Chiara Bonzano1, Barbara Canciani2, Sara Olivari3, Marina Papadia4, Alessandro Bagnis3, Carlo Alberto Cutolo3, Elisabetta Bonzano5, Paola Pagani3, Ranieri Cancedda2, Carlo Enrico Traverso3. 1. Eye Clinic, DiNOGMI, University of Genoa and IRCCS San Martino Polyclinic Hospital, Genoa, Italy oculistabonzano@gmail.com. 2. Laboratory of Regenerative Medicine, Department of Oncology, Biology and Genetics, IRCCS San Martino Polyclinic Hospital, Genoa, Italy. 3. Eye Clinic, DiNOGMI, University of Genoa and IRCCS San Martino Polyclinic Hospital, Genoa, Italy. 4. Italian Auxologic Institute, Milan, Italy. 5. School of Experimental Medicine, University of Pavia and Department of Radiation Oncology, IRCCS San Matteo Polyclinic Foundation, Pavia, Italy.
Abstract
AIM: To develop a method capable of identifying human corneal limbal stem cells (LSCs) and follow their proliferation and migration in the epithelium. MATERIALS AND METHODS: Ten fresh matched pairs of cadaveric normal human corneas were obtained from donors. Carboxyfluorescein diacetate succinimidyl ester (CFSE) was used to target LSCs. The distribution of CFSE-positive cell clusters was analyzed by fluorescence microscopy by counterstaining with 4',6-diamidino-2-phenylindole (DAPI). Fluorescence was digitally recorded for seven days, and the rate of cell movement was determined. RESULTS: CFSE-labeled cells were tracked in corneas. Analysis of time sequences revealed that they moved centripetally. Daily average CFSE-labeled LSC movement was 0.073±0.01 cm (±SD). CONCLUSION: CFSE allowed us to identify LSCs and to track their centripetal migration from the limbal basal layer to the anterior ocular surface. This experimental system appears to be a valuable tool for further studies on corneal epithelial cell migration and proliferation. Copyright
AIM: To develop a method capable of identifying human corneal limbal stem cells (LSCs) and follow their proliferation and migration in the epithelium. MATERIALS AND METHODS: Ten fresh matched pairs of cadaveric normal human corneas were obtained from donors. Carboxyfluorescein diacetate succinimidyl ester (CFSE) was used to target LSCs. The distribution of CFSE-positive cell clusters was analyzed by fluorescence microscopy by counterstaining with 4',6-diamidino-2-phenylindole (DAPI). Fluorescence was digitally recorded for seven days, and the rate of cell movement was determined. RESULTS:CFSE-labeled cells were tracked in corneas. Analysis of time sequences revealed that they moved centripetally. Daily average CFSE-labeled LSC movement was 0.073±0.01 cm (±SD). CONCLUSION:CFSE allowed us to identify LSCs and to track their centripetal migration from the limbal basal layer to the anterior ocular surface. This experimental system appears to be a valuable tool for further studies on corneal epithelial cell migration and proliferation. Copyright