State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University , Nanning, People's Republic of China .
Stem Cells Dev. 2012 Sep 1;21(13):2485-94. doi: 10.1089/scd.2012.0018. Epub 2012 May 14.
Ectopically, expression of defined factors could reprogram mammalian somatic cells into induced pluripotent stem cells (iPSCs), which initiates a new strategy to obtain pluripotent stem cell lines. Attempts have been made to generate buffalo pluripotent stem cells by culturing primary germ cells or inner cell mass, but the efficiency is extremely low. Here, we report a successful method to reprogram buffalo fetal fibroblasts (BFFs) into pluripotent stem cells [buffalo induced pluripotent stem cell (biPSCs)] by transduction of buffalo defined factors (Oct4, Sox2, Klf4, and c-Myc) using retroviral vectors. The established biPSCs displayed typical morphological characteristics of pluripotent stem cells, normal karyotype, positive staining of alkaline phosphatase, and expressed pluripotent markers including Oct4, Sox2, Nanog, Lin28, E-Cadherin, SSEA-1, SSEA-4, TRA-1-81, STAT3, and FOXD3. They could form embryoid bodies (EBs) in vitro and teratomas after injecting into the nude BALB/C mice, and 3 germ layers were identified in the EBs and teratomas. Methylation assay revealed that the promoters of Oct4 and Nanog were hypomethylated in biPSCs compared with BFFs and pre-biPSCs, while the promoters of Sox2 and E-Cadherin were hypomethylated in both BFFs and biPSCs. Further, inhibiting p53 expression by coexpression of SV40 large T antigen and buffalo defined factors in BFFs or treating BFFs with p53 inhibitor pifithrin-a (PFT) could increase the efficiency of biPSCs generation up to 3-fold, and nuclear transfer embryos reconstructed with biPSCs could develop to blastocysts. These results indicate that BFFs can be reprogrammed into biPSCs by buffalo defined factors, and the generation efficiency of biPSCs can be increased by inhibition of p53 expression. These efforts will provide a feasible approach for investigating buffalo stem cell signal pathways, establishing buffalo stem cell lines, and producing genetic modification buffaloes in the future.
在异位情况下，特定因子的表达可以将哺乳动物体细胞重编程为诱导多能干细胞（iPSCs），这开创了获得多能干细胞系的新策略。人们尝试通过培养原始生殖细胞或内细胞团来生成水牛多能干细胞，但效率极低。在这里，我们报告了一种通过逆转录病毒载体转导水牛定义因子（Oct4、Sox2、Klf4 和 c-Myc）将水牛胎儿成纤维细胞（BFFs）重编程为多能干细胞[水牛诱导多能干细胞（biPSCs）]的成功方法。所建立的 biPSCs 显示出多能干细胞的典型形态特征、正常核型、碱性磷酸酶的阳性染色，并表达多能标记物，包括 Oct4、Sox2、Nanog、Lin28、E-Cadherin、SSEA-1、SSEA-4、TRA-1-81、STAT3 和 FOXD3。它们可以在体外形成胚状体（EBs），并在注射到裸鼠 BALB/C 后形成畸胎瘤，在 EBs 和畸胎瘤中鉴定出 3 个胚层。甲基化测定显示，与 BFFs 和前 biPSCs 相比，biPSCs 中 Oct4 和 Nanog 的启动子呈低甲基化，而 Sox2 和 E-Cadherin 的启动子在 BFFs 和 biPSCs 中均呈低甲基化。此外，通过共表达 SV40 大 T 抗原和水牛定义因子抑制 BFFs 中的 p53 表达或用 p53 抑制剂 pifithrin-a（PFT）处理 BFFs 可以将 biPSCs 的生成效率提高 3 倍，并用 biPSCs 重构的核移植胚胎可以发育为囊胚。这些结果表明，BFFs 可以通过水牛定义因子重编程为 biPSCs，并且抑制 p53 表达可以提高 biPSCs 的生成效率。这些努力将为未来研究水牛干细胞信号通路、建立水牛干细胞系和生产基因修饰水牛提供可行的方法。
