Neurointegration of Assembling Human Salivary Stem/Progenitor Cells Assists Neotissue Formation

Objectives: Patients undergoing radiation therapy for head and neck cancer often lose salivary gland function and develop hyposalivation disorders. Cell-based bioengineering strategies to develop a permanent tissue replacement seek to restore native tissue functions. The nervous system influences salivary gland morphogenesis and innervation helps produce a functional gland. We examined the impact of neural progenitor populations on human salivary stem progenitor cell (hS/PC) growth and differentiation in migration-permissive hyaluronate-based hydrogels. Next, we examined ingrowth of host nerve fibers into cell-laden hydrogels implanted in a mini-swine model. Third, we examined hS/PC production of the pro-neural growth factor neurturin.
Methods: We used retinoic acid to drive murine neuroprogenitor cells (D3s) towards the neuronal lineage and co-cultured these with hS/PCs in hydrogels to study their ability to influence maturation of functional salivary microstructures. Immunofluorescent visualization of lineage-based biomarkers assessed differentiation status of hS/PC populations towards an acinar, saliva-producing phenotype and D3 populations towards a neuronal phenotype. Live/dead analysis measured cell viability and phase-contrast images assessed microstructure sizes. Time-dependent ingrowth of host nerve fibers into implanted cell-laden hydrogels in vivo was examined similarly. Neurturin production by hS/PCs was measured using a commercial ELISA kit.
Results: Co-culture of D3s and hS/PCs produced larger microstructures than hS/PCs alone, with axonal extensions enveloping assembled salivary microstructures. Co-culture revealed acinar polarization, indicated by luminal orientation of α-amylase without adding exogenous neurotransmitters. Both cell populations were viable in co-culture for several weeks. Freshly resected hydrogels containing hS/PCs implanted in mini-swine for 4 weeks showed evidence of long nerve fibers penetrating toward salivary microstructures.
Conclusions: 1) Co-culture of hS/PCs and nerve fosters salivary neotissue maturation and function; 2) the hydrogel permits ingrowth of host nerves when implanted as an engineered salivary replacement; 3) neurturin production by hS/PCs, if retained after implantation, augments neurointegration of functional salivary neotissues.