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Gonocytes are germline stem cells in the neonatal testis with important potential applications in fertility restoration and transgenesis. Using stepwise experiments, we examined the effects of different media combined with fetal bovine serum (FBS) and/or knockout serum replacement (KSR) on the in vitro proliferation, colony-formation, ultrastructure, and expression of pluripotency markers of porcine gonocytes. Testis cells from 1-wk-old piglets were cultured for 28 days in 6 different culture media (DMEM, DMEM/F12, GMEM, α-MEM, StemPro, and RPMI), each supplemented with 5%, 10%, or 15% FBS and/or 5%, 10%, or 15% KSR. The media and FBS/KSR combination leading to the maximum number of gonocytes, and their colonies were selected for further analyses. KSR supplementation resulted in a reduced somatic cell propagation and increased gonocyte colony formation (P < 0.001). Culturing in DMEM+15%FBS led to the greatest number of gonocytes (P < 0.001), while the largest diameter and greatest number of colonies were formed in DMEM+5%FBS+10%KSR cultures (P < 0.001). Gonocytes and their colonies in DMEM+15%FBS expressed all the examined gonocyte and pluripotency markers. KSR alone did not support gonocyte propagation, likely due to a reduced somatic cell proliferation; however, the combination of FBS and KSR increased gonocyte colony formation and their size. • Optimized culture of germline stem cells has important potential applications for fertility restoration and transgenesis. • Tested the effects of six different media combined with various concentrations of FBS or KSR on proliferation of gonocytes, their colonies, and their pluripotency. • Gonocytes and their colonies in DMEM+15%FBS expressed all the examined gonocyte and pluripotency markers. • KSR alone did not support gonocyte propagation, but the combination of FBS and KSR increased gonocyte colony formation and their size. [ABSTRACT FROM AUTHOR]

Gonocytes are progenitors of spermatogonial stem cells in the neonatal testis. We have previously shown that upon culturing, neonatal porcine gonocytes and their colonies express germ cell and pluripotency markers. The objectives of present study were to investigate in vitro trans-differentiation potential of porcine gonocytes and their colonies into cells from three germinal layers, and to assess pluripotency of cultured gonocytes/colonies in vivo. For osteogenic and tri-lineage differentiation, cells were incubated in regular culture media for 14 and 28 days, respectively. Cells were cultured for an additional 14 days for osteogenic differentiation or 7 days for differentiation into derivates of the three germinal layers. Osteogenic differentiation of cells and colonies was verified by Alizarin Red S staining and tri-lineage differentiation was confirmed using immunofluorescence and gene expression analyses. Furthermore, upon implantation into recipient mice, the cultured cells/colonies developed teratomas expressing markers of all three germinal layers. Successful osteogenic differentiation from porcine germ cells has important implications for bone regeneration and matrix formation studies. Hence, gonocytes emerge as a promising source of adult pluripotent stem cells due to the ability to differentiate into all germinal layers without typical biosafety risks associated with viral vectors or ethical implications. [ABSTRACT FROM AUTHOR]