Identification of human corneal epithelial stem cells on the basis of high ABCG2 expression combined with a large N/C ratio.

ABSTRACT

PURPOSE: Till date there is no exclusive marker for human corneal epithelial stem cells (CESCs). In this study, our strategy is to combine high expression of ABCG2, a putative SC marker with high N/C ratio to develop a specific method for identification of CESCs. METHODS: Limbal/corneal epithelial cells (LECs/CECs) were isolated from cadaver eyes by enzymatic treatment and the cytospin smears/cryosections were immunostained for ABCG2, p63, or connexin-43 (Cx-43) and counterstained with propidium iodide/DAPI. Analysis was carried out for (1) ABCG2 expression, N/C ratio, (2) p63, ABCG2, N/C ratio, and (3) ABCG2, Cx-43, N/C ratio using confocal/fluorescence microscopy. RESULTS: ABCG2 was highly expressed by cells in the basal and suprabasal layers of limbus. The SCs identified on the basis of our earlier method using high levels of p63 expression along with high (>0.7) N/C ratio were strongly positive for ABCG2 and negative for differentiation marker Cx-43. High expression of ABCG2 was also observed in cells with high p63 expression and low (<0.7) N/C ratio; however, they were positive for Cx-43. Combining high ABCG2 expression with large N/C ratio in fluorescence microscopy identified 5.3 ± 3.5% LECs as putative SCs and this was confirmed by confocal microscopic analysis. CONCLUSIONS: We demonstrate the importance of combining ABCG2 expression with N/C ratio for identification and quantification of human CESCs. This method will have application in evaluating the role of niche cells/factors in the maintenance of stemness.