Single-cell transcriptome of the mouse retinal pigment epithelium in response to a low-dose of doxorubicin

Abstract

Cellular senescence of the retinal pigment epithelium (RPE) is thought to play an important role in vision-threatening retinal degenerative diseases, such as age-related macular degeneration (AMD). However, the single-cell RNA profiles of control RPE tissue and RPE tissue exhibiting cellular senescence are not well known. We have analyzed the single-cell transcriptomes of control mice and mice with low-dose doxorubicin (Dox)-induced RPE senescence (Dox-RPE). Our results have identified 4 main subpopulations in the control RPE that exhibit heterogeneous biological activities and play roles in ATP synthesis, cell mobility/differentiation, mRNA processing, and catalytic activity. In Dox-RPE mice, cellular senescence mainly occurs in the specific cluster, which has been characterized by catalytic activity in the control RPE. Furthermore, in the Dox-RPE mice, 6 genes that have not previously been associated with senescence also show altered expression in 4 clusters. Our results might serve as a useful reference for the study of control and senescent RPE.

Supramolecular Senolytics via Intracellular Oligomerization of Peptides in Response to Elevated Reactive Oxygen Species Levels in Aging Cells

“…Then, the major cell types were assigned to each cluster based on the expression levels of known marker genes (Supplementary Figure 32b, Supplementary Data 1). , Since gene expression was affected little by treatment with Mito-K2 in the control mice (Supplementary Figure 34f), the control, Alu , and Mito-K2-groups were included in the subsequent analysis. Among all the cells, 10,531 RPE cells were identified according to the expression of RPE-specific markers, including Rpe65 (Con-RPE: 3424, Alu -RPE: 3587, and Mito-K2-RPE, 3520).…”

“…Therefore, to better characterize the cellular senescence of RPE cells in a GA mouse model induced by Alu RNA and appropriate monitoring of the senescent RPE cells clearance, we tried to establish a panel of genes. First, considering the enormous molecular diversity between cell types, we included genes that were significant in our recent study using Dox-induced RPE senescence model . The twelve ‘senescence/RPE panel’ genes identified in the analysis were as follows: a senescence core gene (Cdkn1a), genes associated with cellular senescence (A2m, Ntrk2, Malat1), SASP component genes (Serping1, Tmem176b, Gas6), genes associated with the regulation of apoptosis (Ckmt1, Ccng1), genes related to the extracellular matrix (ECM) or epithelial-mesenchymal transition (EMT) of RPE (Timp1, Vim), and a gene associated with drusen biogenesis in AMD (Clu) .…”

“…Among all the cells, 10,531 RPE cells were identified according to the expression of RPE-specific markers, including Rpe65 (Con-RPE: 3424, Alu -RPE: 3587, and Mito-K2-RPE, 3520). The other cells mainly corresponded to known cell types in the retina and choroidal tissues. , The RPE cells from each group were further subdivided into 7 clusters in the UMAP plot (Figure a, Table ), which showed heterogeneity with differentially expressed genes (DEGs) (Supplementary Data 2). To investigate whether specific functions could be assigned to each cluster, Gene Ontology (GO) analysis was performed using the cluster-specific markers found by the FindAllMarkers­() function in the Seurat package in R (Supplementary Data 3, 4, and 5).…”

“…To overcome this issue and to generate datasets consisting of a sufficient amount of RPE cells, we meticulously prepared single cells from the retina/RPE/choroid tissues of 10 eyeballs per group, as previously established in our work. 46 All of the RPE cells were then pooled, and 1/5 of the retinal cells were mixed to make a retina/RPE sample for each group. Analysis of 83,289 cells yielded a uniform manifold approximation and projection (UMAP) plot featuring 8 clusters with distinct expression profiles (Supplementary Figure 32a).…”

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

“…Our present transcriptomic analysis demonstrated an incomplete ability to separate the RPE cell transcriptomic profiles based solely on time, pointing out the need to dissect RPE cellular heterogeneity at each timepoint. Recent studies using single cell approaches have reported multiple RPE subpopulations within the stem cell-derived, native human and mouse RPE with unique transcriptional and morphological characteristics, indicating that the RPE cell layer is composed of heterogenous cell populations that may have differing functional and clinical capabilities (Lee et al, 2022; Petrus-Reurer et al, 2022; Voigt et al, 2019; Xu et al, 2021). Identification of the subpopulations responsible for vision rescue can improve regulatory evaluation and improve RPE cell products to result in better transplantation outcomes.…”