Quantified F-Actin Morphology Is Predictive of Phagocytic Capacity of Stem Cell-Derived Retinal Pigment Epithelium

Abstract

Summary: With stem cell-derived retinal pigment epithelial (RPE) replacement therapies in clinical testing, establishing potency of RPE prior to transplantation is imperative. Phagocytosis of photoreceptor outer segment fragments (POS) is a key indicator of RPE functionality. Comparing RPE derived from different donor human adult RPE stem cell lines, we found that cells were either high-phagocytic or low-phagocytic despite sharing phagocytic receptors and ligands, junctional ZO-1, and lack of epithelial-mesenchymal transition. We found that low-phagocytic cells harbored F-actin stress fibers but lacked contiguous lateral circumferential F-actin and ezrin-rich microvilli of high-phagocytic cells. Rho kinase inhibition reversed the F-actin phenotype and restored phagocytic capacity to low-phagocytic RPE. Conversely, RhoA activation induced stress fiber formation and reduced phagocytic function of high-phagocytic RPE. These results demonstrate that a stress fiber-rich microfilament cytoskeleton causes phagocytic dysfunction of RPE cells. We propose F-actin assessment as a rapid, sensitive, and quantitative test to identify RPE populations lacking phagocytic capacity. : Here, Finnemann and colleagues demonstrate that F-actin stress fibers in adult stem cell-derived retinal pigment epithelial (RPE) cells predict and cause poor phagocytic activity, a cardinal RPE function. ROCK inhibitor treatment of differentiated RPE monolayers suffices to revert F-actin phenotype and restore phagocytic function. The authors propose F-actin phenotype scoring as a rapid, sensitive, and quantitative assessment of RPE quality. Keywords: adult RPE stem cells, cytoskeleton, F-actin, phagocytosis, retinal pigment epithelium, RPE, RhoA/ROCK, stress fibers