Neuronal and Progenitor/Stem Cell Function During Salivary Gland Development

National Institute Of Dental & Craniofacial Research

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Abstract

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. Recently, we showed that recombinant neurturin, a neurotrophic factor, improves epithelial regeneration of mouse salivary glands in ex vivo culture after irradiation by reducing apoptosis of parasympathetic neurons. We generated a neurturin-expressing adenovirus for gene therapy in vivo to protect parasympathetic neurons. First, we used ex vivo fetal salivary gland culture to compare the neurturin adenovirus to recombinant neurturin, showing they both improve growth after irradiation by reducing neuronal apoptosis and increasing innervation. Then, the neurturin adenovirus was delivered to mouse salivary glands in vivo, 24 h before irradiation and compared to a control adenovirus. The control-treated glands have 50% reduction in salivary flow 60 days post-irradiation whereas, neurturin-treated glands have similar flow to nonirradiated glands. Further, markers of parasympathetic function, including vesicular acetylcholine transporter, decreased with irradiation but not with neurturin treatment. Our findings suggest that in vivo neurturin gene therapy prior to irradiation protects parasympathetic function and prevents irradiation-induced hypofunction. Cell transplantation of autologous adult biopsies, grown ex vivo as epithelial organoids or expanded as spheroids, are also proposed treatments to regenerate damaged salivary glands. However, it is not clear whether transplantation of adult organoids or spheroids alone is sufficient to initiate a fetal-like program of branching morphogenesis in which coordinated branching of multiple cell types including nerves, mesenchyme and blood vessels occurs. Yet this is an essential concept for the regeneration of branching organs. We maintained and expanded adult murine and human epithelial salivary gland progenitors in non-adherent spheroid cultures, called salispheres. The culture conditions stimulated critical developmental pathways, and increased expression of epithelial progenitor markers such as Keratin5, Keratin14, FGFR2b and KIT. Moreover, physical recombination of adult salispheres in a laminin-111 extracellular matrix with fetal salivary mesenchyme, containing endothelial and neuronal cells, only induced branching morphogenesis when neurturin was added. Neurturin was essential to improve neuronal survival, axon outgrowth, innervation of the salispheres, and resulted in the formation of branching structures with a proximal-distal axis that mimicked fetal branching morphogenesis, thus recapitulating organogenesis. Epithelial progenitors were also maintained, and developmental differentiation programs were initiated, showing that the fetal microenvironment provides a template for adult epithelial progenitors to initiate branching and differentiation. Further delineation of secreted and physical cues from the fetal niche will be useful to develop novel regenerative therapies that instruct adult salispheres to resume a developmental-like program in vitro and to regenerate branching organs in vivo. We are also characterizing cells that express keratin 5 (K5) and Keratin 14 (K14) in salivary glands to identify markers that could be used to isolate specific subpopulations and to understand the relationships among these cells. We use both mice that express transgenic reporters and combine this with single cell transcriptomic analysis of murine salivary development. Our ongoing analysis will characterize the multiple cell populations and identify markers to isolate some of them by FACs. These cells may be useful to expand in vitro for use in regenerative therapy while also providing neurotrophic support to allow their parasympathetic innervation and improve functional salivary gland regeneration.