Isolation And Characterization Of Adult Stem Cells Harboring In Human Minor Salivary Glands And Study On Generation Of Bioengineered Salivary Gland Organoids

BackgroundThe concept of regenerative medicine brings about novel strategy to the treatment of xerostomia, in which the acquisition of seed cells regeneration of salivary gland function play the key role. Minor salivary glands share common structure and functions with major ones, apart from its abundant source and easy accessibility in clinical setting, which makes them ideal source for adult stem cells. So far, not much work have been done on stem cells located in human minor salivary glands. Recombination of epithelial and mesenchymal components is a canonical reconstructive model in vitro and is widely used in organ regeneration and developmental research. Reconstruction of salivary gland orgaoids by seed cells isolated from human minor salivary glands will offer theoretical fundaments and technology support for clinical application and organ regeneration research.Objectives1. Isolate and characterize adult stem cells with self-renewal and multi-potent differential capacity from human minor salivary glands to explore ideal seed cells for bioengineered salivary glands.2. Generate functional bioengineered salivary gland organoids in vitro and prove its possibility of clinical application in vivo.Methods, Results & Conclusions1. Isolation and Characterizaiton of Human Minor Salivary Gland Mesenchymal Sstem Cells(hMSGMSCs)Using the explant culture method, we isolated a population of cells with self-renewal and differentiation capacities harboring that reside in the human minor salivary glands, called human minor salivary gland mesenchymal stem cells (hMSGMSCs). These cells show embryonic stem cell and mesenchymal stem cell phenotypes. Our results demonstrate that hMSGMSCs have the potential to undergo mesodermal, ectodermal and endodermal differentiation in conditioned culture systems in vitro. Furthermore, in vivo transplantation of hMSGMSCs into SCID mice after partial hepatectomy shows that hMSGMSCs are able to survive and engraft, characterized by the survival of labeled cells and the expression of the hepatocyte markers AFP and KRT18. These data demonstrate the existence of hMSGMSCs and suggest their potential in cell therapy and regenerative medicine.2. Gene Expression Profile Comparison among hMSGMSCs, hBMSCs and hASCsTo defining the molecular characteristics of hMSGMSCs, we compared the gene expression profiles among hMSGMSCs, hBMSCs and hASCs by microarray test. Differentially expressed genes (DEGs) were based on the comparison between the expression levels in ADSCs and BMSCs with those in hMSGMSCs. In total, 387 up-regulated and 533 down-regulated genes were detected when comparing hBMSCs with hMSGMSCs. Furthermore,234 up-regulated and 192 down-regulated genes were detected in ASCs compared with hMSGMSCs. According to the clustering analysis results, hMSGMSCs showed a closer relationship to hASCs than hBMSCs. Functional annotations of the 54 common down-regulated genes revealed the enrichment of genes primarily related to the immune response, receptor binding and extracellular regions. Based on the above analyses, we can observe reduced levels of extracellular components and signal transduction in hMSGMSCs.3. Isolation and 3D Culture of Human Minor Salivary Gland Stem/Progenitor CellsHuman minor salivary gland stem/progenitor cells(hMSGSCs) were isolated from human minor salivary glands. hMSGSCs showed typical epithelial lineage characterizations with positive expression of CD49f and cell keratin proteins. hMSGSCs served as adult stem cells in salivary glands and showed capacity differentiating into both acinar cells and duct cells. When added Noggin, CHIR99032 and Wnt3A to the 3D culture system of hMSGSCs, cells showed higher expression of LGR5 and less expression of AMY1B and MUC5B. Thus, by activating Wnt and inhibiting BMP signaling pathway, hMSGSCs were kept at stem cell sate and less differentiated.4. Generation of functional salivary gland organoids from adult human minor salivary gland stem cellsWe labeled two type of stem cells with GFP and RFP respectively and seeded them into Matrigel for 3D culture in vitro. We observed organoids with duct-branching morphology formed in 7 days and hMSGSCs labeled with GFP generated majority part of the organoid including both duct and acinar-like parts. Interestingly, such structure did not form if only hMSGSCs were cultured in Matrigel. Moreover, organoids generated by two cell types showed expression of MUC5B and AMY1B, indicating mature salivary gland function. We then transplanted organoids generated in vitro into mice animal models with submandibular glands surgically removed. We were able to detect expression of KRT19, AQP5 and KRT14 after 2 weeks in vivo. These data suggest that together with tissue-specific mesenchymal stem cells, salivary gland stem cells would generate functional organoids in 3D culture system.