Isolation of Putative Limbal Epithelial Stem Cells by Fluorescein Activated Cell Sorting.
- Author:
Mee Kum KIM
1
;
Kyeong Seon SHIN
;
Gyung Ah JUNG
;
Ki Cheul SHIN
;
Won Ryang WEE
;
Jae Lim LEE
;
Ki Sook PARK
;
Young Sook SON
Author Information
1. Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea. wrwee@snu.ac.kr
- Publication Type:Original Article
- Keywords:
Brd U-retaining cell;
Cornea;
Epithelium;
Hoechst exclusion;
Side population
- MeSH:
Cornea;
Epithelium;
Fluorescein*;
Propidium;
Rabbits;
Stem Cells*;
Verapamil
- From:Journal of the Korean Ophthalmological Society
2005;46(3):521-527
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
PURPOSE: To analyze the isolating pattern of slow cycling cells as putative limbal epithelial stem cells (PLESCs) using Hoechst exclusive cell sorting. METHODS: Rabbits were injected with 5-bromo-2-deoxyuridine (Brd U) 1 month prior to be sacrificed. After obtaining limbal tissues, fluorescence-activated cells were sorted on a Coulter EPICS 753 after they had been incubated with Hoechst 33342 and propidium iodide. Two different methods were applied to sort PLESCs. Side-population(Sp) cells were obtained using gates with dichroic mirror to detect low Hoechst blue and red after verapamil was treated. Hoechst negative cells were obtained using gates exhibiting low Hoechst blue with a 424/44 BP filter. Brd U-retaining cells were counted and their sizes were evaluated in each gated sample to compare isolating pattern of PLESCs in each method. RESULTS: The percentages of Sp cells and of the Hoechst negative fraction were 0.96 +/- 0.79% and 16.01 +/- 13.60%, respectively(p=0.021). Homogeneity and density of the small cells were higher in Hoechst negative fraction than in Sp cells. The percentage of Brd U-retaining cells was 47.36 +/- 10.34% and 47.14 +/- 14.94% in Sp cells and Hoechst negative fraction, respectively(p>0.05), and they were 10 times higher than in non-Sp and Hoechst positive fraction(p=0.000). CONCLUSIONS: Hoechst negative exclusion without verapamil more efficiently isolated PLESCs than Sp did.
