A method to isolate human limbal basal cells enriched for a subset of epithelial cells with a large nucleus/cytoplasm ratio expressing high levels of p63

The objectives were to develop method of isolating viable human limbal basal cells in order to enrich a subset of small cells with a large Nucleus/Cytoplasm (N/C) ratio expressing high levels of p63, nuclear protein. Limbal tissues were treated with trypsin for 50 min at 37 degrees C in an orbital shaker at 100 rpm with epithelial side down followed by additional 5 min with epithelial side up and then with Dispase II to obtain various epithelial fractions. Isolated cell fractions were assessed for colony forming efficiency and DeltaNp63alpha, connexin (Cx43) mRNA levels. Cytospin smears were double-immunostained for p63 and any one of the stem cell (SC) related markers and analyzed using a laser scanning confocal microscope and advanced image analysis software (Leica Confocal software, 2.61 build 1537 version) for quantification of fluorescence intensity. The isolated limbal basal cells were highly positive for DeltaNp63alpha mRNA but expressing low Cx43 mRNA. They gave rise to higher number of large colonies with compact morphology in contrast to the limbal suprabasal/superficial (LS/S) colonies. Furthermore, a subset with a large N/C ratio expressing high levels of p63 was observed, as much as 25% among the limbal basal cell fraction, in contrast to only about 4% in the total limbal epithelial cells. Such cells were positive for K5 and negative for Ki67, Cx43, and 14-3-3s and were absent in the LS/S fraction. These results collectively substantiate our method of isolation of limbal basal layer cells containing an enriched population of cells with SC phenotype.