Ca ++-switch induction of RPE differentiation

Cultured retinal pigment epithelial (RPE) cells are commonly used as a model of the tissue to study their involvement in visual diseases. Unfortunately, cultured RPE often lose their differentiated phenotype reducing their usefulness as a model of the RPE in vivo. In this study, we used a Ca ++-swit...

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Bibliographic Details
Published in:Experimental eye research 2006-04, Vol.82 (4), p.648-656
Main Authors: Rak, Daniel J., Hardy, Katherine M., Jaffe, Glenn J., McKay, Brian S.
Format: Article
Language:English
Subjects:
Adolescent
Adult
Aged
Aged, 80 and over
bestrophin
Bestrophins
Biomarkers - analysis
cadherin
Cadherins - metabolism
Calcium - administration & dosage
Calcium - metabolism
Carrier Proteins - analysis
Cell Differentiation - physiology
Cells, Cultured
cellular retinaldehyde binding protein
Chloride Channels
Cytoskeletal Proteins - analysis
development
Eye Color - physiology
Eye Proteins - analysis
Glycoproteins - analysis
Humans
Microscopy, Electron - methods
Microscopy, Fluorescence - methods
Middle Aged
Models, Biological
Monophenol Monooxygenase - analysis
myocilin
Phenotype
pigment
Pigment Epithelium of Eye - cytology
Pigment Epithelium of Eye - ultrastructure
retinal pigment epithelium
tyrosinase
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Summary:Cultured retinal pigment epithelial (RPE) cells are commonly used as a model of the tissue to study their involvement in visual diseases. Unfortunately, cultured RPE often lose their differentiated phenotype reducing their usefulness as a model of the RPE in vivo. In this study, we used a Ca ++-switch protocol to initiate the patterned expression of several phenotypic and functional markers of RPE differentiation. Cultured RPE cells from adult donors were maintained through at least six serial passages prior to assay to minimize their differentiated properties. The cells were then subjected to the Ca ++-switch protocol and maintained at confluence for up to 4 months. Paired control and Ca ++-switch cells were examined for phenotype, pigmentation, and the expression of tyrosinase, CRABP, myocilin, and bestrophin by western blot analysis. The Ca ++-switch protocol led to a rapid restriction of N-cadherin to lateral cell borders, and to expression of tyrosinase by day 4. After 8 weeks, the experimental RPE monolayers began to accumulate visible pigment, and after 12 weeks CRABP expression was observed. Myocilin was observed at 4 months after the Ca ++-switch but bestrophin was not detected at any time point. Our results suggest this protocol may drive epithelial morphogenesis in RPE cells. We note two specific differences in cells plated in low Ca ++, reduced spreading on the substrate and coordinated development of cadherin adhesion when the Ca ++-concentration is returned to normal. Thus, we suggest that this method produces phenotypic changes through multiple cell signalling pathways.
ISSN:0014-4835
1096-0007
DOI:10.1016/j.exer.2005.09.002