximately 30% expressed VASA positivity, indicating further progression into meiosis. Cells cultured in media conditioned by adult interstitial cells had greater expression of DAZL and VASA than those cultured with neonatal interstitial cells. The optimal condition was determined to be a 1:5 ratio of adult cells to mESCs. DAZL and VASA expression were negative in the control group, suggesting the important role of the medium conditioned by testicular cells. These results indicate that our novel culture system can promote differentiation of mESCs into PGCLCs and further meiotic differentiation. Initiation of neospermatogenesis using mESCs can be optimized in the presence of factors secreted from Leydig cells derived from adult mouse testes. Reproducing spermatogenesis in vitro may provide valuable information on overcoming male infertility due to spermatogenic arrest or germ cell aplasia.