This project has two parts, one investigating the communication between parasympathetic nerve development and SMG epithelial morphogenesis, and the second identifying mouse salivary gland stem/progenitor cell populations within the gland. The maintenance of progenitors as a reservoir of undifferentiated cells is required for organ development and regeneration. Parasympathetic nerves are a vital component of the progenitor niche during development. Injured adult organs do not regenerate after parasympathectomy, and there are few treatments to improve organ regeneration or prevent damage, particularly that caused by therapeutic irradiation. Restoring parasympathetic function with the neurotrophic factor neurturin increases epithelial organ regeneration after irradiation. We used SMG explant culture and injured the tissue with irradiation. The progenitors survived, parasympathetic function was diminished, and epithelial apoptosis reduced expression of neurturin, which consequently increased neuronal apoptosis. Treatment with neurturin reduced neuronal apoptosis, restored parasympathetic function, and increased epithelial regeneration. Furthermore adult human salivary glands damaged by irradiation also had reduced parasympathetic innervation but an increase in sympathetic innervation. We propose that neurturin will protect the parasympathetic nerves from damage and improve regeneration. We are using viral vectors that express neurotrophic factors to infect salivary glands in vivo and ex vivo to improve the function of surviving nerves in repairing the gland. Previously we discovered that parasympathetic innervation maintains K5+ epithelial progenitor cells during SMG organogenesis. We confirmed that K5+ cells are progenitor cells in the SMG by genetic lineage tracing. It is also known that KIT, a tyrosine kinase receptor, marks another progenitor population in the SMG and that KIT+ cell transplantation regenerates murine SMGs after IR. We previously analyzed the expression of keratins in the KIT+ cells by FACS and identified multiple subpopulations expressing K5, K14, and K19 or combinations of them. Keratins form heterodimers and K5 usually pairs with K14 in epithelia, e.g. in skin and hair follicles. However, in the SMG KIT+ endbuds there are separate subpopulations of K5+ or K14+. We characterized the progenitor potential of K14+ cells by genetic lineage tracing, using a K14Cre mouse crossed with a RosamTmG reporter strain. The K14+ progenitors are multipotent, since their progeny at postnatal day 1 included K5+ ductal cells, amylase-producing acinar cells, and smooth-muscle-actin+ myoepithelial cells. We showed that during development the SMG endbud contains two distinct populations of progenitors, a pool of distal KIT+K14+ progenitors and a pool of proximal KIT+K5+ progenitors. These findings are consistent with the concept that the oral epithelium contains a primitive K5+K14+ progenitor. We propose that once KIT expression occurs, separate lineages of KIT+K5+ and KIT+K14+ progenitors appear in the gland.
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