In search for correlation among markers for limbal stem cells niche

Abstract

Abstract Purpose The corneoscleral limbus is known to be the site of corneal epithelial stem cells (SC). Several molecules have been proposed as SC markers but none of them is able to univocally identify them. The aim of this study was to evaluate co‐expression of different SC markers in human limbus. Methods In this work five corneoscleral specimens from normal human donor eye‐bank eyes (age 52‐73 years) were fixed in formalin, divided in 8 segments, embedded in paraffin and examined by immunohistochemistry and immunofluorescence for p63, vimentin (vim), laminin 5, integrin (Int) α6, Int β1, Int β4, connexin 43, ki67 and N‐cadherin positivity. We firstly analyzed the distribution and the anatomical structure of limbal crypts in each of the segments. Then we evaluated the percentage of positive areas in the niches. Finally we looked for colocalizations and possible correlations among markers. Results We confirmed a different number of niches among the segments of the same corneoscleral rim. Moreover we observed high variability of niches number among patients which interestingly correlates with the percentage of p63 positivity of niche cells. Confocal microscopy double staining for p63 and vim did not show evident colocalization and vim + cells were seen in the superficial layers rather than in the deep layer of crypts. Int β1 staining directly correlated with p63 positivity while the remaining proteins appeared variably and widely distributed Conclusion Colocalization was evident at least for two SC markers (Int β1/p63) within the basal layers, while vim, expressed mainly in the superficial layers could act as late progenitor cell marker.