Derivation Of Induced Pluripotent Stem Cells From Testicular Sertoli Cells Of Neonatal Bulls

As the sole somatic cells in the seminiferous epithelium,Sertoli cells(SCs)play vital roles in regulating the process of spermatogenesis by providing nutrients and support.The isolation and culture of SCs is very important for studying spermatogenesis in vitro.The related researches for human and mouse SCs have made significant progress.In aspects of porcine and bovine SCs,the isolation and culture,formation and development of tight connections,cloning and analysis of related functional genes have been reported.Since it's inconvenient and expensive to get the fresh testicular tissue in cattle as well as the uncertain sampling time,the progress of bovine SCs-related studies have been delayed.To address the above-mentioned questions,here we further investigated the feasibility of SC isolation and enrichment from cryopreserved neonatal bovine testis based on our previous work.The cryopreserved testicular tissues of postnatal 1-day-old bulls were thawed for morphological examination and SC separation.Hematoxylin-eosin(HE)staining of the paraffin sections showed that the seminiferous cords and interstitium were well preserved.The two-step enzymatic digestion showed that the seminiferous epithelia were dissociated completely.The attached primary cells were enriched by differential plating,and expanded subsequently.Most of them were in irregular polygon shape,and grew to confluence within 3 days.RT-PCR analysis revealed that they expressed SC marker Gdnf and Abp but not spermatogonial marker Plzf.Immunofluorescence staining showed that the adherent cells expressed SC protein markers GDNF and ABP.Flow cytometry analysis showed that the enriched cells contained high purified ABP+ cells(99.7%)and GDNF+ cells(99.8%).The above data indicate that highly purified SCs can be obtained from the cryopreserved testicular tissues of neonatal bulls.Induced pluripotent stem cells(iPSCs)are a type of pluripotent cells that have great similarity with(embryonic stem cells)ESCs in morphology,growth pattern and biological characteristics.At present,studies on the induction,establishment,identification and reprogramming mechanism of iPSCs in mice,rats and humans have been reported.However,bovine iPSCs still faced with many problems,such as inconvenient acquisition of donor cells,low induction efficiency,easy differentiation in culture,difficult maintenance of stem cell characteristics,and specific markers are controversial.These problems indicate that the biological characteristics and in vitro culture conditions of bovine iPSCs are quite different from those of rodents and human iPSCs.It is reported that the method of classic four-factor can be used to reprogram mouse SCs into iPSCs.According to their biological properties such as rapid proliferation,morphological homogeneity and low immunogenicity,and some undifferentiated characteristics,the immature SCs are suitable for iPSC generation.Accordingly,we explored the induction system from bovine SCs to iPSCs and their freezing conditions.Strong activities of alkaline phosphatase(AP)was detected in bovine iPSCs by AP staining.Immunofluorescence staining showed that the pluripotency markers were expressed in bovine iPSCs.The results of teratoma and embryoid body formation showed that the obtained bovine iPSCs could differentiate into three embryonic germ layers-derived tissues in vitro and in vivo.RNA-seq analysis showed that pluripotency gene(Oct4,Sox2,Nanog,Tet1/3,Klf4)and anti-apoptosis gene(Bcl2)were up-regulated,while cycles-related gene(P21),and apoptotic gene(Casp3)were down-regulated in bovine iPSCs.Additionally,many pathways were also involved in the regulation of the growth,development and apoptosis of bovine iPSCs,such as pluripotency regulation,metabolic pathways,protein binding,cell membrane,nucleus ect.Quantitative PCR results showed that Oct4,Sox2 and pro-apoptosis-related gene Bax were significantly up-regulated,while anti-apoptosis gene Bcl2 was down-regulated,in the iPSCs from the cryopreservation group containing knockout serum replacer(KSR).AP staining revealed that there was a higher expression of AP activity in the embryonic stem cell serum(ES-FBS).The results of teratoma formation assay showed that bovine iPSCs could still form teratoma with structure derived from three embryonic germ layers in vitro after cryopreservation/thawing.In sumarry,here we obtained neonatal bovine testicular SCs with high purity,reprgrammed them into iPSCs using classical four-factor,transcriptome profile the various related pathways involved in the reprogramming from bovine SCs to the iPSCs,finally investigated their cryopreservation conditions and confirmed their pluripotency and differentiation potential after freezing/thawing.Together,we generated bovine SCs-derived iPSCs and investigated the reprogramming mechanism,which laid a foundation for the praticall application of the bovine iPSCs.