Abstract
Purpose :
Current protocols for generating human pluripotent stem cell (hPSC)-derived RPE cells vary in efficiencies across different hPSC lines, have a lengthy culture time, and require serum-containing media. To address these limitations, we have developed a rapid, standardized workflow and culture kit to generate mature and functional RPE cells.
Methods :
RPE cells were generated by mimicking stages of eye development. Four hPSC lines maintained in mTeSR™ Plus, were differentiated sequentially using the STEMdiff™ RPE Differentiation Kit to generate immature RPE cells. The immature RPE cells were exposed to maturation conditions, and differentiation efficiency was assessed by the expression of premelanosome protein (PMEL17) and retinoid isomerohydrolase RPE65 (RPE65). RPE cell polarization was determined by the localization of pigment epithelial-derived factor (PEDF) and vascular endothelial growth factor (VEGF) secretion. Functionality of the RPE cells was measured by trans-epithelial electrical resistance (TEER) and bovine rod outer segment phagocytosis.
Results :
The STEMdiff™ RPE Differentiation Kit generated immature RPE cells rapidly with high efficiency (87.9 ± 1.9% PMEL17, n = 22), which were enriched to a mature population (95.4 ± 0.7% PMEL17; 93.4 ± 1.1% RPE65, n = 14). Mature RPE cells established correct apicobasal polarity, secreting greater apical PEDF (114 ± 11.5 ng/mm2 apical, 5.5 ± 3.0 ng/mm2 basal, n = 8) and greater basal VEGF (17.3 ± 3.0 pg/mm2 apical, 29.6 ± 4.1 pg/mm2 basal, n = 7). Monolayers of RPE cells established a strong barrier with high TEER (479 ± 32 Ω×cm2, n = 8), suggesting RPE cells formed tight junctions and displayed characteristic features required for the visual axis including the ability to phagocytose bovine rod outer segments (90.6 ± 2.7%, n = 4). All data are mean ± sem.
Conclusions :
We have developed the first cell culture kit that efficiently and rapidly generates mature RPE cells using a standardized workflow. The cultured RPE cells display key functional features of in vivo RPE cells, and the kit composition makes it amenable for future pre-clinical and therapeutic studies.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.