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

Abstract

SummaryWith 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.

Phagocytosis by the Retinal Pigment Epithelium: Recognition, Resolution, Recycling

“…Given the amount of new membrane delivered and/or the unfurling of the microvillar membrane that would require substantive F-actin disassembly for ensheathment, it makes sense that PI3K activation is indeed a prerequisite for synchronized POS phagocytosis and that its inhibition leads to increased F-actin at aborted phagocytic cups (71). That Rho inactivation is central to the process is supported by experiments demonstrating decreased phagocytosis when Rho kinase is made to be constitutively active (72).…”

The Cytoskeleton of the Retinal Pigment Epithelium: from Normal Aging to Age-Related Macular Degeneration

“…Besides maintaining cell shape and geometry, the cytoskeleton is also involved in the traffic and transport of intracellular granules. Alterations in the cytoskeleton have been linked to poor phagocytosis, as shown in stem-cell-derived RPE cells [163], and might indicate abnormality of other cell functions. An indispensable pre-requisite for any cell replacement therapy is to deliver intact and functioning RPE cells.…”

A Human Retinal Pigment Epithelium-Based Screening Platform Reveals Inducers of Photoreceptor Outer Segments Phagocytosis

“…To our knowledge, this is the first report of ERMN expression in in vitro cultured RPE cells, which supports the notion that pluripotent stem cell-derived RPE cells are a bettersuited model for phagocytosis assays than commercially available cell lines. F-actin morphology has been shown to be predictive of phagocytic capacity of RPE cells (Müller et al, 2018). Following RM treatment, no stress fibers were detected, as indicated by phalloidin staining, suggesting that RM has a positive effect on actin morphology and dynamics, which are involved in POS phagocytosis.…”