ABSTRACT
Corneal limbal epithelial stem cell transplantation using cultivated human corneal epithelial cell sheets has been used successfully to treat limbal stem cell deficiencies. Here we report an investigation into the quality of cultivated human corneal epithelial cell sheets using time-lapse imaging of the cell culture process every 20 minutes over 14 days to ascertain the level of cell jamming, a phenomenon in which cells become smaller, more rounded and less actively expansive. In parallel, we also assessed the expression of p63, an important corneal epithelial stem cell marker. The occurrence of cell jamming was variable and transient, but was invariably associated with a thickening and stratification of the cell sheet. p63 was present in all expanding cell sheets in the first 9 days of culture, but it's presence did not always correlate with stratification of the cell sheet. Nor did p63 expression necessarily persist in stratified cell sheets. An assessment of cell jamming, therefore, can shed significant light on the quality and regenerative potential of cultivated human corneal epithelial cell sheets.
Subject(s)
Corneal Diseases/therapy , Epithelium, Corneal/cytology , Membrane Proteins/metabolism , Stem Cell Transplantation , Stem Cells/cytology , 3T3 Cells , Animals , Biomedical Engineering/methods , Cell Differentiation/physiology , Cell Line , Cell Proliferation/physiology , Epithelial Cells/cytology , Female , Humans , Limbus Corneae/cytology , Male , Mice , Middle AgedABSTRACT
Activation of bovine pancreatic trypsinogen (BPTG) by trypsin (BPT) was found to be inhibited by d GalN/GalNAc at pH 5.5, the pH of secretory granules in the pancreas. Binding studies with biotinylated sugar-polymers indicated that BPTG and BPT bind to α-GalNAc, α-Man, and α-Gal better at pH 5.5 than at pH 7.5. Ultraviolet-difference spectra indicated that BPTG binding to α-GalNAc differs substantially from BPTG binding to other sugars. The N-α-benzoyl-d,l-arginine-p-nitroanilide hydrochloride-hydrolyzing activity of BPT was only slightly affected by these sugars. The results indicate that the binding of GalNAc - containing glycoconjugates protects BPTG from autoactivation, and this may be a self-defense mechanism against intrapancreatic activation.
