Partially Differentiated Neuroretinal Cells Promote Maturation of the Retinal Pigment Epithelium

Abstract

PURPOSE. Many studies have demonstrated the ability of the retinal pigment epithelium (RPE) to foster the maturation of the developing retina. Few studies have examined the reciprocal effects of developing retina on the RPE. METHODS. RPE isolated from human fetal RPE or differentiated from human stem cells was cultured on Transwell filter inserts. Retinal progenitor cells (RPCs) were differentiated from human stem cells and cultured on a planar scaffold composed of gelatin, chondroitin sulfate, hyaluronic acid, and laminin-521. Cultures were analyzed by quantitative RT-PCR, immunofluorescence, immunoblotting, and transepithelial electrical resistance (TER). RESULTS. RPCs initially differentiated into several retina-like cell types that segregated from one another and formed loosely organized layers or zones. With time, the presumptive photoreceptor and ganglion cell layers persisted, but the intervening zone became dominated by cells that expressed glial markers with no evidence of bipolar cells or interneurons. Co-culture of this underdeveloped retinoid with the RPE resulted in a thickened layer of recoverin-positive cells but did not prevent the loss of interneuron markers in the intervening zone. Although photoreceptor inner and outer segments were not observed, immunoblots revealed that co-culture increased expression of rhodopsin and red/green opsin. Co-culture of the RPE with this underdeveloped retinal culture increased the TER of the RPE and the expression of RPE signature genes. CONCLUSIONS. These studies indicated that an immature neurosensory retina can foster maturation of the RPE; however, the ability of RPE alone to foster maturation of the neurosensory retina is limited.

Retinal Cell Transplantation, Biomaterials, and In Vitro Models for Developing Next-generation Therapies of Age-related Macular Degeneration

“…Recent breakthroughs have overcome the frustrations of past attempts and have already identified potential therapeutic targets. 84 , 86 , 87 , 102 These novel data support continued exploration of RPE-choroid co-cultures by in vivo testing to determine their potential as a tissue-replacement strategy. Further development of these models should also provide a platform to test potential therapeutic agents before committing to expensive animal studies.…”

“…Below the timeline are approximate times for events observed for co-cultures of RPC cultured on the GCH-521 scaffold with RPE cultured on laminin-111 coated PET. 102 Reprinted from Singh et al 102 (CC BY-NC-ND 4.0).…”

“…3 ), RPC were harvested, dissociated, and cultured on a scaffold composed of gelatin, hyaluronic acid, chondrotin sulfate, and laminin-521 (GCH-521). 102 , 104 For co-culture, RPE monolayers (hfRPE or hiPSC-RPE) were established on laminin-111-coated PET. Differentiation of the co-culture is compared to the differentiation of spherical retinal organoids in Fig.…”

A Literature-Derived Knowledge Graph Augments the Interpretation of Single Cell RNA-seq Datasets

“…However, we also identified several CDGs with little or no literature association to RPE cells, including genes encoding ion transporters (SLC6A13, CLIC6) and proteins that modulate Wnt signaling (FRZB, SFRP5). While these Wnt modulators have been infrequently referenced as RPE markers [91,92], and CLIC6 has been detected by proteomics in RPE tissue and cells [93,94], the contribution of these genes to RPE function has never been explored. We confirmed that these markers were also upregulated in RPE cells relative to other retinal cells using the more traditional within-study comparison as well (Figure S10).…”

A literature-derived knowledge graph augments the interpretation of single cell RNA-seq datasets

“…However, we also identified several CDGs with little or no literature association to RPE cells, including genes encoding ion transporters (SLC6A13, CLIC6) and proteins that modulate Wnt signaling (FRZB, SFRP5). While these Wnt modulators have been infrequently referenced as RPE markers [70,71], and CLIC6 has been detected by proteomics in RPE tissue and cells [72,73], the contribution of these genes to RPE function has never been explored.…”