Exosomes uptake by human retinal pigment epithelium cells

Abstract

Purpose : The purpose of these experiments was to test and optimize uptake of mesenchymal stem cells (MSC) exosomes by human retinal pigment epithelium (ARPE-19) cell line utilizing the natural uptake mechanisms of exosomes by ARPE-19 cells.

Methods : We used human mesenchymal stem cells (MSC) exosomes labeled using ExoGlow (red-fluorescent dye) or human embryonic kidney 293 cells (HEK-293) exosomes X-Pack GFP-loaded (green-fluorescent protein). The concentrations of exosomes and the incubation period of transfected ARPE-19 were varied to determine which conditions allow for the highest transfection rate. ARPE-19 were labeled using an Oregon Green Phalloidin or CellTrace calcein red/orange AM and were imaged using an Olympus FV3000 confocal and ImageXpress Micro XL high-content microscopes. Assays were done in 96-well plates with four replicates, and plates were scanned on the ImageXpress Micro XL high-content screening system (Molecular Devices) at UC Davis IDDRC Biological Analysis Core. Additional images were obtained using a confocal microscope Olympus FV3000.

Results : Results indicate that ARPE-19 cells were able to uptake exosomes from two different heterologous sources, human mesenchymal stem cells (MSC) and human embryonic kidney 293 (HEK-293) cells. The highest transfection rate was achieved with 0.20 ug of exosomes added to 1x10^4 ARPE-19 cells/well, after a 5-hour incubation period. Imaging was best within the first 30 hours after exosomes were added to ARPE-19 cells. Signal diminished significantly after 30 hours. A higher quantity of exosomes did not equate to a higher transfection rate.

Conclusions : Exosomes are mediators of long-distance communication among different tissue and cells in the body. Exosomes carry cargo (proteins, mRNA, and microRNAs) to communicate messages to target tissues or cells. Our lab has previously identified a set of microRNAs (miRNAs) dysregulated in DR patients' ocular fluids, that may play a role in diabetic retinopathy (DR) pathogenesis. Our results strongly suggest that exosomes were quickly taken up by naïve recipient ARPE-19 cells by natural mechanisms. Our long-term goal is to develop miRNA-based therapies for DR using exosomes as natural carriers of miRNAs.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.