Gap junction protein connexin 43 serves as a novel negative marker for stem cell–containing population of human corneal epithelial cells

Abstract

Abstract: : Purpose: To evaluate the expression of the gap junction protein connexin (Cx) 43 by human limbal epithelia and test a hypothesis that the Cx 43 could serve as a novel negative cell surface marker to select human corneal epithelial stem cells. Methods:Immunostaining, laser scanning confocal microscopy and semiquantitative RT––PCR were performed to localize Cx 43 expression in human corneal and limbal tissues. Magnetic cell selection was performed on human corneal epithelial cells from fresh limbal tissues and cultures using bispecific tetrameric antibody complexes containing a murine IgG1 monoclonal antibody against Cx 43, magnetic nanoparticles and an EasySep^TM magnet. Cx 43 positive and negative populations were evaluated for their proliferative potential on a mitomycin C–treated 3T3 fibroblast feeder layer and their mRNA and protein expression of stem cell associated and differentiation markers. Results:Immunostaining and confocal microscopy showed that Cx 43 was strongly expressed in the corneal and limbal suprabasal epithelial cells but was negative in the basal layer of the limbus. Cx 43 mRNA was barely detectable in the limbal epithelia and was more abundant in the corneal epithelia. The selected Cx 43 negative population (Cx–), confirmed by barely detectable mRNA and negative immunostaining of Cx 43, showed greater colony forming efficiency (CFE, 104 ± 12.8/well, P<0.01) on a 3T3 feeder layer on day 6 than the Cx 43 positive population (Cx+, CFE, 28 ± 8/well). The expression of stem cell associated markers, such as ρNp63, ABCG2, integrin α9 and α–enolase mRNA, was higher in Cx– cells than Cx+ cells by RT–PCR. Immunostaining and confocal microscopy showed that the Cx– population had higher percentages of positive cells for p63 (67%) and α–enolase (86%) than Cx+ (8% and 10%, respectively), and lower percentages of positive cells for differentiation markers such as involucrin (12%) and K3 (14%) than Cx+ (60% and 37% respectively). Conclusions:Our findings suggest for the first time that the gap junction protein connexin 43 could serve as a novel negative cell surface marker for enrichment for human corneal epithelial stem cells.

Keywords: cornea: epithelium • cornea: basic science • gap junctions/coupling