Project Summary/Abstract: Voice dysfunction impairs the quality of life of affected patients and treating these disorders is associated with substantial and far-ranging social, psychological, and economic costs. Vocal fold (VF) inflammatory lesions cause common voice disorders related to disrupted epithelial homeostasis accompanied by inflammatory infiltrates and changes in the lamina propria. In studies of the epithelium from elsewhere in the body, cell types that originate hyperplasic changes are epithelial stem cells. These cells have the capability to self-renew and give rise to the progeny of differentiated daughter cells which is regulated by the local microenvironment and cell-autonomously via Notch signaling. Inactivation of Notch1 in the presence of inflammatory cytokines can lead to epithelial hyperplasia, which can be modeled using in vitro organoids. The overall objective of this proposal is to provide a comprehensive characterization of VF epithelial stem cells, their requirements for self- renewal and differentiation under physiological conditions and in response to stress factors, namely injury and mechanical load, while also creating VF organoids to elucidate molecular mechanisms that underline aberrant epithelial remodeling as seen in benign inflammatory VF lesions. To achieve our goal we will genetically label epithelial stem cells targeting the Lrig1 gene that has been linked to stemness properties in majority of epithelia and our preliminary data show that Lrig1 is also expressed in human and murine VF epithelial cells. In Aim 1, we will perform transcriptome profiling of murine and human VF Lrig1 cells and measure Lrig1 cell responses to mechanical load during homeostasis. We will delineate the mechanistic role of murine Lrig1 cells in homeostasis, and genetically inactivate Notch1 in murine Lrig1 cells to determine its effect on proliferation and differentiation in vivo. In Aim 2, we will induce VF epithelial injury in a murine model, perform transcriptome profiling of murine Lrig1 cells and measure their responses to mechanical load during epithelial recovery. We will determine the functional role of murine Lrig1 cells and Notch1 signaling in re-epithelization. In Aim 3, we will determine differentiation potential of murine and human VF Lrig1 cells using in vivo subrenal graft assay and in vitro organoids. We will utilize VF organoids to model Notch-mediated epithelial hyperplasia using genetic, pharmacologic approaches, and inflammatory cytokines. We will create a reliable culture system that will improve our understanding of VF epithelial cell biology related to VF inflammatory lesions in the context of personalized medicine.