TGF-ß1 Triggers Salivary Hypofunction via Attenuating Protein Secretion and AQP5 Expression in Human Submandibular Gland Cells.

Aging-related salivary gland degeneration usually causes poor oral health. Periductal fibrosis frequently occurs in the submandibular gland of the elderly. Transforming growth factor ß1 (TGF-ß1) is the primary driving factor for fibrosis, which exhibits an increase in the fibrotic submandibular gland tissue. This study aimed to investigate the effects of TGF-ß1 on the human submandibular gland (HSG) cell secretory function and its influences on aquaporin 5 (AQP5) expressions and distribution. We found that TGF-ß1 reduces the protein secretion amount of HSG and leads to the abundance alteration of 151 secretory proteins. Data are available via ProteomeXchange with the identifier PXD043185. The majority of HSG secretory proteins (84.11%) could be matched to the human saliva proteome. Meanwhile, TGF-ß1 enhances the expression of COL4A2, COL5A1, COL7A1, COL1A1, COL2A1, and α-SMA, hinting that TGF-ß1 possesses the potential to drive HSG fibrosis-related events. Besides, TGF-ß1 also attenuates the AQP5 expression and its membrane distribution in HSGs. The percentage for TGF-ß1-induced AQP5 reduction (52.28%) is much greater than that of the TGF-ß1-induced secretory protein concentration reduction (16.53%). Taken together, we concluded that TGF-ß1 triggers salivary hypofunction via attenuating protein secretion and AQP5 expression in HSGs, which may be associated with TGF-ß1-driven fibrosis events in HSGs.

SPM Receptor Expression and Localization in Irradiated Salivary Glands.

Radiation therapy-mediated salivary gland destruction is characterized by increased inflammatory cell infiltration and fibrosis, both of which ultimately lead to salivary gland hypofunction. However, current treatments (e.g., artificial saliva and sialagogues) only promote temporary relief of symptoms. As such, developing alternative measures against radiation damage is critical for restoring salivary gland structure and function. One promising option for managing radiation therapy-mediated damage in salivary glands is by activation of specialized proresolving lipid mediator receptors due to their demonstrated role in resolution of inflammation and fibrosis in many tissues. Nonetheless, little is known about the presence and function of these receptors in healthy and/or irradiated salivary glands. Therefore, the goal of this study was to detect whether these specialized proresolving lipid mediator receptors are expressed in healthy salivary glands and, if so, if they are maintained after radiation therapy-mediated damage. Our results indicate that specialized proresolving lipid mediator receptors are heterogeneously expressed in inflammatory as well as in acinar and ductal cells within human submandibular glands and that their expression persists after radiation therapy. These findings suggest that epithelial cells as well as resident immune cells represent potential targets for modulation of resolution of inflammation and fibrosis in irradiated salivary glands.

Parasympathetic neurons in the human submandibular ganglion.

The submandibular ganglion (SMG) contains parasympathetic neurons which innervate the submandibular gland. In this study, immunohistochemistry for vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), choline acetyltransferase (ChAT), dopamine ß-hydroxylase (DBH), tyrosine hydroxylase (TH), and the transient receptor potential cation channel subfamily V members 1 (TRPV1) and 2 (TRPV2) was performed on the human SMG. In the SMG, 17.5 % and 8.9 % of parasympathetic neurons were immunoreactive for VIP and TRPV2, respectively. SMG neurons mostly contained ChAT- and DBH-immunoreactivity. In addition, subpopulations of SMG neurons were surrounded by VIP (69.6 %)-, TRPV2 (54.4 %)- and DBH (9.5 %)-immunoreactive (-ir) nerve fibers. SMG neurons with pericellular VIP- and TRPV2-ir nerve fibers were significantly larger than VIP- and TRPV2-ir SMG neurons, respectively. Other neurochemical substances were rare in the SMG. In the human submandibular gland, TRPV1- and TRPV2-ir nerve fiber profiles were seen around blood vessels. Double fluorescence method also demonstrated that TRPV2-ir nerve fiber profiles were located around myoepithelial and acinar cells in the submandibular gland. VIP and TRPV2 are probably expressed by both pre- and post-ganglionic neurons innervating the submandibular and sublingual glands. VIP, DBH and TRPV2 may have functions about regulation of salivary components in the salivary glands and neuronal activity in the SMG.

Specification of the patterning of a ductal tree during branching morphogenesis of the submandibular gland.

The development of ductal structures during branching morphogenesis relies on signals that specify ductal progenitors to set up a pattern for the ductal network. Here, we identify cellular asymmetries defined by the F-actin cytoskeleton and the cell adhesion protein ZO-1 as the earliest determinants of duct specification in the embryonic submandibular gland (SMG). Apical polarity protein aPKCζ is then recruited to the sites of asymmetry in a ZO-1-dependent manner and collaborates with ROCK signaling to set up apical-basal polarity of ductal progenitors and further define the path of duct specification. Moreover, the motor protein myosin IIB, a mediator of mechanical force transmission along actin filaments, becomes localized to vertices linking the apical domains of multiple ductal epithelial cells during the formation of ductal lumens and drives duct maturation. These studies identify cytoskeletal, junctional and polarity proteins as the early determinants of duct specification and the patterning of a ductal tree during branching morphogenesis of the SMG.

CP-25 alleviates antigen-induced experimental Sjögren's syndrome in mice by inhibiting JAK1-STAT1/2-CXCL13 signaling and interfering with B-cell migration.

The etiology of primary Sjögren's syndrome (pSS) remains unknown, and there is no complete curative drug. In this study, we treated a mouse model of the submandibular gland (SG) protein-immunized experimental Sjögren's syndrome (ESS) with paeoniflorin-6'-O-benzene sulfonate (termed CP-25) to evaluate the potential therapeutic effects of CP-25. Through in vivo experiments, we found that CP-25 increased saliva flow, alleviated the salivary gland indexes, and improved tissue integrity in the ESS model. The viability of splenocytes and B-lymphocyte migration from spleen were reduced in ESS mice. Furthermore, CP-25 decreased the expression of IgG antibodies, anti-SSA and anti-SSB antibodies and modulated the levels of cytokines in the serum of SS mice. The numbers of total B lymphocytes, plasma cells (PCs), and memory B cells diminished in the salivary gland. Increased expression of the JAK1-STAT1-CXCL13 axis and IFNα was found in human tissue isolated from pSS patients. In vitro, after stimulation with IFNα, the levels of CXCL13 mRNA and CXCL13 in human salivary gland epithelial cells (HSGEC) increased, while CP-25 counteracted the secretion of CXCL13 and downregulated the expression of CXCL13. IFN-α activated the JAK1-STAT1/2-CXCL13 signaling pathway in HSGEC, which was negatively regulated by additional CP-25. As a consequence, B-cell migration was downregulated in coculture with IFN-α-stimulated HSGEC. Taken together, this study demonstrated that the therapeutic effects of CP-25 were associated with the inhibition of the JAK1-STAT1/2-CXCL13 signaling pathway in HSGEC, which impedes the migration of B cells into the salivary gland. We identified the underlying mechanisms of the therapeutic effect of CP-25 and provided an experimental foundation for CP-25 as a potential drug in the treatment of the human autoimmune disorder pSS.

Cell Sheets Restore Secretory Function in Wounded Mouse Submandibular Glands.

Thermoresponsive cell culture plates release cells as confluent living sheets in response to small changes in temperature, with recovered cell sheets retaining functional extracellular matrix proteins and tight junctions, both of which indicate formation of intact and functional tissue. Our recent studies demonstrated that cell sheets are highly effective in promoting mouse submandibular gland (SMG) cell differentiation and recovering tissue integrity. However, these studies were performed only at early time points and extension of the observation period is needed to investigate duration of the cell sheets. Thus, the goal of this study was to demonstrate that treatment of wounded mouse SMG with cell sheets is capable of increasing salivary epithelial integrity over extended time periods. The results indicate that cell sheets promote tissue organization as early as eight days after transplantation and that these effects endure through Day 20. Furthermore, cell sheet transplantation in wounded SMG induces a significant time-dependent enhancement of cell polarization, differentiation and ion transporter expression. Finally, this treatment restored saliva quantity to pre-wounding levels at both eight and twenty days post-surgery and significantly improved saliva quality at twenty days post-surgery. These data indicate that cell sheets engineered with thermoresponsive cell culture plates are useful for salivary gland regeneration and provide evidence for the long-term stability of cell sheets, thereby offering a potential new therapeutic strategy for treating hyposalivation.

Diverse epithelial cell populations contribute to the regeneration of secretory units in injured salivary glands.

Salivary glands exert exocrine secretory function to provide saliva for lubrication and protection of the oral cavity. Its epithelium consists of several differentiated cell types, including acinar, ductal and myoepithelial cells, that are maintained in a lineage-restricted manner during homeostasis or after mild injuries. Glandular regeneration following a near complete loss of secretory cells, however, may involve cellular plasticity, although the mechanism and extent of such plasticity remain unclear. Here, by combining lineage-tracing experiments with a model of severe glandular injury in the mouse submandibular gland, we show that de novo formation of acini involves induction of cellular plasticity in multiple non-acinar cell populations. Fate-mapping analysis revealed that, although ductal stem cells marked by cytokeratin K14 and Axin2 undergo a multipotency switch, they do not make a significant contribution to acinar regeneration. Intriguingly, more than 80% of regenerated acini derive from differentiated cells, including myoepithelial and ductal cells, that appear to dedifferentiate to a progenitor-like state before re-differentiation into acinar cells. The potential of diverse cell populations serving as a reserve source for acini widens the therapeutic options for hyposalivation.

Sex-dependent Regeneration Patterns in Mouse Submandibular Glands.

Our previous studies indicated that YIGSR-A99 peptides chemically conjugated to fibrin hydrogel (FH) and applied to wounded submandibular gland (SMG) in vivo, formed new organized salivary tissue, whereas wounded SMG treated with FH alone or in the absence of a scaffold showed disorganized collagen formation and poor tissue healing. While these studies indicated that damaged SMG grow and differentiate when treated with FH containing L1 peptide, they were performed only in female mice. However, there is a well-established sexual dimorphism present in mouse SMG (e.g., males develop well-differentiated granular convoluted tubules, but these structures are poorly developed in females) and little is known about how these sex differences influence wound healing events. Therefore, the goal of this study was to conduct comparative analyses of regeneration patterns in male and female mice using L1p-FH in a wounded SMG mouse model. Particularly, we focused on sex-dependent wound healing events such as macrophage polarization, vascularization, tissue organization, and collagen deposition, and how these events affect salivary gland functioning.

The lack of terminal tubule cells in the submandibular gland of mice deficient in submandibular gland protein C.

The submandibular gland (SMG) of newborn mice has no mature acini but has the rudiments of acini called terminal tubules (TT). The TT are composed of TT cells with dark secretory granules and proacinar cells with lighter secretory granules, the latter being considered the immediate precursor of mature acinar cells. TT cells contain a specific secretory protein, submandibular gland protein C (SMGC) and they decrease in number postnatally at a higher rate in males than in females. In the present study, in order to clarify the biological roles of TT cells and their secretory product SMGC, we generated a knockout (KO) mouse strain deficient in SMGC. The KO mice of both sexes grew normally, had normal reproductive capacity and had normal acinar and duct systems in the SMG in adult ages. However, through the neonatal and early postnatal stages, the KO mice were deficient not only in the production of SMGC but also in TT cells. With electron microscopy of the SMG of newborn KO mice, TT cells with characteristic granules were absent and replaced by undifferentiated ductal cells, whereas proacinar cells were normal. These results suggested that the absence of SMGC inhibits the development of TT cells and that the absence of SMGC and TT cells has no notable influence on the postnatal development of the acinar and duct systems in the SMG.

Stress or injury induces cellular plasticity in salivary gland acinar cells.

Salivary gland function is severely disrupted by radiation therapy used to treat patients diagnosed with head and neck cancer and by Sjögren's syndrome. The resulting condition, which results in xerostomia or dry mouth, is due to irreversible loss of the secretory acinar cells within the major salivary glands. There are presently no treatments for the resolution of xerostomia. Cell-based approaches could be employed to repopulate acinar cells in the salivary gland but investigations into potential therapeutic strategies are limited by the challenges of maintaining and expanding acinar cells in vitro. We investigate the encapsulation of salivary gland cell aggregates within PEG hydrogels as a means of culturing secretory acinar cells. Lineage tracing was used to monitor the fate of acinar cells isolated from murine submandibular gland (SMG). Upon initial formation in vitro, SMG aggregates comprise both acinar and duct cells, with the majority cells of acinar origin. With longer culture times, acinar cells significantly decreased the expression of specific markers and activated the expression of keratins normally found in duct cells. A similar acinar-to-duct cell transition was also observed in vivo, following duct ligation injury. These results indicate that under conditions of stress (mechanical and enzymatic isolation from glands) or injury (duct ligation), salivary gland acinar cells exhibit plasticity to adopt a duct cell phenotype.

Aberrant MUC1 accumulation in salivary glands of Sjögren's syndrome patients is reversed by TUDCA in vitro.

OBJECTIVES: Xerostomia in SS patients has been associated with low quality and quantity of salivary mucins, which are fundamental for the hydration and protection of the oral mucosa. The aim of this study was to evaluate if cytokines induce aberrant mucin expression and whether tauroursodeoxycholic acid (TUDCA) is able to counteract such an anomaly. METHODS: Labial salivary glands from 16 SS patients and 15 control subjects, as well as 3D acini or human submandibular gland cells stimulated with TNF-α or IFN-γ and co-incubated with TUDCA, were analysed. mRNA and protein levels of Mucin 1 (MUC1) and MUC7 were determined by RT-qPCR and western blot, respectively. Co-immunoprecipitation and immunofluorescence assays for mucins and GRP78 [an endoplasmic reticulum (ER)-resident protein] were also performed. mRNA levels of RelA/p65 (nuclear factor-κB subunit), TNF-α, IL-1ß, IL-6, SEL1L and EDEM1 were determined by RT-qPCR, and RelA/p65 localization was evaluated by immunofluorescence. RESULTS: MUC1 is overexpressed and accumulated in the ER of labial salivary gland from SS patients, while MUC7 accumulates throughout the cytoplasm of acinar cells; however, MUC1, but not MUC7, co-precipitated with GRP78. TUDCA diminished the overexpression and aberrant accumulation of MUC1 induced by TNF-α and IFN-γ, as well as the nuclear translocation of RelA/p65, together with the expression of inflammatory and ER stress markers in 3D acini. CONCLUSION: Chronic inflammation alters the secretory process of MUC1, inducing ER stress and affecting the quality of saliva in SS patients. TUDCA showed anti-inflammatory properties decreasing aberrant MUC1 accumulation. Further studies are necessary to evaluate the potential therapeutic effect of TUDCA in restoring glandular homeostasis in SS patients.

Slc4a11 disruption causes duct cell loss and impairs NaCl reabsorption in female mouse submandibular glands.

Slc4a11, a member of the Slc4 HCO3- transporter family, has a wide tissue distribution. In mouse salivary glands, the expression of Slc4a11 mRNA was more than eightfold greater than the other nine members of the Slc4 gene family. The Slc4a11 protein displayed a diffuse subcellular distribution in both the acinar and duct cells of mouse submandibular glands (SMG). Slc4a11 disruption induced a significant increase in the Na+ and Cl- concentrations of stimulated SMG saliva, whereas it did not affect the fluid secretion rate in response to either ß-adrenergic or cholinergic receptor stimulation. Heterologous expressed mouse Slc4a11 acted as a H+ /OH- transporter that was uncoupled of Na+ or Cl- movement, and this activity was blocked by ethyl-isopropyl amiloride (EIPA) but not 4,4'-Diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS). Slc4a11 disruption revealed that Slc4a11 does not play a major role in intracellular pH regulation in mouse salivary gland cells. In contrast, NaCl reabsorption was impaired in the SMG saliva of female compared to male Slc4a11 null mice, which correlated with the loss of duct cells and a decrease in expression of the duct-cell-specific transcription factor Ascl3. Together, our results suggest that Slc4a11 expression regulates the number of ducts cells in the mouse SMG and consequently NaCl reabsorption.

Physical exercise stimulates salivary secretion of cystatins.

Physical exercise is known to activate the sympathetic nervous system, which influences the production of saliva from salivary glands. Our examination of saliva collected from highly trained athletes before and after a number of physical competititions showed an increase in the secretion of S-type cystatins and cystatin C as a subacute response to aerobic and anaerobic exercise. The elevation in salivary cystatins was transient and the recovery time course differed from that of amylase and other salivary proteins. An in vitro assay was developed based on a cell line from a human submandibular gland (HSG) that differentiated into acinus-like structures. Treatments with the ß-adrenergic agonist isoproterenol caused a shift in the intracellular distribution of S-type cystatins and cystatin C, promoting their accumulation at the outer regions of the acinus prior to release and suggesting the activation of a directional transport involving co-migration of both molecules. In another treatment using non-differentiated HSG cells, it was evident that both expression and secretion of cystatin C increased upon addition of the ß-adrenergic agonist, and these effects were essentially eliminated by the antagonist propranolol. The HSG cell line appears to have potential as a model for exploring the mechanism of cystatin secretion, particularly the S-type cystatins that originate primarily in the submandibular glands.

Cholesterol metabolism plays a crucial role in the regulation of autophagy for cell differentiation of granular convoluted tubules in male mouse submandibular glands.

It has been long appreciated that sex hormone receptors are expressed in various non-gonadal organs. However, it remains unclear how sex hormones regulate the morphogenesis of these non-gonadal organs. To address this issue, we used a male mouse model of androgen-dependent salivary gland morphogenesis. Mice with excessive cholesterol synthesis in the salivary glands exhibited defects in the maturation of granular convoluted tubules (GCTs), which is regulated through sex hormone-dependent cascades. We found that excessive cholesterol synthesis resulted in autophagy failure specifically in the duct cells of salivary glands, followed by the accumulation of NRF2, a transcription factor known as one of the specific substrates for autophagy. The accumulated NRF2 suppressed the expression of Foxa1, which forms a transcriptional complex with the androgen receptor to regulate target genes. Taken together, our results indicate that cholesterol metabolism plays a crucial role in GCT differentiation through autophagy.

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands.

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (αSMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

Establishment of a Murine Pro-acinar Cell Line to Characterize Roles for FGF2 and α3ß1 Integrins in Regulating Pro-acinar Characteristics.

Radiation therapy for head and neck cancers results in permanent damage to the saliva producing acinar compartment of the salivary gland. To date, a pure pro-acinar cell line to study underlying mechanisms of acinar cell differentiation in culture has not been described. Here, we report the establishment of a pro-acinar (mSG-PAC1) and ductal (mSG-DUC1) cell line, from the murine submandibular salivary gland (SMG), which recapitulate developmental milestones in differentiation. mSG-DUC1 cells express the ductal markers, keratin-7 and keratin-19, and form lumenized spheroids. mSG-PAC1 cells express the pro-acinar markers SOX10 and aquaporin-5. Using the mSG-PAC1 cell line, we demonstrate that FGF2 regulates specific steps during acinar cell maturation. FGF2 up-regulates aquaporin-5 and the expression of the α3 and α6 subunits of the α3ß1 and α6ß1 integrins that are known to promote SMG morphogenesis and differentiation. mSG-DUC1 and mSG-PAC1 cells were derived from genetically modified mice, homozygous for floxed alleles of the integrin α3 subunit. Similar to SMGs from α3-null mice, deletion of α3 alleles in mSG-PAC1 cells results in the up-regulation of E-cadherin and the down-regulation of CDC42. Our data indicate that mSG-DUC1 and mSG-PAC1 cells will serve as important tools to gain mechanistic insight into salivary gland morphogenesis and differentiation.

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers.

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sjögren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

Fibronectin-induced ductal formation in salivary gland self-organization model.

BACKGROUND: Recent advances in tissue regeneration approaches including 3D organoids, were based on various 3D organogenesis models. However, 3D models are generally technique-sensitive and time-consuming. Thus, we utilized an existing model of submandibular salivary gland (SMG) to modify a simple and highly reproducible in vitro 3D culture model of primary SMG cells self-organization into a well-developed cell spheroid inside Matrigel substrate. We used this model to observe the collective multicellular behavior during spheroid formation. Further, we applied various quantitative approaches including real-time live imaging and immune histochemical image analysis to dissect the cellular dynamics during tissue patterning. RESULTS: On a time-scale of hours, we observed marked size and shape transformations in the developed 3D spheroid which resulted in a spatially-controlled growth differential from the canter to the periphery of the formed aggregates. Moreover, we investigated the effect of fibronectin (FN) on SMG cells self-organization using our simplified culture model. Interestingly, we discovered a novel role of FN in inducing duct-like elongation during initial stages of SMG bud formation. CONCLUSION: This in vitro model provides an excellent tool for analyzing the intercellular dynamics during early SMG tissue development as well as revealing a novel role of FN in SMG ductal expansion.

Sox9 regulates the luminal stem/progenitor cell properties of salivary glands.

Exocrine glands share a common morphology consisting of ductal, acinar, and basal/myoepithelial cells, but their functions and mechanisms of homeostasis differ among tissues. Salivary glands are an example of exocrine glands, and they have been reported to contain multipotent stem cells that differentiate into other tissues. In this study, we purified the salivary gland stem/progenitor cells of adult mouse salivary glands using the cell surface marker CD133 by flow cytometry. CD133+ cells possessed stem cell capacity, and the transplantation of CD133+ cells into the submandibular gland reconstituted gland structures, including functional acinar. CD133+ cells were sparsely distributed in the intercalated and exocrine ducts and expressed Sox9 at higher levels than CD133- cells. Moreover, we demonstrated that Sox9 was required for the stem cell properties CD133+ cells, including colony and sphere formation. Thus, the Sox9-related signaling may control the regeneration salivary glands.

Salispheres from Different Major Salivary Glands for Glandular Regeneration.

Dysfunctional salivary glands (SGs) are a clinical challenge due to the lack of effective treatments. Cell therapy with stem/progenitor cells may improve this situation by providing promising therapeutic solutions. Therefore, exploring abundant cellular sources is important. Three major pairs of SGs are located in different anatomic regions: the parotid glands, the submandibular glands, and the sublingual glands. Although SG stem/progenitor cells can be isolated and cultivated from all major SGs as salispheres, the differences among SG origins remain unclear. In this study, salispheres were successfully isolated from all major SGs. The salispheres demonstrated unique cellular features that originated from their native tissues. The characteristic expression profiles and cellular features of SG stem cells were demonstrated in all salispheres. When they were transplanted into irradiated animals, the salispheres were all capable of improving the saliva secretion that was disrupted by irradiation. Typical histologic structures could be observed in most parts of the treated glands, and the fibrotic environments of irradiated submandibular glands were remodeled by all salispheres regardless of origins. This study characterized the cellular features and in vivo effects of salispheres that were derived from different anatomic origins. The results suggest the possibility of functional redundancy among distinct pairs of major SGs, which is useful for the design of cell therapy to treat dysfunctional glandular organs.