Optimization Of Porcine Spermatogonial Stem Cell Culture System And Detection Of Xenograft Characteristics

The culture of porcine spermatogonial stem cells(pSSCs)is very important as it is the only somatic stem cell type that could pass genetic information to the next generation.For its increasing importance in the stem cell research,we need to prolong the passage of pSSCs and explore its potential fate in vitro.Peptide-coating 2D as a component of the feeder-free system and some small chemical molecules including Chir99021,Repsox,Vatamin C,Folic Acid,and CD Lipid Concentrate were used to prolong the culture time of pSSCs.The characteristics were analyzed by morphology and immunofluorescence.Then the pSSCs were labeled with PKH26 and transplanted into the testis of mice treated with busulfan.Finally,we analyzed the fate of pSSCs in the testis of recipient mice by immunofluorescence.After screening different extra-cellular matrix and some small chemical molecules,we prolong the culture time of pSSCs to at least 3 months using peptide-coating 2D matrix and lipid.Besides,we observed two different kinds of clone morphology during the culture process.One is the traditional grape-like SSC-like cluster,and the other is similar with the typical mouse embryonic stem(mES)cell clone,which might be the pES-like cells.Moreover,we found that pSSCs could proliferate and self-renew in infertile mouse seminiferous tubules.Peptide-coating 2D is beneficial to the longtime culture of porcine male germ cell-derived clone(pGDCs)and lipid are effective to prolong the culture time of pGDCs in vitro.pSSCs could self-renew and proliferate in mouse seminiferous tubules,but they could not further perform meiosis.Our study shows that this feeder-free culture system will serve as a basic refinement in future studies and facilitate studies on SSC biology and genetic manipulation of germ cells.