Abstract

Simple Summary The primary function of spermatogonial stem cells (SSC) in the testis is to produce germ cells (GC) and sperm. This function may be recreated under laboratory conditions using in vitro three-dimensional (3D) cell culture systems. This technology may be used for the treatment of men infertility and in animals for dissemination of elite genetics and conservation of endangered species; however, SSC are scarce and difficult to culture. Potentially, mesenchymal stem cells (MSC) derived from peripheral blood (PB-MSC) may also be candidates for GC production, due to their simplicity for culture and differentiation capacity. The aim of the present study was to compare the potential for production of GC of bull PB-MSC and SSC using an in vitro 3D co-culture system with Sertoli cells (SC). Samples of PB-MSC or SSC co-cultures with SC were collected on days 0, 7, 14 and 21 and analyzed for pluripotency, GC and mesenchymal gene expression. Co-culture of bull PB-MSC or SSC+SC in 3D resulted in differential expression of genes associated to pluripotency and GC differentiation. In conclusion, profiles of expression of pluripotency and GC genes indicate that SSC display a more robust GC differentiation profile compared to PB-MSC, when co-cultured in a 3D system with SC. Although spermatogonial stem cells (SSC) constitute primary candidates for in vitro germ cell (GC) derivation, they are scarce and difficult to maintain in an undifferentiated state. Alternatively, mesenchymal stem cells (MSC) are also candidates for GC derivation due to their simplicity for culture and multipotential for transdifferentiation. The aim of the present study was to compare the GC differentiation potentials of bull peripheral blood-derived MSC (PB-MSC) and SSC using an in vitro 3D co-culture system with Sertoli cells (SC). Samples of PB-MSC or SSC co-cultures with SC were collected on days 0, 7, 14 and 21 and analyzed for pluripotency, GC and mesenchymal marker expression. Co-culture of PB-MSC+SC resulted in down-regulation of NANOG and up-regulation of OCT4 at day 7. In comparison, co-culture of SSC+SC resulted in consistent expression of NANOG, OCT4 and SOX2 at day 14. During co-culture, SSC+SC increased the expression of DAZL, PIWIL2, FRAGILIS and STELLA and activated the expression of STRA8, whereas co-culture of PB-MSC+SC only increased the expression of DAZL and PIWIL2. Thus, co-culture of bull PB-MSC+SC and SSC+SC in 3D SACS results in differential expression of pluripotency and GC markers, where bull SSC display a more robust GC differentiation profile compared to PB-MSC.