Direct identification and enrichment of retinal stem cells/progenitors by Hoechst dye efflux assay.
OBJECTIVE
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, RT-PCR, 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 mitogen-exposed 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.
Bhattacharya
Sumitra
S
Department of Ophthalmology, University of Nebraska Medical Center, Omaha, Nebraska 68198-7691, USA.
Jackson
John D
JD
Das
Ani V
AV
Thoreson
Wallace B
WB
Kuszynski
Charles
C
James
Jackson
J
Joshi
Shantaram
S
Ahmad
Iqbal
I
D000818:Animals; D001562:Benzimidazoles; D015415:Biomarkers / Q000032:analysis; D018929:Cell Culture Techniques; D002469:Cell Separation; D018920:Coculture Techniques; D004594:Electrophysiology; D004622:Embryo, Mammalian; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D008856:Microscopy, Fluorescence; D010641:Phenotype; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D014700:Verapamil / Q000494:pharmacology
D001562:Benzimidazoles; D015415:Biomarkers; D005456:Fluorescent Dyes; D014700:Verapamil; C017807:bisbenzimide ethoxide trihydrochloride