Fig. 1

LESCs express HA in vitro. A hLESCs were cultured in vitro and the expression and localization of HA (red) and Phalloidin (white) investigated. Scale bar represents 50 μm (B) hLESCs were cultured in vitro and the expression and localization of HA (green) and K15 (red) investigated showing there is a strong correlation between the expression of putative limbal stem cell marker K15 and HA. Specifically, 97% of cells expressing K15 also express HA. C and D The expression of HA was also analyzed by flow cytometry after cells were maintained on PLL (C) or PLL/HA (D) coated dishes. There is a linear relationship between the expression of HA and K15, therefore cells that express high levels of K15 also express HA and cells that do not express K15 also do not express HA. E, H The expression of an HA matrix by hLESCs was analyzed using the red blood exclusion assay and the exclusion space measured. This assay evidences that a subset of both circular and non-circular cells express a thick glycocalyx and that the removal of HA from the ECM precludes the formation of this glycocalyx, indicating HA is necessary for the formation of a hLESC specific ECM (glycocalyx). *Represents p ≤ 0.05, scale bar represents 1000 μm