title: Markers for distinguishing cultured human corneal endothelial cells from corneal stromal myofibroblasts.
abstract: OBJECTIVE
  Eliminating contamination by corneal stromal cells is critical when preparing cultured human corneal endothelial cells (CECs) transplantation. We investigated markers for the purification of cultured human CECs and markers for excluding cultured human corneal stromal myofibroblasts (CSMFs) from cultured human CECs.
  
  
  METHODS
  CECs and CSMFs were obtained from human donor corneas by culturing separately in serum-containing medium. Candidate markers of CECs and CSMFs were screened with microarray analysis in the fourth passaged CECs and CSMFs. Then, selected factors were evaluated in reverse transcription polymerase chain reaction (RT-PCR), western blot, immunocytochemistry, and flow cytometry to investigate differential markers for each cell.
  
  
  RESULTS
  Among the genes identified by microarray analysis, cultured human CECs, but not CSMFs, expressed integrin alpha 3 (ITGA3 and CD49c) protein according to immunocytochemistry and western blotting. Iroquois homeobox 2 (IRX2) gene was a marker that distinguished CSMFs from cultured human CECs by RT-PCR. The IRX2 gene can be used as a marker of CSMFs contaminating cultured CECs.
  
  
  CONCLUSIONS
  These molecules could be important markers for the production of highly purified cultured CECs for regenerative medicine.
authors.affiliation: a Corneal Regeneration Research Team, Foundation for Biomedical Research and Innovation , Kobe , Japan .|b Department of Ophthalmology , Juntendo University School of Medicine , Tokyo , Japan .
authors.forename: Masahiro
authors.initials: M
authors.lastname: Yamaguchi
authors.affiliation: a Corneal Regeneration Research Team, Foundation for Biomedical Research and Innovation , Kobe , Japan .|c Department of Ophthalmology , University of Tokyo Graduate School of Medicine , Tokyo , Japan .
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Shima
authors.affiliation: a Corneal Regeneration Research Team, Foundation for Biomedical Research and Innovation , Kobe , Japan .
authors.forename: Miwa
authors.initials: M
authors.lastname: Kimoto
authors.affiliation: b Department of Ophthalmology , Juntendo University School of Medicine , Tokyo , Japan .|d Department of Ophthalmology , Juntendo University Urayasu Hospital , Chiba , Japan and.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Ebihara
authors.affiliation: b Department of Ophthalmology , Juntendo University School of Medicine , Tokyo , Japan .
authors.forename: Akira
authors.initials: A
authors.lastname: Murakami
authors.affiliation: a Corneal Regeneration Research Team, Foundation for Biomedical Research and Innovation , Kobe , Japan .|e Corneal Transplantation Section, University of Tokyo Graduate School of Medicine , Tokyo , Japan.
authors.forename: Satoru
authors.initials: S
authors.lastname: Yamagami
mesh_terms: D000368:Aged; D015415:Biomarkers / Q000378:metabolism*; D015153:Blotting, Western; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000166:cytology* / Q000378:metabolism; D004728:Endothelium, Corneal / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D039422:Integrin alpha3 / Q000378:metabolism; D008875:Middle Aged; D058628:Myofibroblasts / Q000166:cytology* / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014019:Tissue Donors; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D015415:Biomarkers; D018398:Homeodomain Proteins; C501595:IRX2 protein, human; C000599264:ITGA3 protein, human; D039422:Integrin alpha3; D014157:Transcription Factors
keywords: Corneal endothelial cell; differential marker; keratocyte; myofibroblast; regenerative medicine