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诱导多能干细胞来源的视网膜色素上皮细胞:细胞系与分化方法的比较研究

Induced Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium: A Comparative Study Between Cell Lines and Differentiation Methods.

作者信息

Leach Lyndsay L, Croze Roxanne H, Hu Qirui, Nadar Vignesh P, Clevenger Tracy N, Pennington Britney O, Gamm David M, Clegg Dennis O

机构信息

1 Center for Stem Cell Biology and Engineering, University of California , Santa Barbara, California.

2 Neuroscience Research Institute, University of California , Santa Barbara, California.

出版信息

J Ocul Pharmacol Ther. 2016 Jun;32(5):317-30. doi: 10.1089/jop.2016.0022. Epub 2016 May 16.

Abstract

PURPOSE

The application of induced pluripotent stem cell-derived retinal pigmented epithelium (iPSC-RPE) in patients with retinal degenerative disease is making headway toward the clinic, with clinical trials already underway. Multiple groups have developed methods for RPE differentiation from pluripotent cells, but previous studies have shown variability in iPSC propensity to differentiate into RPE.

METHODS

This study provides a comparison between 2 different methods for RPE differentiation: (1) a commonly used spontaneous continuously adherent culture (SCAC) protocol and (2) a more rapid, directed differentiation using growth factors. Integration-free iPSC lines were differentiated to RPE, which were characterized with respect to global gene expression, expression of RPE markers, and cellular function.

RESULTS

We found that all 5 iPSC lines (iPSC-1, iPSC-2, iPSC-3, iPSC-4, and iPSC-12) generated RPE using the directed differentiation protocol; however, 2 of the 5 iPSC lines (iPSC-4 and iPSC-12) did not yield RPE using the SCAC method. Both methods can yield bona fide RPE that expresses signature RPE genes and carry out RPE functions, and are similar, but not identical to fetal RPE. No differences between methods were detected in transcript levels, protein localization, or functional analyses between iPSC-1-RPE, iPSC-2-RPE, and iPSC-3-RPE. Directed iPSC-3-RPE showed enhanced transcript levels of RPE65 compared to directed iPSC-2-RPE and increased BEST1 expression and pigment epithelium-derived factor (PEDF) secretion compared to directed iPSC-1-RPE. In addition, SCAC iPSC-3-RPE secreted more PEDF than SCAC iPSC-1-RPE.

CONCLUSIONS

The directed protocol is a more reliable method for differentiating RPE from various pluripotent sources and some iPSC lines are more amenable to RPE differentiation.

摘要

目的

诱导多能干细胞衍生的视网膜色素上皮细胞(iPSC-RPE)在视网膜退行性疾病患者中的应用正朝着临床应用迈进,临床试验已经在进行中。多个研究小组已经开发出了从多能细胞分化出RPE的方法,但先前的研究表明,iPSC分化为RPE的倾向存在差异。

方法

本研究对两种不同的RPE分化方法进行了比较:(1)一种常用的自发连续贴壁培养(SCAC)方案;(2)一种使用生长因子的更快速、定向分化方法。无整合的iPSC系被分化为RPE,并对其进行了全基因组表达、RPE标记物表达和细胞功能方面的表征。

结果

我们发现,所有5个iPSC系(iPSC-1、iPSC-2、iPSC-3、iPSC-4和iPSC-12)使用定向分化方案都能产生RPE;然而,5个iPSC系中的2个(iPSC-4和iPSC-12)使用SCAC方法未能产生RPE。两种方法都能产生表达标志性RPE基因并执行RPE功能的真正RPE,且与胎儿RPE相似但不完全相同。在iPSC-1-RPE、iPSC-2-RPE和iPSC-3-RPE之间的转录水平、蛋白质定位或功能分析中,未检测到方法之间的差异。与定向分化的iPSC-2-RPE相比,定向分化的iPSC-3-RPE显示RPE65的转录水平增强,与定向分化的iPSC-1-RPE相比,BEST1表达增加且色素上皮衍生因子(PEDF)分泌增加。此外,SCAC iPSC-3-RPE分泌的PEDF比SCAC iPSC-1-RPE更多。

结论

定向方案是一种从各种多能来源分化RPE更可靠的方法,并且一些iPSC系更适合RPE分化。

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