Abstract
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.
Highlights
Organ replacement regenerative therapy based on human adult stem cells may be effective for salivary gland hypofunction
After incubation at 37 °C in 5% CO2 for 4 h, epithelial cells were cultured in keratinocyte medium (KM; Sciencell) with supplements (KM-S) containing 5 μg/mL bovine serum albumin (BSA; Sigma-Aldrich), 5 ng/mL fibroblast growth factor-2 (FGF2; Sigma-Aldrich), 1 ng/mL epidermal growth factor (EGF; Sigma-Aldrich), 5 μg/mL insulin (Sigma-Aldrich), 5 μg/mL transferrin (Sigma-Aldrich), and 0.5 μg/mL hydrocortisone (SigmaAldrich); mesenchymal cells were cultured in Dulbecco’s modified Eagle’s medium with supplements (DMEM-S), Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Invitrogen)
All the cultured cells were positive for Keratin 19 (K19), and ductal cells were detected in Human submandibular gland (hSMG) tissue, indicating a ductal origin (Fig. 1d)
Summary
Organ replacement regenerative therapy based on human adult stem cells may be effective for salivary gland hypofunction. The generated tissues are immature because the signaling factors that induce the differentiation of human salivary gland stem cells into salivary glands are unknown. There are three major salivary glands—the parotid, submandibular, and sublingual glands—and minor salivary glands. They produce an average of 1–1.5 L of saliva daily, which is essential to the health and function of the gastrointestinal tract and oral cavity, including digestion of starch, normal speech, taste, mastication, swallowing, and the maintenance of teeth through its digestion antimicrobial, cleansing, lubricating, and buffering functions [1, 2]. Stem cell-based regenerative medicine holds the promise for treating salivary gland hypofunction fundamentally
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