title: A novel method for a high enrichment of human corneal epithelial stem cells for genomic analysis.
abstract: Understanding the molecular mechanisms that regulate the corneal epithelial stem cells (CESCs) in maintaining corneal homeostasis remains elusive largely due to the lack of a specific marker for their isolation. This study aims to enrich CESCs from human donor limbal epithelium and to evaluate the level of enrichment based on expression of ΔNp63α, a putative CESC marker. A two-stage enrichment of CESCs was carried out. (a) The limbal basal epithelial cells were isolated by differential enzymatic treatment and five-fold enrichment was achieved from 2% of CESCs present in the total limbal epithelium. The CESCs were quantified on the basis of two parameters-high expression of p63/ABCG2 and nucleus to cytoplasmic (N/C) ratio ≥0.7. (b) Cytospin smears of isolated basal cells were Giemsa stained and cells with N/C ratio ≥0.7 were separated by laser capture microdissection. This strategy resulted in an enrichment of CESCs to 78.57% based on two-parameter analysis using p63 and 76.66% using ABCG2. RT-PCR was carried out for ΔNp63 isoforms (α, β, and γ) and connexin-43, with GAPDH for normalization. The expression of ΔNp63α was restricted to the enriched population of CESCs in contrast to its absence in limbal basal cells with N/C ratio <0.7 and CCECs. The unique expression of ΔNp63α and 5.9-fold reduced connexin-43 expression in the enriched population of CESCs indicates its high purity. Further analysis of these cells will help in elucidating the molecular mechanisms associated with stemness and also in identifying a specific marker for CESCs.
authors.affiliation: Department of Immunology and Stem Cell Biology, Aravind Medical Research Foundation, Dr. G. Venkataswamy Eye Research Institute, Madurai, Tamil Nadu, India.
authors.forename: Jhansi Rani
authors.initials: JR
authors.lastname: Kasinathan
authors.affiliation: Cornea Clinic, Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Madurai, Tamil Nadu, India.
authors.forename: Venkatesh Prajna
authors.initials: VP
authors.lastname: Namperumalsamy
authors.affiliation: Department of Immunology and Stem Cell Biology, Aravind Medical Research Foundation, Dr. G. Venkataswamy Eye Research Institute, Madurai, Tamil Nadu, India.
authors.forename: Muthukkaruppan
authors.initials: M
authors.lastname: Veerappan
authors.affiliation: Department of Immunology and Stem Cell Biology, Aravind Medical Research Foundation, Dr. G. Venkataswamy Eye Research Institute, Madurai, Tamil Nadu, India.
authors.forename: Gowri Priya
authors.initials: GP
authors.lastname: Chidambaranathan
mesh_terms: D002467:Cell Nucleus / Q000502:physiology; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000502:physiology; D004847:Epithelial Cells / Q000166:cytology*; D019573:Epithelium, Corneal / Q000166:cytology; D005819:Genetic Markers / Q000235:genetics; D023281:Genomics / Q000379:methods*; D006801:Humans; D061346:Laser Capture Microdissection / Q000379:methods*; D016850:Limbus Corneae / Q000166:cytology*; D016133:Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology*
chemical_list: D005819:Genetic Markers
keywords: high N/C ratio; laser capture microdissection; limbal basal epithelial cells