Abstract
Purpose :
Controversy exits regarding the role of Notch signaling on limbal epithelial stem/progenitor cells (LSCs). We investigated the inhibition of Notch signaling pathway on the phenotype of LSCs in vitro.
Methods :
Primary human LSCs were cultured as single cells on a monolayer of 3T3 feeder cells in SHEM medium. Notch signaling was inhibited using the small molecule DAPT to prevent the release of the active Notch intracellular domain (NICD). DAPT titration at 1, 5, 10 and 20 µM was performed. Notch inhibition was confirmed by the absence of the NICD in the nucleus by immunocytochemistry. The phenotype of cultured LSCs was analyzed by assessing cell morphology, colony forming efficiency (CFE), cell expansion rate, cell size and expression level of putative LSC markers both at the mRNA and protein level. Differences in stratification and differentiation in the presence of DAPT were also analyzed after air-lifting induction.
Results :
Morphology and CFE of LSCs cultured in the presence of DAPT were similar to those in the control. The cell expansion rate was reduced at high concentrations of DAPT (10 and 20µM, p<0.05). An increase in the percentage of small cells (≤12 µm) was detected in the presence of DAPT at 20µM (p<0.05). The percentage of p63α bright cells was comparable to that in the control (p>0.05). The percentage of K12+ cells was significantly reduced at high concentrations of DAPT (10 and 20µM, p<0.05). Upon air-lifting induction, LSCs in the presence of DAPT showed no significant differences in the stratification and differentiation capacity compared to the control.
Conclusions :
Inhibition of Notch signaling using DAPT appears to help maintain the LSC phenotype in vitro.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.