Abstract
Notch signaling has been shown to regulate the homeostasis and wound healing of the corneal epithelium. We investigated the effect of Notch inhibition in the human limbal stem/progenitor cells (LSCs) in vitro by using small molecules. Treatment of the LSCs with DAPT and SAHM1 reduced the proliferation rate and maintained the undifferentiated state of the LSCs in a concentration dependent manner. Stratification and differentiation of the corneal epithelium were not reduced after Notch inhibition, indicating that the function of the corneal basal cells is retained. Our findings suggest that Notch signaling plays a role in the proliferation and maintenance of LSCs.
Highlights
Approaches to maintain the undifferentiated state of limbal stem/progenitor cells (LSCs) ex vivo are focused on emulating the in vivo microenvironment
At the mRNA level, Notch[1] (N1), Notch[2] (N2) and HES1 were expressed in the epithelium at a similar level in both the central cornea and limbus; instead, HEY1 showed a higher expression in the limbus (Fig. 1A)
The expression of the Notch[1] intracellular domain (N1IC), HES1 and HEY1 was detected in primary human limbal epithelial cells by Western blot (Fig. 1B and Fig. S3)
Summary
Approaches to maintain the undifferentiated state of LSCs ex vivo are focused on emulating the in vivo microenvironment. DAPT and SAHM1 inhibit Notch signaling in the limbal epithelial cells. The inhibition of Notch signaling in the freshly isolated primary limbal epithelial cells was assessed after 24 hours of the incubation with DAPT or SAHM1 at different concentrations by analyzing (1) the expression of Notch target genes HES1, HES5 and HEY1 at the mRNA level and (2) expression and cellular localization of the N1IC at the protein level.
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