May 2005
Volume 46, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2005
Characterization of Human Limbal Side Population (SP) Cells
Author Affiliations & Notes
  • H. Miyashita
    Cornea Center, Physiology,
    Tokyo Dental College, Ichikawa, Japan
  • S. Shimmura
    Ophthalmology, Ophthalmology,
    Tokyo Dental College, Ichikawa, Japan
  • Y. Matsuzaki
    Cornea Center, Physiology,
    Keio University School of Medicine, Tokyo, Japan
  • K. Higa
    Cornea Center, Physiology,
    Tokyo Dental College, Ichikawa, Japan
  • H. Okano
    Cornea Center, Physiology,
    Keio University School of Medicine, Tokyo, Japan
  • J. Shimazaki
    Ophthalmology, Ophthalmology,
    Tokyo Dental College, Ichikawa, Japan
  • K. Tsubota
    Ophthalmology, Ophthalmology,
    Keio University School of Medicine, Tokyo, Japan
  • Footnotes
    Commercial Relationships  H. Miyashita, None; S. Shimmura, None; Y. Matsuzaki, None; K. Higa, None; H. Okano, None; J. Shimazaki, None; K. Tsubota, None.
  • Footnotes
    Support  None.
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 2085. doi:
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      H. Miyashita, S. Shimmura, Y. Matsuzaki, K. Higa, H. Okano, J. Shimazaki, K. Tsubota; Characterization of Human Limbal Side Population (SP) Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2085.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: To investigate the molecular marker expression profile and colony forming efficiency of limbal epithelial SP cells. Methods: Four to ten limbal segments from human eyebank corneas were treated enzymatically to obtain single cell suspensions. Cells were incubated with Hoechst 33342 solution (4 ∼ 5 µg / ml) with or without reserpine at 37 °C for 60 min, stained with propidium iodide (PI), and analyzed by flow cytometry. Dead (PI +) cells were gated out. SP cells were defined as reserpine sensitive Hoechst blue (low) and red (low) population. Cytospin samples of SP cells and main population (MP) cells were stained with anti–keratin 19, anti–keratin 3, anti–vimentin, and anti–MART1 (melanocyte marker) antibodies. Unsorted limbal cells were used as control. SP and MP cells were cultured with mitomycin C treated 3T3 cells to observe colony formation. Results: Percentage of SP cells in viable (PI–) cells ranged from 0.01 % to 0.92 % (average=0.25 % ± 0.32 %, n= 10). K19, K3, vimentin, and MART1 positive cells were 52.8 %, 10.8 %, 19.3 %, and 6.4 % in SP cells, 53.8 %, 26.6 %, 8.5 %, and 5.0 % in MP cells, and 44.1%, 44.6 %, 9.7 %, and 2.0 % in unsorted limbal cells, respectively. Colony forming efficiency (CFE) of SP and MP cells was 2.7 % and 2.0 %, respectively. Conclusions: SP fraction of limbal epithelial cells contained fewer K3 (+) cells and higher levels of vimentin (+) cells compared with the MP fraction, however, there was no difference observed in CFE.

Keywords: flow cytometry • cornea: epithelium • cornea: basic science 
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