Induced pluripotent stem cells (iPSC) have successfully been derived from somatic fibroblasts through transfection of synthetic modified mRNA encoding transcription factors. This technique obviates the use of recombinant DNA and viral vectors in cellular reprogramming. The present study derived iPSC from adipose-derived mesenchymal stem cells (of a 50-year-old female patient) by utilizing a similar technique, but with defined culture medium without feeder cells, during both reprogramming and propagation. Clonal selection was performed to yield 12 putative iPSC lines from individual colonies of nascent reprogrammed cells, starting from 150,000 cells. However, only seven lines maintained their undifferentiated state after 10 continuous serial passages. These seven lines were then subjected to a rigorous battery of analyses to confirm their identity as iPSC. These tests included immunostaining, flow cytometry, qRT-PCR, in vitro differentiation assay, and teratoma formation assay within SCID mice. Positive results were consistently observed in all analyses, thus verifying the cells as fully reprogrammed iPSC. While all 7 iPSC lines displayed normal karyogram up to passage 13, chromosomal anomalies occurred in 4 of 7 lines with extended in vitro culture beyond 24 serial passages. Only three lines retained normal karyotype of 46,XX. The remaining four lines displayed mosaicism of normal and abnormal karyotypes. Hence, this study successfully derived iPSC from abundant and easily accessible adipose tissues of a middle-aged patient; utilizing a mRNA-based integration-free technique under feeder-free conditions. This is a step forward in translating iPSC into personalized regenerative medicine within the clinic.

译文

通过转染合成的编码转录因子的修饰mRNA,已成功地从体细胞成纤维细胞中衍生出诱导多能干细胞 (iPSC)。该技术避免了在细胞重编程中使用重组DNA和病毒载体。本研究通过使用类似的技术从脂肪来源的间充质干细胞 (一名50岁女性患者) 中衍生出iPSC,但在重编程和繁殖过程中使用了没有饲养细胞的确定培养基。进行克隆选择,以从150,000细胞开始,从新生重编程细胞的各个菌落产生12个推定的iPSC系。但是,在连续10次连续传代之后,只有7条线保持了其未分化状态。然后对这七个行进行了严格的分析,以确认其作为iPSC的身份。这些测试包括免疫染色,流式细胞术,qRT-PCR,体外分化测定和SCID小鼠内的畸胎瘤形成测定。在所有分析中始终观察到阳性结果,从而验证了细胞为完全重新编程的iPSC。尽管所有7个iPSC系在第13代之前均显示正常的核子图,但7个系中的4个发生了染色体异常,其体外培养范围超过24个连续传代。只有三个系保留了正常的核型46,XX。其余四行显示出正常和异常核型的镶嵌。因此,这项研究成功地从中年患者丰富且易于获取的脂肪组织中提取了iPSC; 在无饲养者条件下利用基于mRNA的无整合技术。这是在临床中将iPSC转化为个性化再生医学的一步。

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