Abstract
Purpose :
Notch signaling plays a pivotal role in regulating stem cell maintenance, differentiation and homeostasis. We investigated the role of Jagged1-mediated Notch signaling activation in the regulation of human limbal stem/progenitor cells (LSCs) in vitro.
Methods :
The expression of several molecules involved in Notch signaling pathway was analyzed on human sclerocorneal tissue at the mRNA and protein level (n=4). Primary human LSCs were cultured on Jagged1-coated (Jag1-Fc) plates (n=4). Notch activation was confirmed by the presence of the nuclear Notch1 intracellular domain (N1IC) in rat gut Notch1 transfected cells (LN1-7 control cells; n=3) and LSCs (n=3) by immunocytochemistry. LSC phenotype was analyzed by assessing cell morphology, cell size, and expression level of the putative LSC markers K14 and p63α, and the differentiation marker K12. Differences in the stratification and differentiation in the presence of Jag1 were analyzed in air-lifting experiments (n=3).
Results :
The expression of Notch receptors (Notch1, Notch2), ligands (Jagged1, Jagged2, Delta-like1) and target genes (Hes1, Hey1) was detected in the human corneal epithelium at the mRNA and protein level. When Notch signaling was activated by Jag1 in the cultivated LSCs, cell morphology was less compact, cell growth was reduced by 1.6-fold (P=0.04), and the percentage of small cells (≤12 µm) was 1.4-fold decreased (P=0.03) compared to the control. No significant differences were found in the expression of K14; however, a lower percentage of p63α high-expressing cells (p63αbright) was found in the cells cultured with Jag1 (12.8% ± 2.3% versus 17 ± 2.1% in the control; P=0.002). Upon air-lifting, Jag1 activated cultures showed reduced stratification (P<0.001), lower density of basal cells (P=0.01), and the amount of K12+ cells was increased (P<0.001) with regards to the control.
Conclusions :
Jagged1-mediated Notch signaling activation appears to induce differentiation of LSCs in vitro.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.