Temporal Regulation of Notch Signaling and Its Influence on the Differentiation of Ex Vivo Cultured Limbal Epithelial Cells

Purpose: Notch signaling plays a vital role in the differentiation and proliferation of corneal epithelial cells from limbal stem cells. The temporal regulation of Notch signaling during this differentiation remains unknown. Hence, we investigated the importance of temporal activation/blockage of Notch signaling during corneal differentiation.

Methods: Human limbal epithelial cultures were established with and without Notch activators (rec-Human Jagged1 Fc chimera) and pharmacological blockers (LY-411575). The modulation of Notch signaling was done at different time points during cell differentiation, which were collected on Day 14 for further analysis of differentiation, proliferation, maturation and apoptosis using RT-qPCR and immunofluorescence staining.

Results: The activation of Notch signaling at Day 8 resulted in the highest number of mature corneal epithelial cells (p = .008) and pro-apoptosis marker BAX (p = .0001) with no increase in the number of corneal progenitors, and proliferation marker Ki67 compared to untreated controls. Cultures grown in the presence of Notch signaling blockers showed a significantly higher number of corneal progenitors (p = .0001) and proliferation marker Ki67 (p = .02) but lower corneal epithelial marker CK3/CK12 (p = .0007) and no difference in the pro-apoptotic marker BAX compared to untreated controls.

Conclusion: During the differentiation of limbal epithelial cells to the corneal epithelial cell type, Day 8 seems to be a crucial window to modulate Notch signaling for a customized outcome.