title: The side population cells in the rabbit limbus sensitively increased in response to the central cornea wounding.
abstract: OBJECTIVE
  Side population (SP) cells are known to reside in the limbus as putative corneal epithelial stem cells. This study was performed to demonstrate the presence and the characteristics of SP cells in the rabbit limbal epithelium and explore their sensitivity in response to the central cornea wounding.
  
  
  METHODS
  To sort out the SP cells, freshly isolated rabbit limbal and central corneal epithelial cells were subjected to Hoechst 33342 dye efflux assay. For characterization of the sorted SP cells, RT-PCR analysis, semi-dry three-dimensional (3-D) cell culture, and transplantation in nude mice were performed. To explore wound sensitivity of the limbal SP cells, the rabbit central cornea was wounded by direct contact of a 6-mm paper disk soaked with 1 N NaOH, and changes in the population size of the SP cells and the colony-forming efficiency (CFE) was monitored on days 1, 2, and 5 after wounding.
  
  
  RESULTS
  The SP cells were present in the rabbit limbal epithelium with an incidence of 0.73% +/- 0.14% (n = 8) and were smaller in cell size than the major population (MP) cells, quiescent in the cell cycle, and in the undifferentiated state. The SP cells were able to regenerate the cornea-like structure with basal enrichment of p63-positive cells by in vitro 3-D culture and in vivo transplantation, all of which were best achieved by the whole population (WP) of cells comprising SP and MP cells. After central cornea wounding, this rare population of the limbal SP cells increased in size fivefold on day 1 and then decreased on day 2. The transient increase in the SP cells was subsequently followed by the propagation of an increase in CFE in the limbal MP cells on day 2 and then in the corneal MP cells on day 5. In the hematopoietic colony-forming assay, the limbal SP cells gave approximately eightfold higher CFU than the limbal MP cells.
  
  
  CONCLUSIONS
  The SP cells identified in the rabbit limbus are an undifferentiated and noncycling rare epithelial cell population, which sensitively respond to the central cornea wounding by their transient increase in the population size.
authors.affiliation: R&D Institute, Modern Cell & Tissue Technologies, Inc., Seoul, Korea.
authors.forename: Ki-Sook
authors.initials: KS
authors.lastname: Park
authors.forename: Chae Ho
authors.initials: CH
authors.lastname: Lim
authors.forename: Byung-Moo
authors.initials: BM
authors.lastname: Min
authors.forename: Jae Lim
authors.initials: JL
authors.lastname: Lee
authors.forename: Hee-Yong
authors.initials: HY
authors.lastname: Chung
authors.forename: Choun-Ki
authors.initials: CK
authors.lastname: Joo
authors.forename: Chan-Woong
authors.initials: CW
authors.lastname: Park
authors.forename: Youngsook
authors.initials: Y
authors.lastname: Son
mesh_terms: D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D002057:Burns, Chemical / Q000378:metabolism*; D018929:Cell Culture Techniques; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D003114:Colony-Forming Units Assay; D019573:Epithelium, Corneal / Q000166:cytology* / Q000187:drug effects; D005126:Eye Burns / Q000139:chemically induced* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D016850:Limbus Corneae / Q000166:cytology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012972:Sodium Hydroxide; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000502:physiology*
chemical_list: D001562:Benzimidazoles; D018797:Cell Cycle Proteins; D005456:Fluorescent Dyes; D012972:Sodium Hydroxide; C017807:bisbenzimide ethoxide trihydrochloride