title: Identification of human corneal epithelial stem cells on the basis of high ABCG2 expression combined with a large N/C ratio.
abstract: OBJECTIVE
  Till date there is no exclusive marker for human corneal epithelial stem cells (CESCs). In this study, our strategy is to combine high expression of ABCG2, a putative SC marker with high N/C ratio to develop a specific method for identification of CESCs.
  
  
  METHODS
  Limbal/corneal epithelial cells (LECs/CECs) were isolated from cadaver eyes by enzymatic treatment and the cytospin smears/cryosections were immunostained for ABCG2, p63, or connexin-43 (Cx-43) and counterstained with propidium iodide/DAPI. Analysis was carried out for (1) ABCG2 expression, N/C ratio, (2) p63, ABCG2, N/C ratio, and (3) ABCG2, Cx-43, N/C ratio using confocal/fluorescence microscopy.
  
  
  RESULTS
  ABCG2 was highly expressed by cells in the basal and suprabasal layers of limbus. The SCs identified on the basis of our earlier method using high levels of p63 expression along with high (>0.7) N/C ratio were strongly positive for ABCG2 and negative for differentiation marker Cx-43. High expression of ABCG2 was also observed in cells with high p63 expression and low (<0.7) N/C ratio; however, they were positive for Cx-43. Combining high ABCG2 expression with large N/C ratio in fluorescence microscopy identified 5.3 ± 3.5% LECs as putative SCs and this was confirmed by confocal microscopic analysis.
  
  
  CONCLUSIONS
  We demonstrate the importance of combining ABCG2 expression with N/C ratio for identification and quantification of human CESCs. This method will have application in evaluating the role of niche cells/factors in the maintenance of stemness.
authors.affiliation: Department of Immunology, Stem Cell Biology, Aravind Medical Research Foundation, Dr. G. Venkataswamy Eye Research Institute, Madurai, Tamil Nadu, India.
authors.forename: Chidambaranathan Gowri
authors.initials: CG
authors.lastname: Priya
authors.forename: Tilak
authors.initials: T
authors.lastname: Prasad
authors.forename: Namperumalsamy Venkatesh
authors.initials: NV
authors.lastname: Prajna
authors.forename: Veerappan
authors.initials: V
authors.lastname: Muthukkaruppan
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis*; D019573:Epithelium, Corneal / Q000166:cytology*; D015870:Gene Expression; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009363:Neoplasm Proteins / Q000096:biosynthesis*; D013234:Stem Cells / Q000502:physiology*
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D009363:Neoplasm Proteins