Centre for Reproduction and Development, Monash Institute of Medical Research, Melbourne, Victoria, Australia.
Cell Transplant. 2010;19(5):525-36. doi: 10.3727/096368910X491374. Epub 2010 Feb 8.
Ectopic expression of key reprogramming transgenes in somatic cells enables them to adopt the characteristics of pluripotency. Such cells have been termed induced pluripotent stem (iPS) cells and have revolutionized the field of somatic cell reprogramming, as the need for embryonic material is obviated. One of the issues facing both the clinical translation of iPS cell technology and the efficient derivation of iPS cell lines in the research laboratory is choosing the most appropriate somatic cell type for induction. In this study, we demonstrate the direct reprogramming of a defined population of neural stem cells (NSCs) derived from the subventricular zone (SVZ) and adipose tissue-derived cells (ADCs) from adult mice using retroviral transduction of the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc, and compared the results obtained with a mouse embryonic fibroblast (mEF) control. We isolated mEFs, NSCs, and ADCs from transgenic mice, which possess a GFP transgene under control of the Oct4 promoter, and validated GFP expression as an indicator of reprogramming. While transduction efficiencies were not significantly different among the different cell types (mEFs 68.70 +/- 2.62%, ADCs 70.61 +/- 15.4%, NSCs, 68.72 +/- 3%, p = 0.97), the number of GFP-positive colonies and hence the number of reprogramming events was significantly higher for both NSCs (13.50 +/- 4.10 colonies, 0.13 +/- 0.06%) and ADCs (118.20 +/- 38.28 colonies, 1.14 +/- 0.77%) when compared with the mEF control (3.17 +/- 0.29 colonies, 0.03 +/- 0.005%). ADCs were most amenable to reprogramming with an 8- and 38-fold greater reprogramming efficiency than NSCs and mEFs, respectively. Both NSC iPS and ADC iPS cells were demonstrated to express markers of pluripotency and could differentiate to the three germ layers, both in vitro and in vivo, to cells representative of the three germ lineages. Our findings confirm that ADCs are an ideal candidate as a readily accessible somatic cell type for high efficiency establishment of iPS cell lines.
体细胞中转录因子的异位表达使其获得多能性特征。这些细胞被称为诱导多能干细胞（iPS 细胞），它彻底改变了体细胞重编程领域，因为它避免了对胚胎材料的需求。面临的问题之一是 iPS 细胞技术的临床转化和研究实验室中 iPS 细胞系的高效诱导，即选择最合适的诱导体细胞类型。在这项研究中，我们使用逆转录病毒转导 Yamanaka 因子 Oct4、Sox2、Klf4 和 c-Myc，直接重编程来自脑室下区（SVZ）的定义群体的神经干细胞（NSC）和来自成年小鼠的脂肪组织衍生细胞（ADCs），并将结果与小鼠胚胎成纤维细胞（mEF）对照进行比较。我们从携带 GFP 转基因的转基因小鼠中分离出 mEFs、NSCs 和 ADCs，该 GFP 转基因受 Oct4 启动子的控制，并验证 GFP 表达作为重编程的指标。虽然不同细胞类型之间的转导效率没有显著差异（mEFs 68.70 +/- 2.62%，ADCs 70.61 +/- 15.4%，NSCs 68.72 +/- 3%，p = 0.97），但 GFP 阳性集落的数量，即重编程事件的数量，在 NSCs（13.50 +/- 4.10 集落，0.13 +/- 0.06%）和 ADCs（118.20 +/- 38.28 集落，1.14 +/- 0.77%）中均显著高于 mEF 对照（3.17 +/- 0.29 集落，0.03 +/- 0.005%）。与 NSCs 和 mEFs 相比，ADCs 最适合重编程，其重编程效率分别高出 8 倍和 38 倍。证明 NSC iPS 和 ADC iPS 细胞均表达多能性标志物，并在体外和体内分化为三个胚层，形成代表三个胚系的细胞。我们的发现证实，ADCs 是作为高效建立 iPS 细胞系的理想候选体细胞类型，因为它易于获得。
