[Improvement in the method of cultivated corneal epithelial sheet generation]

Stromal cells and/or soluble factors such as growth factors have been considered until now as the main niche of corneal epithelial stem cells. We found that cultivated epithelial cells had epithelial clusters when cultured, maintaining the sheet structure. Therefore, we hypothesized that the epithelial cells themselves might be a niche of the stem cells, and the cell clusters could be maintained during the gentle enzymatic digestion of the limbal epithelial sheets. The main methods of generating cultivated limbal epithelial sheet for clinical application are, the cell suspension method (complete enzymatic digestion of limbal epithelia), and the explant method (cells grown from limbal explants). For cell suspension, it is good to expand cells and generating sheets quickly, but it is not clear wheather these sheets maintain the normal homeostatic levels of the progenitor cells. Explants maintain homeostatic levels more similar to in vivo homeostasis, but the low proliferative speed of epithelial expansion and reproducibility are the problem. In this review, we hypothesize that to generate cultivated epithelial sheets while maintaining a corneal epithelial stem cells' niche is a better way to proceed as it includes the advantages of both the cell suspension and explant methods. The growth potential, colony-forming efficiency of p63-positive cell clusters, and immunostaining by differentiation/ undifferentiation markers, demonstrated that progenitor cells could be maintained by these gentle enzymatic digestion maintaining cell clusters.