Generation of Induced Pluripotent Stem Cells From Buffalo (Bubalus bubalis) Fetal Fibroblasts with Buffalo Defined Factors
Generation of Induced Pluripotent Stem Cells From Buffalo (Bubalus bubalis) Fetal Fibroblasts with Buffalo Defined Factors
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使用水牛确定因子从水牛 (Bubalus bubalis) 胎儿成纤维细胞中生成诱导多能干细胞
DOI：
10.1089/scd.2012.0018
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发表时间：
2012-09-01
影响因子：
4
通讯作者：
Shi, Deshun
中科院分区：
文献类型：
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作者：
Deng, Yanfei;Liu, Qingyou;Shi, Deshun
异位表达特定因子可将哺乳动物体细胞重编程为诱导性多能干细胞（induced pluripotent stem cells，iPSCs），这为获得多能干细胞系开辟了新的途径。已经尝试通过培养原代生殖细胞或内细胞团来产生布法罗多能干细胞，但效率极低。在这里，我们报告了一种成功的方法，重新编程布法罗胎儿成纤维细胞（BFF）到多能干细胞[布法罗诱导的多能干细胞（biPSC）]的转导布法罗定义的因素（Oct 4，Sox 2，Klf 4，和c-Myc）使用逆转录病毒载体。所建立的biPSC显示多能干细胞的典型形态学特征、正常核型、碱性磷酸酶阳性染色，并表达多能标记物，包括Oct 4、Sox 2、Nanog、Lin 28、E-Cadherin、SSEA-1、SSEA-4、TRA-1-81、STAT 3和FOXD 3。它们在体外可形成拟胚体（EBs），注射入裸鼠体内可形成畸胎瘤，并在EBs和畸胎瘤中鉴定出3个胚层。甲基化分析显示，与BFF和pre-biPSC相比，Oct 4和Nanog的启动子在biPSC中低甲基化，而Sox 2和E-Cadherin的启动子在BFF和biPSC中均低甲基化。此外，通过在BFF中共表达SV 40大T抗原和布法罗限定因子来抑制p53表达或用p53抑制剂匹非亭-a（PFT）处理BFF可以将biPSC产生的效率提高高达3倍，并且用biPSC重建的核移植胚胎可以发育成囊胚。这些结果表明，BFF可以通过布法罗确定的因子重编程为biPSC，并且通过抑制p53表达可以提高biPSC的生成效率。这些工作为今后研究布法罗干细胞信号通路、建立布法罗干细胞系和生产转基因水牛提供了可行的途径。
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.