Abstract
PurposeTo establish a novel protocol for differentiation of retinal pigment epithelium (RPE) with high purity from mouse induced pluripotent stem cells (iPSC).MethodsRetinal 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.ResultsWe 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.ConclusionWe 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.
Highlights
Regenerative therapy using differentiated cells derived from stem cells is drawing attention worldwide
We successfully developed a differentiation/purification protocol to obtain mouse iPS-retinal pigment epithelium (RPE)
We have been conducting a clinical study on the autologous transplantation of retinal pigment epithelium (RPE) derived from induced pluripotent stem cells in a patient with age-related macular degeneration
Summary
Retinal progenitor cells were differentiated from mouse iPSC, and RPE differentiation was 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 evaluated
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