Multiple stimulation with spheroids comprising salivary gland and adipose-derived stem cells enhances regeneration of radiation-damaged salivary glands

[Display omitted] •Spheroids are more effective than monolayer cultures in salivary gland regeneration.•Salivary gland stem cells (SGSCs) and adipose-derived stem cells (ASCs) were used.•SGSCs combined with ASCs induced greater regeneration than SGSCs alone.•Signaling pathways related to angiogenesis and anti-apoptosis are likely involved in the effect. Tissue engineering approaches using a single stem cell type have been unsuccessful in achieving regeneration of radiation-damaged salivary glands (SGs). Therefore, we combined adult SG stem cells (SGSCs) and adipose-derived stem cells (ASCs) in optimized spheroids to enhance the regeneration of damaged SGs in mice. SGSC/ASC spheroids survived longer and secreted more vascular endothelial growth factor (VEGF) than did SGSC spheroids. To enhance cell viability and differentiation, SGSC/ASC spheroids were stimulated with the Wnt/β-catenin signaling activator CHIR99021 and retinoic acid inhibitor BMS431 to maintain stemness. FGF7/10 induced the differentiation of multiple stimulated SGSC/ASC spheroids. SGSC/ASC spheroids exhibited improved viability, calcium channel function, differentiation, and VEGF secretion after multiple, sequential stimulation. The 21-day survival rate of multiply stimulated SGSC/ASC spheroids was two-fold that of non-treated SGSC/ASC spheroids. Optimized SGSC/ASC spheroids enhanced the in vivo regeneration and functional recovery of SG tissue in mice by promoting neovascularization. Genes related to VEGF, such as Vegfa and Vegfb, were highly expressed 2∼6 times for in vitro and 1.5 times for in vivo experiment in the multiply stimulated SGSC/ASC spheroids. Therefore, SGSC/ASC spheroids that have undergone multiple stimulation may be useful for healing radiation-damaged SGs.