Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences at Kagawa, Tokushima Bunri University, 1314-1 Shido, Sanuki, Kagawa, 769-2193, Japan.
Hum Cell. 2011 Jun;24(2):96-103. doi: 10.1007/s13577-011-0016-1. Epub 2011 May 12.
Normal human cells have a replicative life span and therefore senesce. Usually, normal human cell strains are differentiated cells and reach a terminally differentiated state after a number of cell divisions. At present, definitive differences are not known between replicative senescence and terminal differentiation. TIG-1 is a human fibroblast strain established from fetal lung and has been used extensively in studies of cellular senescence, and numerous data were accumulated at the molecular level. Recently, a method for generating induced pluripotent stem cells (iPSCs) was developed. Using the method, we introduced four reprogramming genes to TIG-1 fibroblasts and succeeded in isolating colonies that had embryonic stem cell (ESC)-like morphologies. They showed alkaline phosphatase activity and expressed ESC markers, as shown by immunostaining of OCT4, SOX2, SSEA4, and TRA-1-81 as well as reverse-transcription polymerase chain reaction (RT-PCR) for OCT4 and NANOG transcripts. Thus, we succeeded in establishing iPSC clones from TIG-1. The iPSC clones could differentiate to cells originated from all three germ-cell layers, as shown by RT-PCR, for messenger RNA (mRNA) expression of α-fetoprotein (endoderm), MSX1 (mesoderm) and microtubule-associated protein 2 (ectoderm), and by immunostaining for α-fetoprotein (endoderm), α-smooth muscle actin (mesoderm), and β-III-tubulin (ectoderm). The iPSCs formed teratoma containing the structures developed from all three germ-cell layers in severe combined immune-deficiency mice. Thus, by comparing the aging process of parental TIG-1 cells and the differentiation process of iPSC-derived fibrocytes to fibroblasts, we can reveal the exact differences in processes between senescence and terminal differentiation.
正常人类细胞具有复制寿命，因此会衰老。通常，正常人类细胞株是分化细胞，在经过多次细胞分裂后达到终末分化状态。目前，复制性衰老和终末分化之间没有明确的区别。TIG-1 是人胚肺来源的成纤维细胞株，广泛用于细胞衰老研究，积累了大量分子水平的数据。最近，开发了一种诱导多能干细胞（iPSC）的生成方法。我们使用该方法将四个重编程基因引入 TIG-1 成纤维细胞中，并成功分离出具有胚胎干细胞（ESC）样形态的集落。它们表现出碱性磷酸酶活性，并通过 OCT4、SOX2、SSEA4 和 TRA-1-81 的免疫染色以及 OCT4 和 NANOG 转录物的逆转录聚合酶链反应（RT-PCR）显示 ESC 标志物的表达。因此，我们成功地从 TIG-1 建立了 iPSC 克隆。iPSC 克隆可以分化为来自三个胚层的细胞，如 RT-PCR 所示，用于α-胎蛋白（内胚层）、MSX1（中胚层）和微管相关蛋白 2（外胚层）的信使 RNA（mRNA）表达，以及α-胎蛋白（内胚层）、α-平滑肌肌动蛋白（中胚层）和β-III-微管蛋白（外胚层）的免疫染色。iPSCs 在严重联合免疫缺陷小鼠中形成含有来自三个胚层的结构的畸胎瘤。因此，通过比较亲本 TIG-1 细胞的衰老过程和 iPSC 衍生的纤维细胞向成纤维细胞的分化过程，我们可以揭示衰老和终末分化过程之间的确切差异。
