Ca++-switch induction of RPE differentiation

Daniel J. Rak, Katherine M. Hardy, Glenn J. Jaffe, Brian S Mckay

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)648-656
Number of pages9
JournalExperimental Eye Research
Volume82
Issue number4
DOIs
StatePublished - Apr 2006

Fingerprint

Retinal Pigments
Monophenol Monooxygenase
Epithelial Cells
Cadherins
Serial Passage
Phenotype
Pigmentation
Morphogenesis
Western Blotting
Tissue Donors

Keywords

  • Bestrophin
  • Cadherin
  • Cellular retinaldehyde binding protein
  • Development
  • Myocilin
  • Pigment
  • Retinal pigment epithelium
  • Tyrosinase

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems

Cite this

Ca++-switch induction of RPE differentiation. / Rak, Daniel J.; Hardy, Katherine M.; Jaffe, Glenn J.; Mckay, Brian S.

In: Experimental Eye Research, Vol. 82, No. 4, 04.2006, p. 648-656.

Research output: Contribution to journalArticle

Rak, Daniel J. ; Hardy, Katherine M. ; Jaffe, Glenn J. ; Mckay, Brian S. / Ca++-switch induction of RPE differentiation. In: Experimental Eye Research. 2006 ; Vol. 82, No. 4. pp. 648-656.
@article{dac66e63e5724a56a50d3887c1fab5c4,
title = "Ca++-switch induction of RPE differentiation",
abstract = "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.",
keywords = "Bestrophin, Cadherin, Cellular retinaldehyde binding protein, Development, Myocilin, Pigment, Retinal pigment epithelium, Tyrosinase",
author = "Rak, {Daniel J.} and Hardy, {Katherine M.} and Jaffe, {Glenn J.} and Mckay, {Brian S}",
year = "2006",
month = "4",
doi = "10.1016/j.exer.2005.09.002",
language = "English (US)",
volume = "82",
pages = "648--656",
journal = "Experimental Eye Research",
issn = "0014-4835",
publisher = "Academic Press Inc.",
number = "4",

}

TY - JOUR

T1 - Ca++-switch induction of RPE differentiation

AU - Rak, Daniel J.

AU - Hardy, Katherine M.

AU - Jaffe, Glenn J.

AU - Mckay, Brian S

PY - 2006/4

Y1 - 2006/4

N2 - 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.

AB - 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.

KW - Bestrophin

KW - Cadherin

KW - Cellular retinaldehyde binding protein

KW - Development

KW - Myocilin

KW - Pigment

KW - Retinal pigment epithelium

KW - Tyrosinase

UR - http://www.scopus.com/inward/record.url?scp=31044449888&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=31044449888&partnerID=8YFLogxK

U2 - 10.1016/j.exer.2005.09.002

DO - 10.1016/j.exer.2005.09.002

M3 - Article

C2 - 16289163

AN - SCOPUS:31044449888

VL - 82

SP - 648

EP - 656

JO - Experimental Eye Research

JF - Experimental Eye Research

SN - 0014-4835

IS - 4

ER -