Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Muller C

PROVIDER: S-EPMC5918243 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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

Müller Claudia C Charniga Carol C Temple Sally S Finnemann Silvia C SC

Stem cell reports 20180215 3

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- ...[more]

PMID: 29456184

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Project description:Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.

Dataset Information

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

Publications

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

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