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PLoS One. 2016 Jul 6;11(7):e0158282. doi: 10.1371/journal.pone.0158282. eCollection 2016.

Differentiation/Purification Protocol for Retinal Pigment Epithelium from Mouse Induced Pluripotent Stem Cells as a Research Tool.

Author information

1
Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Kobe, Hyogo, Japan.
2
Department of Ophthalmology & Visual Science, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan.
3
Ultrastructural Research Team, RIKEN Center for Life Science Technologies, Kobe, Hyogo, Japan.
4
Department of Cell Biology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan.

Abstract

PURPOSE:

To establish a novel protocol for differentiation of retinal pigment epithelium (RPE) with high purity from mouse induced pluripotent stem cells (iPSC).

METHODS:

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.

RESULTS:

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.

CONCLUSION:

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.

PMID:
27385038
PMCID:
PMC4934919
DOI:
10.1371/journal.pone.0158282
[Indexed for MEDLINE]
Free PMC Article

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