Protocol for Obtaining Mouse iPS-RPE
Iwasaki, Yuko RIKEN|Tokyo Medical and Dental University
Sugita, Sunao RIKEN
Mandai, Michiko RIKEN
Yonemura, Shigenobu RIKEN|Tokushima University Tokushima University Educator and Researcher Directory KAKEN Search Researchers
Onishi, Akishi RIKEN
Ito, Shin-ichiro RIKEN
Mochizuki, Manabu Tokyo Medical and Dental University
Ohno-Matsui, Kyoko Tokyo Medical and Dental University
Takahashi, Masayo RIKEN
To establish a novel protocol for differentiation of retinal pigment epithelium (RPE) with high purity from mouse induced pluripotent stem cells (iPSC).
Retinal progenitor cells were differentiated from mouse iPSC, and RPE differentiation was then enhanced by activation of the Wnt signaling pathway, inhibition of the fibroblast growth factor signaling pathway, and inhibition of the Rho-associated, coiled-coil containing protein kinase signaling pathway. Expanded pigmented cells were purified by plate adhesion after Accutase® treatment. Enriched cells were cultured until they developed a cobblestone appearance with cuboidal shape. The characteristics of iPS-RPE were confirmed by gene expression, immunocytochemistry, and electron microscopy. Functions and immunologic features of the iPS-RPE were also evaluated.
We obtained iPS-RPE at high purity (approximately 98%). The iPS-RPE showed apical-basal polarity and cellular structure characteristic of RPE. Expression levels of several RPE markers were lower than those of freshly isolated mouse RPE but comparable to those of primary cultured RPE. The iPS-RPE could form tight junctions, phagocytose photoreceptor outer segments, express immune antigens, and suppress lymphocyte proliferation.
We successfully developed a differentiation/purification protocol to obtain mouse iPS-RPE. The mouse iPS-RPE can serve as an attractive tool for functional and morphological studies of RPE.
Copyright: © 2016 Iwasaki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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pone_11_7_e0158282.pdf 12.7 MB