Human Salivary Gland Stem Cells Research Articles

Basement Membrane Mimetic Hydrogel Cooperates with Rho-Associated Protein Kinase Inhibitor to Promote the Development of Acini-Like Salivary Gland Spheroids.

Successful engineering of functional salivary glands necessitates the creation of cell-instructive environments for ex vivo expansion and lineage specification of primary human salivary gland stem cells (hS/PCs). Herein, basement membrane mimetic hydrogels were prepared using hyaluronic acid, cell adhesive peptides, and hyperbranched polyglycerol (HPG), with or without sulfate groups, to produce "hyperGel+" or "hyperGel", respectively. Differential scanning fluorescence experiments confirmed the ability of the sulphated HPG precursor to stabilize fibroblast growth factor 10. The hydrogels were nanoporous, cytocompatibile and cell-permissive, enabling the development of multicellular hS/PC spheroids in 14 days. Incorporation of sulfated HPG species in the hydrogel enhanced cell proliferation. Culture of hS/PCs in hyperGel+ in the presence of a Rho kinase inhibitor, Y-27632 (Y-27), led to the development of spheroids with a central lumen, increased the expression of acinar marker aquaporin-3 at the transcript level (AQP3), and decreased the expression of ductal marker keratin 7 at both the transcript (KRT7) and the protein levels (K7). Reduced expression of transforming growth factor beta (TGF-β) targets SMAD2/3 was also observed in Y27-treated cultures, suggesting attenuation of TGF-β signaling. Thus, hyperGel+ cooperates with the ROCK inhibitor to promote the development of lumened spheroids with enhanced expression of acinar markers.

Matrix Degradability Contributes to the Development of Salivary Gland Progenitor Cells with Secretory Functions.

Synthetic matrices that are cytocompatible, cell adhesive, and cell responsive are needed for the engineering of implantable, secretory salivary gland constructs to treat radiation induced xerostomia or dry mouth. Here, taking advantage of the bioorthogonality of the Michael-type addition reaction, hydrogels with comparable stiffness but varying degrees of degradability (100% degradable, 100DEG; 50% degradable, 50DEG; and nondegradable, 0DEG) by cell-secreted matrix metalloproteases (MMPs) were synthesized using thiolated HA (HA-SH), maleimide (MI)-conjugated integrin-binding peptide (RGD-MI), and MI-functionalized peptide cross-linkers that are protease degradable (GIW-bisMI) or nondegradable (GIQ-bisMI). Organized multicellular structures developed readily in all hydrogels from dispersed primary human salivary gland stem cells (hS/PCs). As the matrix became progressively degradable, cells proliferated more readily, and the multicellular structures became larger, less spherical, and more lobular. Immunocytochemical analysis showed positive staining for stem/progenitor cell markers CD44 and keratin 5 (K5) in all three types of cultures and positive staining for the acinar marker α-amylase under 50DEG and 100DEG conditions. Quantitatively at the mRNA level, the expression levels of key stem/progenitor markers KIT, KRT5, and ETV4/5 were significantly increased in the degradable gels as compared to the nondegradable counterparts. Western blot analyses revealed that imparting matrix degradation led to >3.8-fold increase in KIT expression by day 15. The MMP-degradable hydrogels also promoted the development of a secretary phenotype, as evidenced by the upregulation of acinar markers α-amylase (AMY), aquaporin-5 (AQP5), and sodium-potassium chloride cotransporter 1 (SLC12A2). Collectively, we show that cell-mediated matrix remodeling is necessary for the development of regenerative pro-acinar progenitor cells from hS/PCs.

Generation of functional salivary gland tissue from human submandibular gland stem/progenitor cells

BackgroundOrgan replacement regenerative therapy based on human adult stem cells may be effective for salivary gland hypofunction. However, the generated tissues are immature because the signaling factors that induce the differentiation of human salivary gland stem cells into salivary glands are unknown.MethodsIsolated human submandibular gland stem/progenitor cells (hSMGepiS/PCs) were characterized and three-dimensionally (3D) cultured to generate organoids and further induced by fibroblast growth factor 10 (FGF10) in vitro. The induced spheres alone or in combination with embryonic day 12.5 (E12.5) mouse salivary gland mesenchyme were transplanted into the renal capsules of nude mice to assess their development in vivo. Immunofluorescence, quantitative reverse transcriptase-polymerase chain reaction, calcium release analysis, western blotting, hematoxylin–eosin staining, Alcian blue–periodic acid-Schiff staining, and Masson’s trichrome staining were performed to assess the structure and function of generated tissues in vitro and in vivo.ResultsThe isolated hSMGepiS/PCs could be long-term cultured with a stable genome. The organoids treated with FGF10 [FGF10 (+) group] exhibited higher expression of salivary gland–specific markers; showed spatial arrangement of AQP5+, K19+, and SMA+ cells; and were more sensitive to the stimulation by neurotransmitters than untreated organoids [FGF10 (−) group]. After heterotopic transplantation, the induced cell spheres combined with mouse embryonic salivary gland mesenchyme showed characteristics of mature salivary glands, including a natural morphology and saliva secretion.ConclusionFGF10 promoted the development of the hSMGepiS/PC-derived salivary gland organoids by the expression of differentiation markers, structure formation, and response to neurotransmitters in vitro. Moreover, the hSMGepiS/PCs responded to the niche in mouse embryonic mesenchyme and further differentiated into salivary gland tissues with mature characteristics. Our study provides a foundation for the regenerative therapy of salivary gland diseases.

Stable Acinar Progenitor Cell Model Identifies Treacle-Dependent Radioresistance.

Radiotherapy for head and neck cancers can result in extensive damage to the salivary glands, significantly affecting patient quality of life. However, the salivary gland can recover in patients receiving lower doses of radiation. In addition, there is considerable interest in delineating the mechanisms by which stem cells survive radiation exposure and promote tissue regeneration. In this study, we isolated stable radioresistant acinar progenitor cells from the submaxillary gland of the Sprague Dawley rat. Progenitor cells are characterized as c-Kithigh/alpha-amylase+ and are resistant to X rays (≤5 Gy).We further isolated a radiosensitive acinar counterpart, characterized as c-Kitlow/alpha-amylase+, which is effectively killed by exposure to 2 Gy X ray of radiation. Phosphopeptides with homology to the treacle protein (TCOF1) were disproportionately increased in progenitor cells, compared to their radiosensitive counterparts. Silencing of TCOF1 expression (shRNA) radiosensitized progenitor cells, a response conserved in human cells with TCOF1 knockdown. Collectively, these observations indicate that radiation resistance is an intrinsic property of c-Kithigh salivary gland progenitor cells. Since human salivary gland stem cells with c-Kit expression are believed to have enhanced regenerative potencies, our model system provides a stable platform to investigate molecular features associated with c-Kit expression that may contribute to protection or stabilization of the stem cell niche.

Human Salivary Gland Stem Cells Functionally Restore Radiation Damaged Salivary Glands.

Adult stem cells are often touted as therapeutic agents in the regenerative medicine field, however data detailing both the engraftment and functional capabilities of solid tissue derived human adult epithelial stem cells is scarce. Here we show the isolation of adult human salivary gland (SG) stem/progenitor cells and demonstrate at the single cell level in vitro self-renewal and differentiation into multilineage organoids. We also show in vivo functionality, long-term engraftment, and functional restoration in a xenotransplantation model. Indeed, transplanted human salisphere-derived cells restored saliva production and greatly improved the regenerative potential of irradiated SGs. Further selection for c-Kit expression enriched for cells with enhanced regenerative potencies. Interestingly, interaction of transplanted cells with the recipient SG may also be involved in functional recovery. Thus, we show for the first time that salispheres cultured from human SGs contain stem/progenitor cells capable of self-renewal and differentiation and rescue of saliva production. Our study underpins the therapeutic promise of salisphere cell therapy for the treatment of xerostomia.

Human salivary gland stem cells ameliorate hyposalivation of radiation-damaged rat salivary glands

Salivary function in mammals may be defective for various reasons, such as aging, Sjogren's syndrome or radiation therapy in head and neck cancer patients. Recently, tissue-specific stem cell therapy has attracted public attention as a next-generation therapeutic reagent. In the present study, we isolated tissue-specific stem cells from the human submandibular salivary gland (hSGSCs). To efficiently isolate and amplify hSGSCs in large amounts, we developed a culture system (lasting 4–5 weeks) without any selection. After five passages, we obtained adherent cells that expressed mesenchymal stem cell surface antigen markers, such as CD44, CD49f, CD90 and CD105, but not the hematopoietic stem cell markers, CD34 and CD45, and that were able to undergo adipogenic, osteogenic and chondrogenic differentiation. In addition, hSGSCs were differentiated into amylase-expressing cells by using a two-step differentiation method. Transplantation of hSGSCs to radiation-damaged rat salivary glands rescued hyposalivation and body weight loss, restored acinar and duct cell structure, and decreased the amount of apoptotic cells. These data suggest that the isolated hSGSCs, which may have characteristics of mesenchymal-like stem cells, could be used as a cell therapy agent for the damaged salivary gland.

Isolation of Human Salivary Gland Stem Cells from Submandibular Glands

Isolation of Human Salivary Gland Stem Cells from Submandibular Glands

Isolation and characterization of human salivary gland cells for stem cell transplantation to reduce radiation-induced hyposalivation

Background Recently, we showed that transplantation of 100–300 c-Kit + stem cells isolated from cultured salispheres ameliorates radiation-damage in murine salivary glands. The aim of this study is to optimize and translate these findings from mice to man. Methods Mouse and human non-malignant parotid and submandibular salivary gland tissue was collected and enzymatically digested. The remaining cell suspension was cultured according to our salisphere culture method optimized for murine salispheres. Salisphere cells were tested using 3D matrix culturing for their in vitro stem cell characteristics such as the potential to differentiate into tissue specific cell types. Several potential mouse and human salivary gland stem cells were selected using FACS. Results In human salivary gland, c-Kit + cells were only detected in excretory ducts as shown previously in mice. From both human parotid and submandibular gland cell suspensions salispheres could be grown, which when placed in 3D culture developed ductal structures and mucin-expressing acinar-like cells. Moreover, cells dispersed from primary salispheres were able to form secondary spheres in matrigel, a procedure that could be repeated for at least seven passages. Approximately 3000 c-Kit + cells could be isolated from primary human salispheres per biopsy. Conclusion Human salivary glands contain a similar ‘putative’ stem cell population as rodents, expressing c-kit and capable of in vitro differentiation and self-renewal. In the future, these cells may have the potential to reduce radiotherapy-induced salivary gland dysfunction in patients.