The epithelial-mesenchymal transition of human RPEs: an evaluation of transcriptomic and functional implications

Abstract

Purpose : The epithelial-mesenchymal transition (EMT) of retinal pigmented epithelial cells (RPE) has been implicated in retinal diseases including age-related macular degeneration and proliferative vitreoretinopathy. Although several in vitro models of RPE-EMT have been developed, the functional implications and transcriptomic changes occurring during RPE-EMT require further investigation. In this study, we aimed to characterize these changes comprehensively with single-cell transcriptomic profiling and multiple functional readouts.

Methods : To induce EMT, human iPSC-derived RPEs were plated at seven seeding densities and cultured for 14 days in 4 independent experiments. Variations in cell morphology were quantified via Cellpose, a deep-learning cell segmentation algorithm. Changes in gene expression profiles were assessed by single-cell RNA-seq on a total of 50K cells collected across seven seeding densities, while variations in key markers were evaluated at a protein level by immunostaining. Cell Impedance assay, fluorescent live-cell imaging and Luminex assay were carried out to assess the barrier function, phagocytic activity and inflammatory status of the RPEs.

Results : Decreasing cell seeding density resulted in the loss of RPE morphology, including an 8-fold increase in cell size and 24% increase in eccentricity. Changes in morphology were associated with a reduction in RPE markers including a 70% decrease in BEST1 and 50% in MITF, alongside a concurrent increase in mesenchymal markers such as an 8-fold increase in alpha-SMA and 5-fold in PAI-1 at both the mRNA and protein level. These morphological and expression changes are accompanied by compromised barrier function and decreased phagocytosis of photoreceptor outer segments, alongside a significant inflammatory response indicated by a 13-fold increase in CXCL10 and 10-fold increase in CCL2.

Conclusions : Our findings highlighted this in vitro model's utility in studying retinal diseases related to RPEs undergoing EMT. Transcriptomic and functional characterization of this model provide new evidence linking RPE-EMT with functional decline. We envision that this model can be used for genetic or chemical perturbation screens to identify targets that can protect RPE against EMT. Ultimately, this study contributes to the broader effort in developing potential therapeutic approaches for retinal diseases.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.