Masahiro Yamaguchi1,2, Nobuyuki Shima1,3, Miwa Kimoto1, Nobuyuki Ebihara2,4, Akira Murakami2, Satoru Yamagami1,5. 1. a Corneal Regeneration Research Team, Foundation for Biomedical Research and Innovation , Kobe , Japan . 2. b Department of Ophthalmology , Juntendo University School of Medicine , Tokyo , Japan . 3. c Department of Ophthalmology , University of Tokyo Graduate School of Medicine , Tokyo , Japan . 4. d Department of Ophthalmology , Juntendo University Urayasu Hospital , Chiba , Japan and. 5. e Corneal Transplantation Section, University of Tokyo Graduate School of Medicine , Tokyo , Japan.
Abstract
PURPOSE: 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. MATERIALS AND 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. CONCLUSION: These molecules could be important markers for the production of highly purified cultured CECs for regenerative medicine.
PURPOSE: 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. MATERIALS AND METHODS: CECs and CSMFs were obtained from humandonor 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. CONCLUSION: These molecules could be important markers for the production of highly purified cultured CECs for regenerative medicine.
Entities:
Keywords:
Corneal endothelial cell; differential marker; keratocyte; myofibroblast; regenerative medicine