Extracellular Vesicles Derived from Salivary Gland Stem Cells Cultured on Microwell Scaffolds Loaded with Wnt3a Promote the Recovery of Salivary Gland Function Damaged by Radiation Via the Ywhaz-Pi3k-Akt Pathway

Abstract

We developed a double-layered microwell scaffold with a biochemical niche to enhance stemness and promote extracellular vesicle (EV) production from salivary gland (SG) tissue stem cells. We created a WNT3A protein-loaded poly(d,l-lactide-co-glycolide) (PLGA) electrospun nanofiber combined with a polycaprolactone (PCL) microwell array for sustained WNT release and three-dimensional (3D) cell culture. Human SG-derived epithelial stem cells (sgEpSCs) were cultured on 2D plastic dishes, 3D PCL microwells, 3D bare PLGA+PCL microwells, or WNT-loaded PLGA+PCL microwells. We examined the regenerative effects of sgEpSC-derived EVs on SG dysfunction using irradiated murine SG in mice and a human SG organoid model. PCL microwells coupled with a WNT3A-loaded PLGA electrospun nanofiber scaffold facilitated transformation of sgEpSCs into 3D spheroids, sustainably released WNT3A, and increased EV production. sgEpSC-derived EVs from 3D spheroids cultured in WNT3A-releasing microwells (3DWNT-EVs) injected into the SG ducts of irradiated mice reduced SG epithelial and progenitor cell death and preserved SG function. In in vitro organoid culture, 14-3-3 protein zeta/delta, highly expressed within 3DWNT-EVs, significantly boosted the proliferation of SG progenitor cells and increased the expression of phosphorylated phosphoinositide 3-kinases/protein kinase B. Growing 3D spheroids in WNT3A-releasing microwell scaffolds may help restore SG function after radiation exposure.

Keywords: Salivary gland, 3D spheroid culture, Nanofibrous scaffold, Extracellular vesicle, Salivary organoid