Background: Several studies have demonstrated the derivation of multi- or pluripotent stem cells from testicular cells of both newborn and adult mice by a spontaneous conversion process, when these cells are cultured in vitro for an extended time. To obtain a better and robust derivation, we have attempted to identify small molecules (SMs) that induce reprogramming of testicular cells in culture into germline-derived pluripotent stem cells (gPSCs).
Methods: We tested several SMs based on previous reports that have shown enhancement of establishment of induced pluripotent stem cells or embryonic stem cells (ESCs) on mouse NMRI (outbred strain) and C57BL/6 (inbred strain) testicular cells. After appearance of ESC-like colonies at Day 6, they were passaged on mitotically arrested mouse embryonic fibroblasts in mouse ESC medium in the absence or presence of SMs up to Day 14. The generated cells were characterized using a variety of experimental approaches.
Results: The application of several SMs involved in pluripotent reprogramming led to the discovery that CHIR99021 (CHIR), a glycogen synthase kinase-3 (GSK-3) inhibitor, promotes efficient derivation of gPSCs from neonatal mouse NMRI and C57BL/6 testes. The pluripotency of the generated cell lines has been confirmed by in vitro spontaneous and direct differentiation toward cardiac and neural lineages, and formation of chimeras after injection of gPSCs into blastocysts. We have shown that the generated gPSCs could be maintained and expanded under chemically defined serum and feeder-free conditions by inhibition of both the extracellular signal-regulated kinases (Erk1/2) and GSK-3.
Conclusions: To our knowledge, this is the first report of a simple and efficient protocol to reprogram gPSCs from testicular cells solely by inhibition of GSK-3 in two strains of mice with different genetic backgrounds. Additionally, this brings us closer to eliminating the need for genetic modification in pluripotent reprogramming. Future studies will determine whether the inhibition of GSK-3 could affect the generation of naïve gPSCs lines in other mammals.