Derivation, characterization and retinal differentiation of induced pluripotent stem cells
Derivation, characterization and retinal differentiation of induced pluripotent stem cells
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DOI：
10.1007/s12038-012-9296-1
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发表时间：
2013-03-01
影响因子：
2.9
通讯作者：
Mariappan, Indumathi
中科院分区：
文献类型：
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作者：
Mekala, Subba Rao;Vauhini, Vasundhara;Mariappan, Indumathi
全世界有数百万人因视网膜营养不良而患有视力残疾，视网膜营养不良可能与年龄有关，也可能是导致视网膜色素上皮（RPE）细胞和光感受器逐渐变性的遗传性疾病。因此，细胞替代疗法在治疗这些疾病方面提供了很大的希望。由于成人视网膜没有任何干细胞，替代干细胞来源如胚胎干细胞（ESC）和诱导多能干细胞（iPSC）为产生不同类型的视网膜细胞提供了很大的希望。在这里，我们报告了使用携带Oct 4，Sox 2，Klf 4和cMyc基因的重组逆转录病毒的混合物从小鼠胚胎成纤维细胞（MEFs）中衍生出四个iPSC系。通过免疫细胞化学、FACS和RT-PCR分析进一步表征iPS克隆MEF-4F 3的干性标记表达和稳定重编程。nanog启动子的甲基化分析证实了重编程的表观遗传状态。多能性通过胚状体（EB）形成和谱系特异性标志物表达来证实。此外，在视网膜分化时，在6周内产生具有与RPE细胞相似的典型鹅卵石表型的色素细胞斑，并且它们表达ZO-1（紧密连接蛋白）、RPE 65和雌激素样蛋白（成熟RPE标记物），并通过摄取荧光乳胶珠显示吞噬活性。
Millions of people world over suffer visual disability due to retinal dystrophies which can be age-related or a genetic disorder resulting in gradual degeneration of the retinal pigmented epithelial (RPE) cells and photoreceptors. Therefore, cell replacement therapy offers a great promise in treating such diseases. Since the adult retina does not harbour any stem cells, alternative stem cell sources like the embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer a great promise for generating different cell types of the retina. Here, we report the derivation of four iPSC lines from mouse embryonic fibroblasts (MEFs) using a cocktail of recombinant retroviruses carrying the genes for Oct4, Sox2, Klf4 and cMyc. The iPS clone MEF-4F3 was further characterized for stemness marker expression and stable reprogramming by immunocytochemistry, FACS and RT-PCR analysis. Methylation analysis of the nanog promoter confirmed the reprogrammed epigenetic state. Pluripotency was confirmed by embryoid body (EB) formation and lineage-specific marker expression. Also, upon retinal differentiation, patches of pigmented cells with typical cobble-stone phenotype similar to RPE cells are generated within 6 weeks and they expressed ZO-1 (tight junction protein), RPE65 and bestrophin (mature RPE markers) and showed phagocytic activity by the uptake of fluorescent latex beads.