Isolation,identification And Establishment Of Short-term Culture System In Vitro Of Porcine Stem Leydig Cells (SLCs)

Stem Leydig cells(SLCs)is adult stem cells and can differentiate into adult leydig cell(ALCs),so as to regulating the spermatogenesis.Pig is an important economic animal as well as an ideal mammalian model,while there is no study about pig SLCs.Therefore,the objective of this research was that 7 d pig testes were used to isolate and enrich porcine SLCs,and explore the culture system of the primary SLCs.The main research results are as follows:1.Histological identification of porcine SLCsA number of spindle-shaped cells were found in the testicular interstitium of the postnatal 7 days and 2 months old porcine testes.Furthermore,PDGFR? was mainly expressed in the testicular interstitium in postnatal 7 days old pigs,while the expression of PDGFR? was low in the 2 months old porcine testicular interstitium.Moreover,the expression of Nestin in the 7 days old porcine testes was higher than that in the 2 months old testes.Based on these results,we chose to collect SLCs from 7 days old pigs rather than 2months old pigs.2.The isolation and identification of porcine SLCsThe primary LCs were obtained by digestion method.The isolated LCs expressed SLCs or pluripotency stem cell markers(Nestin,PDGFR?,Oct4 and LIFR).Moreover,markers of Sertoli cells(SOX9)and spermatogonial stem cells(PLZF)were not detected,indicating no contamination with these cells in these LCs.The results demonstrated that this digestion method was useful in removing the seminiferous tubules.Moreover,mRNA expressions of LIFR and PDGFR? in the LCs were significantly higher than that in the porcine testes,indicating that this method was able to enrich SLCs from porcine testes.3.The culture system of porcine SLCsEven though the primary SLCs were isolated,their culture system was yet to be determined.In the current study,pTF was used as the main component in the medium.Seven days later,a number of clones were formed,which grew larger following 2 weeks of culture.Immunofluorescent analysis showed that the clones were PDGFR? positive(PDGFR?+).The m RNA expressions of both Nestin and LIFR were higher in porcine SLCs cultured with pTF medium compared to in SLCs without culture,indicating that pTF was able to sustain thestem cell potential of SLCs.However,the isolated cells cultured with a basic medium did not form clones after 2 weeks,and expressed CYP17A1,a marker of pig differentiated LCs.To summarize,we isolated porcine SLCs and identified some of their basic characteristics.The pTF were vital for maintaining the features of stem cells.These findings might help us to understand the regulatory mechanisms of proliferation and differentiation of SLCs as well as to accumulate the scientific data for the isolation and culture of human SLCs.