ackground Cutaneous wound healing is a significant health issue in the US, often requiring skin grafts. StrataGraft (Stratatech Corporation, Madison, WI), a second-generation living human skin substitute created from NIKS human keratinocyte progenitors, was recently found to be a promising skin graft in phase I/II safety and efficacy clinical trial. NIKS proliferation is optimal in the presence of epidermal growth factor (EGF). Our preliminary data suggested that Notch signaling also plays a role in NIKS keratinocyte proliferation. Therefore, we hypothesized that EGF might stimulate NIKS proliferation by regulating Notch1 signaling. Method Notch1 messenger RNA (mRNA) levels from NIKS cells in monolayer culture were assessed by real-time polymerase chain reaction and Notch1 protein levels were detected by Western blot. To determine the role of EGF on Notch1 regulation, cells were incubated in basal media and then treated with EGF (10 ng/mL). A 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay was performed to test NIKS cell proliferation. Cells were grown in basal media supplemented with EGF for 72 h in the presence or absence of N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S- phenylglycine t-butyl ester (DAPT) (0-30 μM), an inhibitor of Notch1 signaling. Results Notch1 mRNA levels were cell confluence-dependent, being more abundant in a subconfluent cell monolayer. We detected a 2-fold decrease in Notch1 mRNA expression and a reduction in active Notch1 protein level in response to EGF. EGF treatment stimulated NIKS cellular proliferation. However, co-treatment with DAPT inhibited NIKS proliferation to basal levels. Blocking Notch1 activation by DAPT alone inhibited NIKS cellular proliferation (P < 0.01%). Conclusion Our results suggest that Notch1 is an essential downstream mediator of NIKS cellular proliferation via the EGF signaling pathway. © 2013 Elsevier Inc. All rights reserved.