Muñiz Alberto, Ramesh Kaini R, Greene Whitney A, Choi Jae-Hyek, Wang Heuy-Ching
Ocular Trauma, U.S. Army Institute of Surgical Research.
Ocular Trauma, U.S. Army Institute of Surgical Research;
J Vis Exp. 2015 Feb 4(96):52262. doi: 10.3791/52262.
Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.
多能干细胞具有无限增殖的能力，并能分化成几乎任何细胞类型。此外，将体细胞重编程为诱导多能干细胞（iPS细胞）的技术发展，引发了人们对定制个性化再生医学可能性的兴趣和兴奋之情。然而，干细胞向所需谱系分化的效率仍然很低。本研究的目的是描述一种通过应用组织工程方法来生成均匀的胚状体（EBs）群体（体外分化过程中的常见中间体），从iPS细胞中获得视网膜色素上皮细胞（RPE）（iPS-RPE）的方案。该方案利用微孔板技术来形成特定大小的EBs。还解释了通过免疫细胞化学和逆转录聚合酶链反应（RT-PCR）鉴定RPE的蛋白质和基因标志物的方法。最后，描述了通过对RPE标志物进行荧光激活细胞分选（FACS）分析来监测不同大小EBs中分化效率的情况。这些技术将有助于iPS细胞分化为RPE以供未来应用。
