Human Adult Retinal Pigment Epithelial Stem Cell-Derived RPE Monolayers Exhibit Key Physiological Characteristics of Native Tissue.

PURPOSE

We tested what native features have been preserved with a new culture protocol for adult human RPE.

METHODS

We cultured RPE from adult human eyes. Standard protocols for immunohistochemistry, electron microscopy, electrophysiology, fluid transport, and ELISA were used.

RESULTS

Confluent monolayers of adult human RPE cultures exhibit characteristics of native RPE. Immunohistochemistry demonstrated polarized expression of RPE markers. Electron microscopy illustrated characteristics of native RPE. The mean transepithelial potential (TEP) was 1.19 ± 0.24 mV (mean ± SEM, n = 31), apical positive, and the mean transepithelial resistance (RT) was 178.7 ± 9.9 Ω·cm2 (mean ± SEM, n = 31). Application of 100 μM adenosine triphosphate (ATP) apically increased net fluid absorption (Jv) by 6.11 ± 0.53 μL·cm2·h-1 (mean ± SEM, n = 6) and TEP by 0.33 ± 0.048 mV (mean ± SEM, n = 25). Gene expression of cultured RPE was comparable to native adult RPE (n = 5); however, native RPE RNA was harvested between 24 and 40 hours after death and, therefore, may not accurately reflect healthy native RPE. Vascular endothelial growth factor secreted preferentially basally 2582 ± 146 pg/mL/d, compared to an apical secretion of 1548 ± 162 pg/mL/d (n = 14, P < 0.01), while PEDF preferentially secreted apically 1487 ± 280 ng/mL/d compared to a basolateral secretion of 864 ± 132 ng/mL/d (n = 14, P < 0.01).

CONCLUSIONS

The new culture model preserves native RPE morphology, electrophysiology, and gene and protein expression patterns, and may be a useful model to study RPE physiology, disease, and transplantation.