The retinal pigment epithelium: an important player of retinal disorders and regeneration.

https://eprints.ncl.ac.uk/file_store/production/229439/64E03BC6-140A-4F3C-AB48-F9F8F983997D.pdf

3D culture of human pluripotent stem cells in RGD-alginate hydrogel improves retinal tissue development.

The mechanisms of cell fate diversification in the retina are not fully understood. The seven principal cell types of the neural retina derive from a population of multipotent progenitors during development. These progenitors give rise to multiple cell types concurrently, suggesting that progenitors are a heterogeneous population. It is thought that differences in progenitor gene expression are responsible for differences in progenitor competence (i.e. potential) and, subsequently, fate diversification. To elucidate further the mechanisms of fate diversification, we assayed the expression of three transcription factors made by retinal progenitors: Ascl1 (Mash1), Ngn2 (Neurog2) and Olig2. We observed that progenitors were heterogeneous, expressing every possible combination of these transcription factors. To determine whether this progenitor heterogeneity correlated with different cell fate outcomes, we conducted Ascl1- and Ngn2-inducible expression fate mapping using the CreER™/LoxP system. We found that these two factors gave rise to markedly different distributions of cells. The Ngn2 lineage comprised all cell types, but retinal ganglion cells (RGCs) were exceedingly rare in the Ascl1 lineage. We next determined whether Ascl1 prevented RGC development. Ascl1-null mice had normal numbers of RGCs and, interestingly, we observed that a subset of Ascl1+ cells could give rise to cells expressing Math5 (Atoh7), a transcription factor required for RGC competence. Our results link progenitor heterogeneity to different fate outcomes. We show that Ascl1 expression defines a competence-restricted progenitor lineage in the retina, providing a new mechanism to explain fate diversification.

Ascl1 Expression Defines A Subpopulation Of Lineage-restricted Progenitors In The Mammalian Retina

Replacing defective retinal pigment epithelial (RPE) cells with those derived from human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs) is a potential strategy for treating retinal degenerative diseases. Early clinical trials have demonstrated that hESC-derived or hiPSC-derived RPE cells can be delivered safely as a suspension to the human eye. The next step is transplantation of hESC/hiPSC-derived RPE cells as cell sheets that are more physiological. We have developed a tissue-engineered product consisting of hESC-derived RPE cells grown as sheets on human amniotic membrane as a biocompatible substrate. We established a surgical approach to engraft this tissue-engineered product into the subretinal space of the eyes of rats with photoreceptor cell loss. We show that transplantation of the hESC-RPE cell sheets grown on a human amniotic membrane scaffold resulted in rescue of photoreceptor cell death and improved visual acuity in rats with retinal degeneration compared to hESC-RPE cells injected as a cell suspension. These results suggest that tissue-engineered hESC-RPE cell sheets produced under good manufacturing practice conditions may be a useful approach for treating diseases of retinal degeneration.

Human ESC–derived retinal epithelial cell sheets potentiate rescue of photoreceptor cell loss in rats with retinal degeneration

The retinal pigment epithelium (RPE) plays a key role in the development of many eye diseases leading to visual impairment and even blindness. Cell culture models of pathological changes in the RPE make it possible to study factors responsible for these changes and signaling pathways coordinating cellular and molecular mechanisms of cell interactions under pathological conditions. Moreover, they give an opportunity to reveal target cells and develop effective specific treatment for degenerative and dystrophic diseases of the retina. In this review, data are presented on RPE cell sources for culture models, approaches to RPE cell culturing, phenotypic changes of RPE cells in vitro, the role of signal pathways, and possibilities for their regulation in pathological processes.

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Cell Models to Study Regulation of Cell Transformation in Pathologies of Retinal Pigment Epithelium

Immunoperoxidase and molecular genetic analysis showed that retinal pigment epithelial cells from adult human eye undergo morphogenetic changes in vitro. They lose expression of tissuespecific protein RPE65 and start to express stem cell markers: Oct4 (POU5F1), Nanog, Prox1, Musashi 1, and Pax6, which indicates their differentiation. Expression of Musashi 1 and Pax6 attest to neural differentiation, which is also confirmed by the expression of βIII-tubulin, a neuroblast marker, and markers of differentiated neuronal cells, tyrosine hydroxylase and neurofilament proteins. These findings attest to the capacity of retinal pigment epithelium from adult human eye to transdifferentiation into neural lineage cells, which makes them an interesting object for cell therapy in neurodegeneration.

Expression of multipotent and retinal markers in pigment epithelium of adult human in vitro.

Phenotypic plasticity of retinal pigment epithelial cells from adult human eye was studied by immunohistochemical methods under different culturing conditions. It was found that retinal pigment epithelium in adult human eye is a heterogeneous population of cells demonstrating different behavior in vitro. Some cells retain epithelial morphology for a long time in culture, while others are rapidly transformed into fibroblast-like cells and synthesize proteins typical of proneural, neural, glial, and photoreceptor cells. However, irrespective of initial morphological features differentiation of retinal pigment cells can be modulated by varying culturing conditions.

Phenotypic Plasticity of Retinal Pigment Epithelial Cells from Adult Human Eye In Vitro

The retinal pigment epithelium (RPE), as well as the neural retina, develops from the neuroectoderm and plays a key role in photoreceptor functions. Several degenerative eye diseases, e.g., macular degeneration or retinitis pigmentosa, associated with an impaired RPE function cause the loss of the photoreceptor and partial or complete blindness. Cultured RPE cells obtained from human cadaver eyes could be a valuable source for transplantation to cure retinal degenerative diseases. The paper describes RPE cell isolation, maintenance in culture, and immunohistochemical characteristics of dedifferentiated cells. It was found that RPE cells from human adults exhibit neural cell properties in vitro.

Human adult retinal pigment epithelial cells as potential cell source for retina recovery

We studied the effect of bFGF on human retinal pigment epithelial cells in vitro In ARPE-19 cells, enhanced expression of KLF4 mRNA and reduced expression of PAX6, MITF, and OTX2 mRNA specific for retinal pigment epithelium were observed after bFGF application. The expression of KLF4 mRNA peaked in 72 h after bFGF application and then sharply decreased, which was accompanied by a 3-fold increase in TUBB3 mRNA expression (neuronal marker). Immunocytochemical analysis showed that in the presence of bFGF, some cells retained epithelial properties and showed positive staining for connexin-43, while others had long axon-like processes and demonstrated positive staining for βIII-tubulin, which attests to their neuronal transdifferentiation. Despite the prevalence of the epithelial properties, ARPE-19 cells under the influence of bFGF can show proneuronal properties.

A. V. Kuznetsova

Papers

Cell Models to Study Regulation of Cell Transformation in Pathologies of Retinal Pigment Epithelium

Expression of multipotent and retinal markers in pigment epithelium of adult human in vitro.

Phenotypic Plasticity of Retinal Pigment Epithelial Cells from Adult Human Eye In Vitro

Human adult retinal pigment epithelial cells as potential cell source for retina recovery

Reprogramming of Human Retinal Pigment Epithelial Cells under the Effect of bFGF In Vitro