In Vitro Phenotypic Modifi cation of Pigmented Epithelium Cells from Human Eye at Early Stages of Development

Abstract

Multipotent characteristics of human fetal (9-11.5 weeks) pigmented epithelial retinal cells and capacity to transdifferentiation in neuronal direction were studied in vitro under different culturing conditions. The cultures were analyzed using a wide spectrum of antibodies. It was found that pigmented epithelium of human eye is a heterogeneous cell population with three subtypes differing by adhesion characteristics, migration, transdifferentiation potential, and reaction to microenvironmental factors. Subtype 1 cells steadily retain their epithelial characteristics, subtype 2 cells change their morphotype and produce neuroblast and photoreceptor cell proteins, and subtype 3 cells form free floating spheres and are capable to multipotent differentiation.

Reprogramming of Differentiated Mammalian and Human Retinal Pigment Epithelium: Current Achievements and Prospects

“…In cell culture, RPE cells lose their original features, such as pigmentation, significantly reduce the expression of specific markers RPE65, MITF, and CRALBP and acquire features of neural cells on markers MUSASHI1, NESTIN, βIII-TUBULIN, GFAP, DOUBLECORTIN, and NF 68 and 200 kDa. Our studies also demonstrate that RPE cells of the human embryo and adult in vitro in media with the addition of morphogens and growth factors lose their pigment granules, dedifferentiate, proliferate, and exhibit markers of several types of neural and glial cells (Milyushina et al, 2009(Milyushina et al, , 2011(Milyushina et al, , 2012Kuznetsova et al, 2014;Kuznetsova et al, 2015Kuznetsova et al, , 2019. At the dedifferentiation stage, cells acquire stem/neuroepithelial cell traits by expressing OCT4, NANOG, KLF4, OTX2, PAX6, and NESTIN (Milyushina et al, 2009Kuznetsova et al, 2014Kuznetsova et al, , 2015Kuznetsova et al, , 2019aKuznetsova et al, , 2019b.…”

Recent Achievements in the Heterogeneity of Mammalian and Human Retinal Pigment Epithelium: In Search of a Stem Cell

“…In the general population of adult human eye RPE cells in vitro, the expression of stem cell pluripotency genes NANOG, OCT4, SOX2, SSEA-4, KLF4, C-MYC, and LIN-28 was detected [100,105,106,108,110]. In another study, OCT4 was not detected in the RPE of the adult human eye [100].…”

“…Furthermore, naturally, we also detected markers of redifferentiation to the original phenotype (RPE65, PEDF, CRALB) [105,106,[108][109][110]. Based on these data, we hypothesized that the human RPE may contain highly plastic cells that have (or acquire during the process of dedifferentiation) the SC phenotype with a dual potential.…”

“…The proliferation of human RPESCs was stimulated by cultivating them in a freefloating state with the addition of growth factors, similar to neural stem cells (NSCs) [100,105,106,110]. The mouse RPE cells were cultivated in the form of spheres to obtain induced iRPESCs [96].…”

“…In our laboratory, using qPCR analysis and immunostaining, it was shown that under in vitro differentiation conditions, the RPE cells of embryonic and adult human eyes express markers of progenitor and neural cells [105,106,[108][109][110]. Other researchers have found photoreceptor markers in RPE cell lines and primary RPE cultures, such as Crx10 [117], Opn3, Opn4, Nrl, Crx, Opn1mw/1w, Sag, Nr2e3, and recoverin [118].…”