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

IF 0.5 Q4 REPRODUCTIVE BIOLOGY
R. K. Pramod, Deepthi Varughese, A. J. Jameel, Bhisma Narayan Panda, Soma Goswami, Abhijit Mitra
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Abstract

To assess the effect of cryopreservation on stemness and proliferation potential of sheep spermatogonial stem cells (SSCs) in vitro. 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. 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. 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.
低温保存的绵羊精原干细胞在体外保持干性和集落形成能力
评估低温保存对体外绵羊精原干细胞(SSCs)的干性和增殖潜力的影响。 分离绵羊睾丸细胞,并通过基于层粘连素的差分平板法富集推定的精原干细胞。将推定的SSCs与基于曼陀罗凝集素(DSA-lectin)方法制备的Sertoli细胞供养体共同培养。培养出的推定 SSCs 被冷冻保存在含 10%二甲基亚砜(DMSO)或 10%二甲基亚砜加 200 mM 曲哈糖的杜氏改良鹰培养基-10%胎牛血清混合液(DMEM-10% FBS)培养基中。利用体外培养和免疫细胞化学方法对冷冻保存的推定 SSCs 的增殖潜力和干性进行评估。最后,分析了冷冻保存的推定 SSCs 的转染能力。 我们从绵羊睾丸中分离出了91%存活的睾丸细胞。大部分富含层粘连蛋白的细胞表达了与SSC相关的标志物ITGA6。绵羊推定SSC细胞与Sertoli细胞饲养剂共培养可产生稳定的集落,SSC标记的表达在多次传代后仍能保持。在含10%二甲基亚砜和200毫摩尔曲哈洛糖的培养基中冷冻保存的SSC与仅含10%二甲基亚砜的培养基相比,能存活的推定SSC数量明显更高(P<0.01)。冷冻保存的假定 SSCs 形成了菌落,并显示出与非冷冻保存的假定 SSCs 相似的 SSC 标记表达。Sertoli细胞饲养器上出现绿色荧光菌落表明冷冻保存的绵羊SSCs转染成功。 低温保存的推定造血干细胞可保持其干性、集落形成能力和体外转染效率。我们的研究可能有助于种质的有效保存和转基因绵羊的产生。
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来源期刊
Asian Pacific Journal of Reproduction
Asian Pacific Journal of Reproduction Veterinary-Veterinary (all)
CiteScore
1.70
自引率
0.00%
发文量
588
审稿时长
9 weeks
期刊介绍: The journal will cover technical and clinical studies related to health, ethical and social issues in field of Gynecology and Obstetrics. Articles with clinical interest and implications will be given preference.
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