Direct identification and enrichment of retinal stem cells/progenitors by hoechst dye efflux assay

Sumitra Bhattacharya, John D. Jackson, Ani V. Das, Wallace B Thoreson, Charles Kuszynski, Jackson James, Shantaram S Joshi, Iqbal Ahmad

Research output: Contribution to journalArticle

91 Citations (Scopus)

Abstract

PURPOSE. The present study describes a method for isolating neural stem cells/progenitors directly from the freshly dissociated embryonic retina (prospective identification) and compares their characteristics with those enriched from mitogen-exposed embryonic retinal cell culture. METHODS. Cell dissociates from embryonic rat retina and mitogen-exposed embryonic retinal cultures were stained with Hoechst 33342 fluorescent dye. The emission patterns of cells were analyzed in both blue and red wavelength using flow cytometry to enrich cells that retained or excluded the dye. The phenotype characteristics and differentiation potential of enriched cells were analyzed by immunocytochemical, RTPCR, and electrophysiological analyses. RESULTS. The Hoechst dye efflux assay identified a minor population of cells, called side population (SP) cells, in fresh retinal dissociates. These cells that preferentially excluded the Hoechst 33342 fluorescent dye were proliferative and expressed both neural progenitor and retinal progenitor markers. The retinal SP cells generated functional neurons and glia and possessed the ability to differentiate along lineages of different late-born retinal cell types. Cells of similar phenotypes and potential were observed in the SP obtained from mitogenexposed retinal culture. CONCLUSIONS. The Hoechst dye efflux assay represents an effective method for direct identification of retinal stem cells/progenitors. These results demonstrate that the prospectively isolated retinal stem cells/progenitors and those enriched as SP cells from mitogen-exposed retinal cell culture may be similar in their properties and potential.

Original languageEnglish (US)
Pages (from-to)2764-2773
Number of pages10
JournalInvestigative Ophthalmology and Visual Science
Volume44
Issue number6
DOIs
StatePublished - Jun 1 2003

Fingerprint

Coloring Agents
Stem Cells
Side-Population Cells
Mitogens
Fluorescent Dyes
Retina
Cell Culture Techniques
Phenotype
Neural Stem Cells
Neuroglia
Population
Flow Cytometry
Neurons
HOE 33342

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Direct identification and enrichment of retinal stem cells/progenitors by hoechst dye efflux assay. / Bhattacharya, Sumitra; Jackson, John D.; Das, Ani V.; Thoreson, Wallace B; Kuszynski, Charles; James, Jackson; Joshi, Shantaram S; Ahmad, Iqbal.

In: Investigative Ophthalmology and Visual Science, Vol. 44, No. 6, 01.06.2003, p. 2764-2773.

Research output: Contribution to journalArticle

Bhattacharya, Sumitra ; Jackson, John D. ; Das, Ani V. ; Thoreson, Wallace B ; Kuszynski, Charles ; James, Jackson ; Joshi, Shantaram S ; Ahmad, Iqbal. / Direct identification and enrichment of retinal stem cells/progenitors by hoechst dye efflux assay. In: Investigative Ophthalmology and Visual Science. 2003 ; Vol. 44, No. 6. pp. 2764-2773.
@article{b7c753c7315149f19bfec18d1c157e5d,
title = "Direct identification and enrichment of retinal stem cells/progenitors by hoechst dye efflux assay",
abstract = "PURPOSE. The present study describes a method for isolating neural stem cells/progenitors directly from the freshly dissociated embryonic retina (prospective identification) and compares their characteristics with those enriched from mitogen-exposed embryonic retinal cell culture. METHODS. Cell dissociates from embryonic rat retina and mitogen-exposed embryonic retinal cultures were stained with Hoechst 33342 fluorescent dye. The emission patterns of cells were analyzed in both blue and red wavelength using flow cytometry to enrich cells that retained or excluded the dye. The phenotype characteristics and differentiation potential of enriched cells were analyzed by immunocytochemical, RTPCR, and electrophysiological analyses. RESULTS. The Hoechst dye efflux assay identified a minor population of cells, called side population (SP) cells, in fresh retinal dissociates. These cells that preferentially excluded the Hoechst 33342 fluorescent dye were proliferative and expressed both neural progenitor and retinal progenitor markers. The retinal SP cells generated functional neurons and glia and possessed the ability to differentiate along lineages of different late-born retinal cell types. Cells of similar phenotypes and potential were observed in the SP obtained from mitogenexposed retinal culture. CONCLUSIONS. The Hoechst dye efflux assay represents an effective method for direct identification of retinal stem cells/progenitors. These results demonstrate that the prospectively isolated retinal stem cells/progenitors and those enriched as SP cells from mitogen-exposed retinal cell culture may be similar in their properties and potential.",
author = "Sumitra Bhattacharya and Jackson, {John D.} and Das, {Ani V.} and Thoreson, {Wallace B} and Charles Kuszynski and Jackson James and Joshi, {Shantaram S} and Iqbal Ahmad",
year = "2003",
month = "6",
day = "1",
doi = "10.1167/iovs.02-0899",
language = "English (US)",
volume = "44",
pages = "2764--2773",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
number = "6",

}

TY - JOUR

T1 - Direct identification and enrichment of retinal stem cells/progenitors by hoechst dye efflux assay

AU - Bhattacharya, Sumitra

AU - Jackson, John D.

AU - Das, Ani V.

AU - Thoreson, Wallace B

AU - Kuszynski, Charles

AU - James, Jackson

AU - Joshi, Shantaram S

AU - Ahmad, Iqbal

PY - 2003/6/1

Y1 - 2003/6/1

N2 - PURPOSE. The present study describes a method for isolating neural stem cells/progenitors directly from the freshly dissociated embryonic retina (prospective identification) and compares their characteristics with those enriched from mitogen-exposed embryonic retinal cell culture. METHODS. Cell dissociates from embryonic rat retina and mitogen-exposed embryonic retinal cultures were stained with Hoechst 33342 fluorescent dye. The emission patterns of cells were analyzed in both blue and red wavelength using flow cytometry to enrich cells that retained or excluded the dye. The phenotype characteristics and differentiation potential of enriched cells were analyzed by immunocytochemical, RTPCR, and electrophysiological analyses. RESULTS. The Hoechst dye efflux assay identified a minor population of cells, called side population (SP) cells, in fresh retinal dissociates. These cells that preferentially excluded the Hoechst 33342 fluorescent dye were proliferative and expressed both neural progenitor and retinal progenitor markers. The retinal SP cells generated functional neurons and glia and possessed the ability to differentiate along lineages of different late-born retinal cell types. Cells of similar phenotypes and potential were observed in the SP obtained from mitogenexposed retinal culture. CONCLUSIONS. The Hoechst dye efflux assay represents an effective method for direct identification of retinal stem cells/progenitors. These results demonstrate that the prospectively isolated retinal stem cells/progenitors and those enriched as SP cells from mitogen-exposed retinal cell culture may be similar in their properties and potential.

AB - PURPOSE. The present study describes a method for isolating neural stem cells/progenitors directly from the freshly dissociated embryonic retina (prospective identification) and compares their characteristics with those enriched from mitogen-exposed embryonic retinal cell culture. METHODS. Cell dissociates from embryonic rat retina and mitogen-exposed embryonic retinal cultures were stained with Hoechst 33342 fluorescent dye. The emission patterns of cells were analyzed in both blue and red wavelength using flow cytometry to enrich cells that retained or excluded the dye. The phenotype characteristics and differentiation potential of enriched cells were analyzed by immunocytochemical, RTPCR, and electrophysiological analyses. RESULTS. The Hoechst dye efflux assay identified a minor population of cells, called side population (SP) cells, in fresh retinal dissociates. These cells that preferentially excluded the Hoechst 33342 fluorescent dye were proliferative and expressed both neural progenitor and retinal progenitor markers. The retinal SP cells generated functional neurons and glia and possessed the ability to differentiate along lineages of different late-born retinal cell types. Cells of similar phenotypes and potential were observed in the SP obtained from mitogenexposed retinal culture. CONCLUSIONS. The Hoechst dye efflux assay represents an effective method for direct identification of retinal stem cells/progenitors. These results demonstrate that the prospectively isolated retinal stem cells/progenitors and those enriched as SP cells from mitogen-exposed retinal cell culture may be similar in their properties and potential.

UR - http://www.scopus.com/inward/record.url?scp=0038823704&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0038823704&partnerID=8YFLogxK

U2 - 10.1167/iovs.02-0899

DO - 10.1167/iovs.02-0899

M3 - Article

C2 - 12766085

AN - SCOPUS:0038823704

VL - 44

SP - 2764

EP - 2773

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 0146-0404

IS - 6

ER -