Sertoli Cells Induce Bone Marrow Stem Cells To Differentiate Into Spermatogonial Cells

Objective:The aim is to establish the culture systems of sertoli cells(SCs) and bone marrow stem cells(BMSCs) in vitro,explore the proliferation and differentiation of BMSCs indirect co-culture with SCs and the differentiation of BMSCs cultured with all-trans retinoic acid(ATRA).Methods:SCs were isolated from 12-16d SD rat testes and BMSCs were separated from adult male SD rat bone marrow. SCs were obtained from the two-step enzymatic digestion and lower osmosis Tris-HCl treatment and were analysed by immunohistochemistry of FasL.BMSCs were obtained from the adherent cells and nonadherent cells were eliminated. MTT colorimetric assay was used to observe the survival and proliferation of SCs and BMSCs in vitro respectively and of BMSCs in indirect co-culture with SCs.RT-PCR was performed to detect the expression of stra8 mRNA, the RA-regulated gene specifically expressed in premeiotic germ cells and a marker of the stem cells transdifferentiated into male germ cells, and immunohistochemistry staining was utilized to examine the expression of c-Kit in BMSCs cultured with ATRA,indirect co-cultured with SCs and with SCs and ATRA,respectively.Results: 1.SCs were isolated successfully and their viabilities were 92.5% determinated by Trypan blue dye exclusion test. The percentage of SCs expressing FasL was 93.2%. The proliferation of SCs was observed in the DMEM containing 10%NBS; arrived peak at culture 48h and it was sustained from culture 48h to 168h.2.BMSCs were separated susccessfully from the adherent cells. The proliferation of BMSCs was observed in the DMEM containing 10%FCS; arrived peak at culture 144h and it was sustained from culture 144h to 168h.3.The proliferation of BMSCs indirect co-cultured with SCs was enhanced from culture 24 to 120h,but their surveval was inhibited and apoptosis was increased from culture 144h to 168h4.The expressions of stra8 mRNA and c-Kit of BMSCs cultured with ATRA, indirect co-cultured with SCs and with SCs and ATRA all were able to be observed and the expressions indicated that BMSCs could transdifferentiate into male germ cells and SCs could secrete some soluble factors to regulate the proliferation, apoptosis and differentiation of BMSCs.Conclusions: 1.It is an effective method for isolating and purifying SCs by the two-step enzymatic digestion and lower osmosis Tris-HCl treatment. SCs can express FasL, proliferate and surveve in vitro.2.BMSCs can be separated successfully from the adherent cells and can proliferate and survive in vitro.3.SCs could secrete some soluble factors to regulate the growth of BMSCs indirect co-cultured in vitro.4.SCs could secrete some soluble factors to induce the differentiation of BMSCs indirect co-cultured with SCs into spermatogonial cells and ATRA is able to induce the differentiation of BMSCs into spermatogonial cells.