Generation And Characteristics Of Human Sertoli Cell Line Immortalized By Overexpression Of Human Telomerase

Backgrounds and objectives:Sertoli cells are the most important somatic cells for regulating spermatogenesis,and thus studies on Sertoli cells are the basis for both reproductive medicine and biology.Notably,Sertoli cells have great plasticity because they can be reprogrammed to other types of functional cells,including neural stem cells,Leydig cells,and the cells secreting insulin,suggesting that Sertoli cells could have important applications in regenerative medicine because of their great plasticity.However,basic research and application of human Sertoli cells have been handicapped due to the following issue: it is rather difficult to obtain human testicular tissues to obtain primary human Sertoli cells.In this study,we have for the first time established a human Sertoli cell line with long-term proliferation potential and high safety,which could offer sufficient human Sertoli cells for basic research as well as reproductive and regenerative medicine.Methods: Lentivirus was packed by co-transfecting three plasmids,including the vector overexpressing h TERT gene,VSVG and ? 8.9,into 293 T cells using lipofectamine 3000.The lentivirus was collected at 48 hours and 72 hours after transfection,respectively.Human Sertoli cells were separated from testicular tissues of obstructive azoospermic(OA)patients with normal spermatogenesis using a two-step enzymatic digestion and followed by differential plating,and they were infected with the lentivirus carrying h TRET by polybrene.EGFP was utilized as a reporter gene,and EGFP-positive cells(the immortalized Sertoli cells)were isolated by FACS and they were expanded in culture.RT-PCR,immunocytochemistry,and Western blot were utilized to identify the phenotypes of the immortalized Sertoli cells.Cell proliferation assay(CCK-8),PCNA and Ki-67 immunnostaining were performedto assess the proliferation potential of the cell line.We next compared global gene expression profiles between the immortalized human Sertoli cell line and primary human Sertoli cells using microarray analysis.Finally,karyotyping assays,multiplex real-time PCR analysis and tumor formation by xenotransplantation assay were conducted to evaluate the safety of human Sertoli cell line.Results: Western blots showed that the protein of h TERT was stably expressed in human Sertoli cell line at passage 10(P10),P15,and P20.The immortalized human cells express numerous hallmarks for primary human Sertoli cells.RT-PCR revealed that the transcripts of AR,ABP,BMP4,BMP6,CLDN11,GATA4,GDNF,OCLN,SCF,SOX9,WT1 and ZO1 were expressed in the immortalized human cells.In contrast,the m RNA of 3?-HSD,SMA and VASA,markers for Leydig cells,myoid cells and germ cells,respectively,was undetectable in these cells.Immunocytochemistry displayed that the immortalized cells were positive for SOX9,WT1,OCLN,VIM,SCF,BMP4,and GDNF,but not for 3?-HSD,SMA,and VASA.Western blots showed that the proteins of SCF,GDNF,BMP4,WT1 and SOX9 were expressed in human cell line,whereas 3?-HSD,SMA and VASA were undetected in these cells.Western blots demonstrated similar expression level of PCNA protein between human Sertoli cell line and primary human Sertoli cells.Immunocytochemistry revealed that more than70% of human Sertoli cell line was positive for ki-67,suggesting this cell line had a high proliferation capacity.The immortalized human Sertoli cells had been cultured for over 6 months and for 30 passages.Neither Y chromosome microdeletion nor tumorgenesis was detected in this cell line,and only 10% of these cells showed abnormal karyotype with unbalanced translocation,which reflects high safety of the immortalized Sertoli cells.Conclusions: We have established the first human Sertoli cell line with the morphological and phenotypic attributes of primary human Sertoli cells.Notably,this human Sertoli cell line has almost normal karyotype,excludes Y chromosome microdeletions and doesn't form tumor.Moreover,the unlimited proliferative capacity of this cell line ensures us to obtain adequate source of human Sertoli cells foruncovering their roles in regulating the complex human spermatogenesis and the treatments for male infertility as well as other human diseases.