title: Comparative transcriptomic analysis of cultivated limbal epithelium and donor corneal tissue reveals altered wound healing gene expression.
abstract: OBJECTIVE
  The improved surgical outcomes associated with transplantation of cultivated amniotic membrane expanded limbal epithelium (AMLE) compared to traditional donor methods has led to substantial adoption of this technique for treatment of limbal stem cell deficiency.
  
  
  METHODS
  The mRNA expression profiles of AMLE and CE were assayed using microarrays. Transcripts with a 1.5-fold change in either direction in addition to a Bonferroni adjusted P value < 0.05 were considered to be differentially expressed. Expression changes detected by microarray profiling and important corneal-limbal markers were assessed using quantitative real-time PCR (qRT-PCR) and immunofluorescence staining.
  
  
  RESULTS
  A total of 487 probe sets (319 upregulated and 168 downregulated) were found to be differentially expressed between AMLE and CE. Enrichment analysis revealed significant overrepresentation of multiple biological processes (e.g., response to wounding, wound healing, and regulation of cell morphogenesis) within the differentially expressed gene list. The expression of a number of genes that were upregulated (ABCG2, S100A9, ITGA5, TIMP2, FGF5, PDGFC, SEMA3A) and downregulated (KLF4, P63α) in AMLE was confirmed using qRT-PCR. Immunofluorescence confirmed that AMLE cultures were P63α, ABCG2, CK3, CK12, and E-cadherin (E-cad) positive.
  
  
  CONCLUSIONS
  In this study, we have shown that genes associated with wound healing processes are upregulated in AMLE. These gene expression changes may contribute to corneal restoration and the positive outcomes associated with transplantation.
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland.
authors.forename: Clair
authors.initials: C
authors.lastname: Gallagher
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland.
authors.forename: Colin
authors.initials: C
authors.lastname: Clarke
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland.
authors.forename: Sinéad T
authors.initials: ST
authors.lastname: Aherne
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland.
authors.forename: Kishore R
authors.initials: KR
authors.lastname: Katikireddy
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland.
authors.forename: Padraig
authors.initials: P
authors.lastname: Doolan
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland.
authors.forename: Vincent
authors.initials: V
authors.lastname: Lynch
authors.affiliation: Irish Blood Transfusion Service, Dublin, Ireland.
authors.forename: Sandra
authors.initials: S
authors.lastname: Shaw
authors.affiliation: Hermitage Clinic, Dublin, Ireland.
authors.forename: Andra
authors.initials: A
authors.lastname: Bobart-Hone
authors.affiliation: The Royal Victoria Eye and Ear Hospital, Dublin, Ireland Royal College of Surgeons in Ireland, Dublin, Ireland.
authors.forename: Conor
authors.initials: C
authors.lastname: Murphy
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland.
authors.forename: Martin
authors.initials: M
authors.lastname: Clynes
authors.affiliation: The Royal Victoria Eye and Ear Hospital, Dublin, Ireland.
authors.forename: William
authors.initials: W
authors.lastname: Power
authors.affiliation: National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland.
authors.forename: Finbarr
authors.initials: F
authors.lastname: O'Sullivan
mesh_terms: D000650:Amnion / Q000166:cytology; D015415:Biomarkers / Q000378:metabolism; D002102:Cadaver; D002478:Cells, Cultured; D016039:Corneal Transplantation; D019573:Epithelium, Corneal / Q000378:metabolism*; D020869:Gene Expression Profiling; D006801:Humans; D000090062:Kruppel-Like Factor 4; D016850:Limbus Corneae / Q000378:metabolism*; D046228:Microarray Analysis; D012313:RNA / Q000378:metabolism; D000077278:RNA, Mitochondrial; D014945:Wound Healing / Q000502:physiology*
chemical_list: D015415:Biomarkers; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D000077278:RNA, Mitochondrial; D012313:RNA
keywords: corneal epithelial; cultivated; limbal; stem cell; wound healing