Abstract
Purpose :
Despite improvements in methods for generating human pluripotent stem cell (hPSC)-derived retinal pigment epithelial (RPE) cells, these methods are still limited by low cell yield, poor post-thaw cell quality, as well as the need for cell enrichment and long term culture. To overcome this, we have developed a scalable and rapid system for the efficient generation of mature RPE cells that can be cryopreserved to generate large cell banks.
Methods :
To generate mature RPE cells, four hPSCs maintained in mTeSR™ Plus were differentiated using STEMdiff™-ACF RPE Differentiation Kit until day 14, then subcultured in STEMdiff™-XF RPE Maturation Medium supplemented with STEMdiff™-ACF RPE Plating Supplement (Plating Medium) until day 21 and Maturation Medium thereafter. RPE cells were cryopreserved on day 14 in CryoStor® CS10. Expression of key RPE maturity markers (PMEL17, RPE65, EZRIN, CRALBP, ZO1, and BEST1) were assessed by flow cytometry or immunohistochemistry. Apicobasal secretion of pigment epithelial-derived factor (PEDF) and vascular endothelial growth factor (VEGF), transepithelial electrical resistance (TEER), and photoreceptor outer segment (POS) phagocytosis efficiency was determined to assess RPE functionality.
Results :
STEMdiff™-ACF RPE Differentiation Kit efficiently generated high yields of immature RPE cells (92.0 ± 1.2% PMEL17, 1.1 x 106 ± 0.6 x 106 cells/cm2, n = 16 - 20) that was scalable to T-75 cm2 flasks. Plating Medium enhanced post-passage and post-thaw plating efficiencies relative to Maturation Medium alone (3.5 ± 0.6 and 3.2 ± 0.4 fold respectively; n = 12). RPE cells enriched to a mature population by day 49 (99.0 ± 0.2% PMEL17, 94.5 ± 0.7% RPE65, 93.1 ± 1.3% EZRIN, 89.5 ± 1.2% CRALBP; n = 16) and IHC analysis confirmed the correct subcellular expression of BEST1, ZO1, RPE65, and EZRIN. Mature RPE established apicobasal polarity, as confirmed by polarized secretion of PEDF and VEGF (15.1 ± 3.8 and 0.23 ± 0.02 apical/basal ratio, respectively; n = 12), high TEER (749 ± 120 Ωxcm2; n = 12), and efficient phagocytosis of POS (94.5 ± 1.4%; n = 12). All data represent mean + SEM.
Conclusions :
STEMdiff™ RPE Differentiation workflow supports the rapid and robust generation of mature RPE cells. The high compliance and scalability of this culture system makes it amenable for both basic and preclinical research, overcoming many limitations faced by current methods.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.