Identifying Biomarkers of Retinal Pigment Epithelial Cell Stem Cell-derived RPE Cell Heterogeneity and Transplantation Efficacy

Abstract

Transplantation of retinal pigment epithelial (RPE) cells holds great promise for patients with retinal degenerative diseases such as age-related macular degeneration. In-depth characterization of RPE cell product identity and critical quality attributes are needed to enhance efficacy and safety of replacement therapy strategies. Here we characterized an adult RPE stem cell-derived (RPESC-RPE) cell product using bulk and single cell RNA sequencing (sc-RNA-seq), assessing functional cell integration in vitro into a mature RPE monolayer and in vivo efficacy by vision rescue in the Royal College of Surgeons rats. scRNA-seq revealed several distinct subpopulations in the RPESC-RPE product, some with progenitor markers. We identified RPE clusters expressing genes associated with in vivo efficacy and increased cell integration capability. Gene expression analysis revealed a lncRNA (TREX) as a predictive marker of in vivo efficacy. TREX knockdown decreased cell integration while overexpression increased integration in vitro and improved vision rescue in the RCS rats.

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

“…The hereditary nature of RPE cell specialization was confirmed by in vitro experiments [71]. Against the backdrop of actively developing studies of RPE cells derived from embryonic stem cells and induced pluripotent stem cells (iPSCs) (iPSC-RPE), the question arises about the emergence of heterogeneity similar to the native RPE in iPSC-RPE.…”

“…Among these subpopulations, there are ten profiles in number. In these cells, the specific marker EZH2 (a transcription factor involved in histone methylation, self-renewal, and the differentiation of SCs) was isolated, indicating a state similar to stem cells or progenitor cells [71]. This study has confirmed RPE heterogeneity under culture conditions, similar to the native tissue, and answered the previously raised question about the innate differences between RPE cells, and whether a trait can be accurately passed on to daughter cells in the reproducing population [31].…”

“…The heterogeneity of RPE-RPESCs is manifested not only in the functional specialization of cells, but also in the ability of RPE-RPESCs to successfully transplant. Several subpopulations of RPE-RPESCs have been identified that may be potential candidates for effective transplantation [71]. These cells showed the enrichment of signaling pathways associated with cell differentiation and proliferation.…”

“…A clear difference between the transcriptomes of transplant-effective and transplant-ineffective subpopulations of RPE-RPESCs was identified. In addition, molecular pathways associated with graft effectiveness were shown, and potential biomarkers for effective RPE cultures were established [71]. As a primary biomarker of transplantation success, the long noncoding RNA (lncRNA) Three Prime Repair Exonuclease 1 (TREX) was considered, which regulates various cellular processes, including migration and cell survival [71].…”

“…In addition, molecular pathways associated with graft effectiveness were shown, and potential biomarkers for effective RPE cultures were established [71]. As a primary biomarker of transplantation success, the long noncoding RNA (lncRNA) Three Prime Repair Exonuclease 1 (TREX) was considered, which regulates various cellular processes, including migration and cell survival [71]. These data will allow us to identify from the heterogeneous populations of RPESC-RPE, iPSC-RPE, ESC-RPE, and RPE obtained from other possible cell sources a separate subpopulation of cells that is most suitable for successful transplantation.…”