Notch Inhibition Prevents Differentiation of Human Limbal Stem/Progenitor Cells in vitro

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.

Role of Jagged1-mediated Notch Signaling Activation in the Differentiation and Stratification of the Human Limbal Epithelium

“…Understanding the regulation of differentiation and stratification in the limbal epithelium is fundamental to develop new treatment strategies. The findings in the current study are consistent with those previously obtained by our group [ 31 ] and others [ 9 , 10 , 11 , 12 , 13 ] in that Notch signaling pathway is present in the limbal epithelium where LSCs are located.…”

“…Moreover, since DAPT is a broad-spectrum inhibitor, it might be blocking some other signaling pathways, and, hence, being less efficient at blocking Notch signaling. We have previously shown that more specific Notch small molecule inhibitors such as SAMH1 can be more efficient at inhibiting Notch signaling in the LECs at some concentrations [ 31 ]. Moreover, Jag1 in combination with either DAPT or SAHM1 decreased Notch signaling activation in the LN1-7 cells, given by the percentage and localization of the N1IC [ 31 ].…”

Integrated Transcriptome and Proteome Analyses Reveal the Regulatory Role of miR-146a in Human Limbal Epithelium via Notch Signaling

“…As the committed early TA cells migrate laterally and upward, where miR-146a expression declines, Notch-1 expression decreases, which is in agreement with the study, demonstrating that loss of Notch-1 disrupts the barrier repair with subsequent increase of migratory behavior in the corneal epithelium [ 71 ]. Conversely, increased Notch-2 expression coincides with cellular differentiation, which is also in agreement with previous studies [ 14 , 16 , 60 , 61 ]. Therefore, miR-146a may orchestrate differential expressions of Notch-1 and Notch-2 in maintaining the population of stem cells and/or their early differentiation.…”

“…There have been some conflicting reports regarding the regulatory role of Notch signaling in the corneal epithelium. Inhibition of Notch signaling by small molecule inhibitors decreased levels of both differentiation and proliferation markers in rat LECs [ 60 ] and in human limbal stem cells [ 61 ]. In contrast, Ma et al demonstrated that inhibition of Notch signaling resulted in decreased proliferation but increased differentiation in vitro, with no effect in a three-dimensional, stratified corneal epithelium equivalent, whereas Notch activation resulted in decreased differentiation [ 14 ].…”

Top Notch Targeting Strategies in Cancer: A Detailed Overview of Recent Insights and Current Perspectives

“…A synthetic, cell-permeable, α-helical peptide (SAHM1) has been developed that blocks MAML1 recruitment with high affinity for the interface on the Notch-CSL/RBP-J transactivation complex effectively reducing T-ALL cell line proliferation and Notch-driven progression of leukaemia in a mouse model of T-ALL [ 307 ]. While this approach has the potential to be a more specific Notch targeting approach, with efficacy in pathologies such as asthma and eye disorders [ 308 , 309 ], there is currently a lack of pharmacokinetic and pharmacodynamic data to support a role in the cancer setting. Aside from this peptide-based therapeutic approach, a first-in-class small-molecule inhibitor of Mastermind recruitment-1 (IMR-1) has been identified from in-silico screening of over 1.5 million drug-like compounds.…”