Long-term Culture Of Porcine Male Germline Stem Cell In Vitro

Male germline stem cells(MGSCs),a special subpopulation of male adult stem cell in testis,are capable of self-renewal to maintain spermatogenesis. It has great value in the field of stem cell biology, regenerative medicine, generation of transgenic animals and treatment for male infertility. However, the low number and purity of MGSCs has restricted its uses for research. Hence, efficient long-term culture system for MGSCs in vitro is particularly needed. In this study, we optimized the program of differential plating to enrich pMGSCs, studied the effects of knockout serum replacement(KSR) and different combination of growth factors on in vitro culture of pMGSCs. The aim of this study is to establish a long-term culture system for pMGSCs and further research.Differential platingmethod was optimized to enrich pure pMGSCs from fresh testicular cell of 7 days piglets. Immunocytochemical analysis of PLZF, GFRA1 and UCHL1 showed that the ratio of MGSCs was improved from 10.50±0.13% to 72.66±2.96 %, significant better than the canonical program of differential plating(28.71±1.00%)Knockout serum replacement(KSR) was used to compensate the low concentration of FBS in our culture medium. Amongthe serum free supplement tested,10% KSR concentration showed an efficient improvement in the propagation and survival of pMGSCs based on 1%FBS culture medium in vitro.The effects of different combinations of growth factors on pMGSCs self-renew still remain unclear. In this study, different combinations were used to promote the proliferation of pMGSCs. The number and the size of colonies were determined and results indicated that each of GDNF, GFRA1, bFGF anf IGF1 could promote the colonies formation, especially with the combination of GDNF, GFRA1 and bFGF.Based on the above research, pMGSCs could be maintained in long-term culture system for 2 month and can be passages for more than 10 times. Immunocytofluorescense, RT-PCR and western-blot analysis showed that cultured cell colonies still express specific makers for MGSCs such as PLZF, GFRA1 and UCHL1. And chromosomal analysis indicated that most cultured pMGSCs have a normal karyotype(38 chromosomes). Xenotransplantation of pSGCs into the testes of busulfan treated mice revealed the migration of pMGSCs to the seminiferous tubule basement membrane. Colony formation and alignment of pMGSCs were also detected.Conclusively, the results demonstrated that a long-term culture system for porcine MGSCs has been established, which pave the way towards the further research and application of porcine MGSCs.