Cryopreserved ovine spermatogonial stem cells maintain stemness and colony forming ability in vitro  

作　　者：R.Kumar Pramod Deepthi Varughese A.Javed Jameel Bhisma Narayan Panda Soma Goswami Abhijit Mitra 

机构地区：[1]ICMR-National Animal Resource Facility for Biomedical Research,Hyderabad,Telangana,500101,India [2]Inter University Centre for Biomedical Research&Super Speciality Hospital,Kerala,686009,India [3]College of Veterinary and Animal Sciences,Mannuthy,Kerala,680651,India [4]Translational Health Science and Technology Institute,Faridabad,Haryana,121001,India [5]G V Research Platform,Hyderabad,Telangana,500101,India [6]Department of Animal Husbandry&Dairying,MoFAHD,Krishi Bhavan,New Delhi,110001,India

出　　处：《Asian pacific Journal of Reproduction》2023年第6期273-280,共8页亚太生殖杂志（英文版）

摘　　要：Objective:To assess the effect of cryopreservation on stemness and proliferation potential of sheep spermatogonial stem cells(SSCs)in vitro.Methods:Sheep testicular cells were isolated and putative SSCs were enriched by the laminin-based differential plating method.Putative SSCs were co-cultured with the Sertoli cell feeder prepared by the Datura Stramonium Agglutinin(DSA-lectin)-based method.The cultured putative SSCs were cryopreserved in Dulbecco's Modified Eagle Medium-10%fetal bovine serum mixture(DMEM-10%FBS)media containing 10%dimethyl sulfoxide(DMSO)alone or 10%DMSO plus 200 mM trehalose.Cryopreserved putative SSCs were evaluated for their proliferation potential using in vitro culture and stemness by immunocytochemistry.Finally,the transfection ability of cryopreserved putative SSCs was analyzed.Results:We isolated 91%viable testicular cells from sheep testes.The majority of the laminin enriched cells expressed the SSC related marker,ITGA6.Co-culture of sheep putative SSCs with Sertoli cell feeder resulted in the generation of stable colonies,and the expression of SSC marker was maintained after several passages.A significantly higher number of viable putative SSCs was recovered from SSCs cryopreserved in media containing 10%DMSO and 200 mM trehalose compared to 10%DMSO alone(P<0.01).Cryopreserved putative SSCs formed colonies and showed SSC marker expression similar to the non-cryopreserved putative SSCs.The appearance of green fluorescent colonies over the Sertoli cell feeder indicated that cryopreserved sheep SSCs were successfully transfected.Conclusions:Cryopreserved putative SSCs can retain their stemness,colony forming ability,and transfection efficiency in vitro.Our research may help in the effective preservation of germplasm and the generation of transgenic ovine species.

关 键 词：Spermatogonial stem cell Sheep STEMNESS CRYOPRESERVATION TRANSFECTION 

分 类 号：R32[医药卫生—人体解剖和组织胚胎学]