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.
CFSE: A New Method for Identifying Human Limbal Stem Cells and Following Their Migration in Human Cornea
CHIARA BONZANO;BARBARA CANCIANI;SARA OLIVARI;MARINA PAPADIA;ALESSANDRO BAGNIS;CARLO ALBERTO CUTOLO;ELISABETTA BONZANO;RANIERI CANCEDDA;CARLO ENRICO TRAVERSO
2019-01-01
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.
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