Salivary Gland Stem Cells Research Articles

The impact of alternate-day fasting on the salivary gland stem cell compartments in non-obese diabetic mice with newly established Sjögren's syndrome.

Intermittent fasting exerts a profound beneficial influence on a spectrum of diseases through various mechanisms including regulation of immune responses, elimination of senescent- and pathogenic cells and improvement of stem cell-based tissue regeneration in a disease- and tissue-dependent manner. Our previous study demonstrated that alternate-day fasting (ADF) led to alleviation of xerostomia and sialadenitis in non-obese diabetic (NOD) mice, a well-defined model of Sjögren's syndrome (SS). This present study delved into the previously unexplored impacts of ADF in this disease setting and revealed that ADF increases the proportion of salivary gland stem cells (SGSCs), defined as the EpCAMhi cell population among the lineage marker negative submandibular gland (SMG) cells. Furthermore, ADF downregulated the expression of p16INK4a, a cellular senescence marker, which was concomitant with increased apoptosis and decreased expression and activity of NLRP3 inflammasomes in the SMGs, particularly in the SGSC-residing ductal compartments. RNA-sequencing analysis of purified SGSCs from NOD mice revealed that the significantly downregulated genes by ADF were mainly associated with sugar metabolism, amino acid biosynthetic process and MAPK signaling pathway, whereas the significantly upregulated genes related to fatty acid metabolic processes, among others. Collectively, these findings indicate that ADF increases the SGSC proportion, accompanied by a modulation of the SGSC property and a switch from sugar- to fatty acid-based metabolism. These findings lay the foundation for further investigation into the functionality of SGSCs influenced by ADF and shed light on the cellular and molecular mechanisms by which ADF exerts beneficial actions on salivary gland restoration in SS.

Pancreatic Differentiation of Oral Minor Salivary Gland Stem Cells.

Stem cells from various sources including major salivary glands have been used to establish pancreatic differentiation in an attempt to provide new treatment options for patients with diabetes mellitus. In contrast, the potential of using the more easily accessible intraoral minor salivary glands has not been evaluated so far. Salivary stem cells were isolated from normal labial minor salivary glands that were removed during the excision of a mucocele and were attempted to differentiate into pancreatic cell lines using a culture medium enriched with activin A, retinoic acid and GLP-1.Real time RT-PCR was used to evaluate the expression of the genes of pancreatic transcription factors MafA, Ptf1a, Hb9 and Arx. Complementary, 22 labial minor salivary gland paraffin-embedded specimens were examined using immunohistochemistry for the presence of the relevant gene products of the pancreatic transcription factors Arx, MafA, Ptf1a and Pdx1. The differentiated salivary stem cells(cells of passage 3) expressed the genes of the pancreatic transcription factors MafA, Ptf1a, Hb9 and Arx even on the first day of the experiment while immunohistochemistry also confirmed the presence of the protein products of Arx, MafA, Ptf1a as well as Pdx1[> 50% of the specimens for Arx(5/8) and MafA(7/8), < 50% for Ptf1a(5/11) and Pdx1(5/11)] in ducts, mesenchymal connective tissue and acinar cells. Labial minor salivary glands may share gene and protein characteristics with pancreas suggesting a possible usefulness for pancreatic regeneration or substitution in cases of deficiency.

Salivary gland organoid transplantation as a therapeutic option for radiation-induced xerostomia

BackgroundXerostomia is a pathological condition characterized by decreased salivation due to salivary gland dysfunction and is frequently attributed to irreversible damage as a side effect of radiation therapy. Stem cell–derived organoid therapy has garnered attention as a promising avenue for resolving this issue. However, Matrigel, a hydrogel commonly used in organoid culture, is considered inappropriate for clinical use due to its undefined composition and immunogenicity. In this study, we aimed to develop a method for culturing collagen-based human salivary gland organoids (hSGOs) suitable for clinical applications and evaluated their therapeutic effectiveness.MethodsHuman salivary gland stem cells were isolated from the salivary gland tissues and cultured in both Matrigel and collagen. We compared the gene and protein expression patterns of salivary gland–specific markers and measured amylase activity in the two types of hSGOs. To evaluate the therapeutic effects, we performed xenogeneic and allogeneic transplantation using human and mouse salivary gland organoids (hSGOs and mSGOs), respectively, in a mouse model of radiation-induced xerostomia.ResultshSGOs cultured in Matrigel exhibited self-renewal capacity and differentiated into acinar and ductal cell lineages. In collagen, they maintained a comparable self-renewal ability and more closely replicated the characteristics of salivary gland tissue following differentiation. Upon xenotransplantation of collagen-based hSGOs, we observed engraftment, which was verified by detecting human-specific nucleoli and E-cadherin expression. The expression of mucins, especially MUC5B, within the transplanted hSGOs suggested a potential improvement in the salivary composition. Moreover, the allograft procedure using mSGOs led to increased salivation, validating the efficacy of our approach.ConclusionsThis study showed that collagen-based hSGOs can be used appropriately in clinical settings and demonstrated the effectiveness of an allograft procedure. Our research has laid the groundwork for the future application of collagen-based hSGOs in allogeneic clinical trials.

CCL28: A Promising Biomarker for Assessing Salivary Gland Functionality and Maintaining Healthy Oral Environments.

The oral cavity serves as the primary path through which substances from the outside world enter our body. Therefore, it functions as a critical component of host defense. Saliva is essential for maintaining a stable oral environment by catching harmful agents, including pathogens, allergens, and chemicals, in the air or food. CCL28, highly expressed in mucosal tissues, such as the colon and salivary glands, is a chemokine that attracts CCR10/CCR3 expressing cells. However, the role of CCL28 in salivary gland formation remains unclear. In this study, we investigated the salivary gland structure in CCL28-deficient mice. Histological analysis showed decreased staining intensity of Alcian blue, which detects acidic mucous, reduced expression of MUC2, and higher infiltration of gram-positive bacteria in the salivary glands of CCL28-deficient mice. In addition, CCL28-deficient mice contained ectopically MUC2-expressed cells in the ducts and reduced the expression of cytokeratin 18, a marker for ductal cells, within the submandibular glands, resulting in decreased duct numbers. Additionally, the submandibular glands of CCL28-deficient mice showed reduced expression of several stem cell markers. These results suggest that CCL28 regulates saliva production via proper differentiation of salivary gland stem cells and could be a valuable biomarker of salivary gland function.

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.

Injectable decellularized extracellular matrix hydrogel promotes salivary gland regeneration via endogenous stem cell recruitment and suppression of fibrogenesis.

Saliva is key to the maintenance of oral homeostasis. However, several forms of salivary gland (SG) disorders, followed by hyposalivation, often result in dental caries, oral infection, and decreased taste, which dramatically affect the quality of patient's life. Functional biomaterials hold great potential for tissue regeneration in damaged or dysfunctional SGs and maintaining the good health of oral cavity. Herein, we prepared an injectable hydrogel derived from decellularized porcine submandibular glands (pDSG-gel), the material and biological properties of the hydrogel were systematically investigated. First, good biocompatibility and bioactivities of the pDSG-gel were validated in 2D and 3D cultures of primary submandibular gland mesenchymal stem cells (SGMSCs). Especially, the pDSG-gel effectively facilitated SGMSCs migration and recruitment through the activation of PI3K/AKT signaling pathway, suggested by transcriptomic analysis and immunoblotting. Furthermore, proteomic analysis of the pDSG revealed that many extracellular matrix components and secreted factors were preserved, which may contribute to stem cell homing. The recruitment of endogenous SG cells was confirmed in vivo, upon in situ injection of the pDSG-gel into the defective SGs in rats. Acinar and ductal-like structures were evident in the injury sites after pDSG-gel treatment, suggesting the reconstruction of functional SG units. Meanwhile, histological characterizations showed that the administration of the pDSG-gel also significantly suppressed fibrogenesis within the injured SG tissues. Taken together, this tissue-specific hydrogel provides a pro-regenerative microenvironment for endogenous SG regeneration and holds great promise as a powerful and bioactive material for future treatments of SG diseases. STATEMENT OF SIGNIFICANCE: Decellularized extracellular matrix (dECM) has been acknowledged as one of the most promising biomaterials that recapitalizes the microenvironment in native tissues. Hydrogel derived from the dECM allows in situ administration for tissue repair. Herein, a tissue-specific dECM hydrogel derived from porcine salivary glands (pDSG-gel) was successfully prepared and developed for functional reconstruction of defective salivary gland (SG) tissues. The pDSG-gel effectively accelerated endogenous SG stem cells migration and their recruitment for acinar- and ductal-like regeneration, which was attributed to the activation of PI3K/AKT signaling pathway. Additionally, the introduction of the pDSG-gel resulted in highly suppressed fibrogenesis in the defective tissues. These outcomes indicated that the pDSG-gel holds great potential in clinical translation toward SG regeneration through cell-free treatments.

Pathway Analysis of Transcriptional Alterations in the Salivary Gland Following Radiation Exposure

Radiation-induced xerostomia (RIX) is the subjective condition of dry mouth caused by radiation therapy to the head and neck and a result of hyposalivation and altered sialochemistry. Our long-term goal is to develop novel approaches to improve salivary function after radiation therapy. To better understand the molecular alterations induced by radiation in the salivary glands, we performed transcriptional profiling aimed at describing acute and long-term alterations in the murine salivary glands induced by radiation to better design effective regenerative therapies. The submandibular gland (SMG) of male C57Bl/6 mice received 15 Gy in a single fraction using a small animal image-guided irradiator. The mice were followed for 90 days. Saliva was collected at baseline and 7, 60 and 90 days after radiation. Salivary glands were collected for histology 3, 7, 60, and 90 days after radiation. The tissues were analyzed using a multiplex analysis platform processed with the murine whole transcriptome panel to investigate changes in gene expression over time. Using the multiplex analysis platform software, 96 regions of interest corresponding to acini, ducts, and granular convoluted tubules (GCTs) were delineated and gene expression was assessed separately in each area. To confirm the identified alterations, SMG tissue was histologically evaluated for fibrosis (masons tri-chrome), mucin (alcian blue), and amylase (α-amylase). Alterations in the immune microenvironment (CD19, CD3, and F4/80) and salivary gland stem cells (MIST1, SCA, Sox2, and c-Kit) were assessed by immunohistochemistry. Radiation resulted in a significant decrease in salivary production compared to nonirradiated controls (p-value = 0.03). The analysis of the transcriptional pathway identified early alterations in cell communication, DNA damage, and the immune response. Protein metabolism and extracellular matrix remodeling were up-regulated at later time points. Histological evaluation of tissues showed an increase in glandular structure disorganization over time, with a decrease in the size and distribution of the acinar compartments throughout the gland (mean area stained: No RT = 25.81% vs 90d RT = 19.98%, p-value = 0.005). There was an increase in periductal fibrosis within the tissue after 60 days compared to controls. Acutely, there was an increase in the expression of MIST1 and macrophages (mean area stained: No RT = 14.8% vs 7d RT = 18.76%, p-value = 0.0004) within the SMG that had resolved by the 60- and 90-day time points. The results implicate macrophage alterations in radiation-induced salivary gland damage and provide valuable mechanistic insight guiding potential approaches to intervene in RIX. We are pursuing cell therapy-based approaches to prevent and repair damage caused by radiation in order to improve the lives of our patients.

Maintenance of adult stem cells from human minor salivary glands via the Wnt signaling pathway

BackgroundXerostomia is a salivary gland dysfunction that negatively impacts the life quality of patients; however, there is no effective treatment for xerostomia. Bioengineered organs, generated using stem cells obtained from newborn salivary glands and ligated injury models, are a new organ transplantation strategy that could be feasible for xerostomia treatment. Reconstruction of salivary gland organoids by seed cells obtained from human minor salivary glands will offer theoretical fundaments and technology support for clinical application and organ regeneration research. Herein, we aimed to propose a new method for culturing and enriching adult human minor salivary gland stem cells in vitro in a three-dimensional (3D) environment via Wnt signaling activation.MethodsObtained and characterized human minor salivary gland stem cells (hMSGSCs) with self-organization ability were 3D-cultured to generate organoids. We examined hMSGSCs proliferation and colony formation using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Telomerase reverse transcriptase staining, flow cytometry, immunofluorescence assay, RNA isolation, RT-PCR, and qPCR were performed to assess hMSGSCs structure and the function of reconstructive organoids in vitro.ResultshMSGSCs showed typical epithelial-like characteristics, such as positive for CD49f and cell KRT expression. hMSGSCs served as adult stem cells in salivary glands and could differentiate into acinar and duct cells. Upon the addition of Noggin, CHIR99021, and Wnt3A to the 3D culture system, hMSGSCs showed higher LGR5 expression and decreased AMY1B and MUC5B expression. Therefore, the Wnt and bone morphogenetic protein (BMP) pathways are important in regulating hMSGSCs self-organization and differentiation.ConclusionsWe showed that the stem cell properties of hMSGSCs in a 3D culture system can be maintained by activating the Wnt signaling pathway and inhibiting the BMP signaling pathway. Our findings contribute new insights on salivary gland organoid generation in vitro.

Nigella sativa oil mitigates xerostomia and preserves salivary function in radiotherapy-treated mice.

This study aimed to assess if Nigella sativa oil (NSO), a health supplement containing thymoquinone as a major component, can act as a protective agent in salivary gland stem cells following radiotherapy (RT) damage. Forty, 10-week-old, male C3H/HeJ mice were randomized to four experimental groups: sham RT + H2O gavage (control) (N = 4); 15 Gy RT + H2O gavage (N = 12); sham RT + NSO gavage (N = 12); and 15 Gy RT + NSO gavage (N = 12). Weight changes, saliva production, and salivary gland histopathologic staining were recorded for each group over the course of the experiment. All mice in the sham RT + H2O gavage and sham RT + NSO gavage groups demonstrated 100% 60-day survival. RT + H2O compared to RT + NSO gavaged mice were significantly underweight by an average of 6.4 g (p < .001). Salivary output showed significant decline in RT + H2O gavaged mice at days 3 and 16, whereas salivary output in RT + NSO during these same time periods was comparable to the control. At day 60, all mice that survived recovered salivary function regardless of their treatment arm. Salivary specimens from the RT + NSO gavage group demonstrated early signs of recovery of Kr 5+ salivary gland stem cells in both submandibular and sublingual glands at day 16 with complete recovery by day 60, marked by strong histopathologic staining, whereas the RT + H2O gavage group did not recover as effectively. NSO may help preserve salivary function in mice treated with RT and may mitigate xerostomia by accelerating the recovery of salivary gland stem cells. Not applicable.

Amelioration of gamma irradiation-induced salivary gland damage in mice using melatonin.

Salivary gland damage caused by ionizing radiation (IR) severely affects the patient quality of life and influences the efficacy of radiotherapy. Most current treatment modalities are palliative, so effective prevention of damage caused by IR is essential. Melatonin (MLT) is an antioxidant that has been reported to prevent IR-induced damage in a range of systems, including the hematopoietic system and gastrointestinal tract. In this study, we explored the effects of MLT on whole-neck irradiation (WNI)-induced salivary gland damage in mice. The results revealed that by protecting the channel protein AQP-5, MLT not only alleviates salivary gland dysfunction and maintains salivary flow rate, but also protects salivary gland structure and inhibits the WNI-induced reduction in mucin production and degree of fibrosis. Compared with WNI-treated mice, in those receiving MLT, we observed a modulation of oxidative stress in salivary glands via its effects on 8-OHdG and SOD2, as well as an inhibition of DNA damage and apoptosis. With respect to its radioprotective mechanism, we found that MLT may alleviate WNI-induced xerostomia partly by regulating RPL18A. In vitro, we demonstrated that MLT has radioprotective effects on salivary gland stem cells (SGSCs). In conclusion, our data this study indicate that MLT can effectively alleviate radiation-induced damage in salivary glands, thereby providing a new candidate for the prevention of WNI-induced xerostomia.

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.

Salivary gland regeneration: from salivary gland stem cells to three-dimensional bioprinting.

Hyposalivation and severe dry mouth syndrome are the most common complications in patients with head and neck cancer (HNC) after receiving radiation therapy. Conventional treatment for hyposalivation relies on the use of sialogogues such as pilocarpine; however, their efficacy is constrained by the limited number of remnant acinar cells after radiation. After radiotherapy, the salivary gland (SG) secretory parenchyma is largely destroyed, and due to the reduced stem cell niche, this gland has poor regenerative potential. To tackle this, researchers must be able to generate highly complex cellularized 3D constructs for clinical transplantation via technologies, including those that involve bioprinting of cells and biomaterials. A potential stem cell source with promising clinical outcomes to reserve dry mouth is adipose mesenchymal stem cells (AdMSC). MSC-like cells like human dental pulp stem cells (hDPSC) have been tested in novel magnetic bioprinting platforms using nanoparticles that can bind cell membranes by electrostatic interaction, as well as their paracrine signals arising from extracellular vesicles. Both magnetized cells and their secretome cues were found to increase epithelial and neuronal growth of in vitro and ex vivo irradiated SG models. Interestingly, these magnetic bioprinting platforms can be applied as a high-throughput drug screening system due to the consistency in structure and functions of their organoids. Recently, exogenous decellularized porcine ECM was added to this magnetic platform to stimulate an ideal environment for cell tethering, proliferation, and/or differentiation. The combination of these SG tissue biofabrication strategies will promptly allow for in vitro organoid formation and establishment of cellular senescent organoids for aging models, but challenges remain in terms of epithelial polarization and lumen formation for unidirectional fluid flow. Current magnetic bioprinting nanotechnologies can provide promising functional and aging features to in vitro craniofacial exocrine gland organoids, which can be utilized for novel drug discovery and/or clinical transplantation.

Minor salivary gland stem cells: a comparative study of the biological properties under clinical-grade culture conditions.

Development of clinical-grade, cell preparations is central to cGMP (good manufacturing practice compliant) conditions. This study aimed to investigate the potential of two serum/xeno-free, cGMP (StemPro, StemMacs) culture media to maintain "stemness" of human minor salivary gland stem cell (mSG-SC) cultures compared to a complete culture medium (CCM). Overall, StemMacs resulted in higher proliferation rates after p.6 compared to the conventional serum-based medium, while StemPro showed substantial delays in cell proliferation after p.9. The mSG-SCs cultures exhibited two distinct cell populations at early passages a mesenchymal subpopulation and an epithelial-like subpopulation. Expression of several markers (CD146, STRO-1, SSEA-4, CD105, CD106, CD34, K 7/8, K14, K18) variably decreased with prolonged passaging (all three media). The percentage of SA-β-gal positive cells was initially higher for StemMacs compared to StemPro/CCM and increased with prolonged passaging in all cases. The telomere fragment length decreased with prolonged passaging in all three media but more pronouncedly for the CCM. Expansion under serum-free conditions caused pronounced upregulation of ALP and BMP-2, with parallel complete elimination of the baseline expressions of LPL (all three media) and ACAN (serum-free media), therefore, showing a preferential shift of the mSG-SCs towards osteogenic phenotypes. Finally, several markers (Nanog, SOX-2, PDX-1, OTX2, GSC, HCG) decreased with prolonged culture, indicating successive loss of "stemness". Based on the findings, it seems that StemPro preserve stemness of the mSG-SCs after prolonged culture. Nevertheless, there is still a vacant role for the ideal development of clinical-grade culture conditions.

Functional Differences in the Role of Ductal Stem Cells in Mouse Major Salivary Glands.

Salivary gland (SG) stem cells are the only cell population capable of extended growth in organotypic cultures, and thus they are considered a source for cell-based therapies aimed at SG regeneration. Studies in the mouse submandibular gland have identified only one population of tissue stem cells capable of salisphere formation in culture. These cells are actively dividing ductal cells that express epithelial progenitor markers keratin (K) 5/14 and normally function as lineage-restricted stem cells for differentiated ductal cells. In response to severe injury, however, these cells undergo a multipotency switch and contribute to regeneration of multiple cell lineages, including secretory units or acini. Little is known about the mechanism of cell renewal and regeneration in the other major SGs and whether comparable stem cell populations exist in the parotid (PG) and sublingual (SLG) glands. Using in vivo and ex vivo models, we show that both the PG and SLG contain a small population of K14-expressing ductal cells. Although they do not cycle frequently, K14-expressing ductal cells are the source of salisphere-forming cells in these glands. Long-term lineage tracing studies in adult mouse PGs showed a progenitor-progeny relationship between the K14-expressing ductal cells and the K19-expressing ductal cells in the striated ducts. In the SLGs, however, K14-expressing ductal cells did not generate a differentiated cell progeny for a 6-month period of observation and did not make a significant contribution to regeneration of gland after severe injury. These studies reveal the functional similarities and differences in tissue stem cells among the major SGs and have implications for developing strategies for SG regenerative therapies.

Aldehyde dehydrogenase 3A1 deficiency leads to mitochondrial dysfunction and impacts salivary gland stem cell phenotype.

Adult salivary stem/progenitor cells (SSPC) have an intrinsic property to self-renew in order to maintain tissue architecture and homeostasis. Adult salivary glands have been documented to harbor SSPC, which have been shown to play a vital role in the regeneration of the glandular structures postradiation damage. We have previously demonstrated that activation of aldehyde dehydrogenase 3A1 (ALDH3A1) after radiation reduced aldehyde accumulation in SSPC, leading to less apoptosis and improved salivary function. We subsequently found that sustained pharmacological ALDH3A1 activation is critical to enhance regeneration of murine submandibular gland after radiation damage. Further investigation shows that ALDH3A1 function is crucial for SSPC self-renewal and survival even in the absence of radiation stress. Salivary glands from Aldh3a1–/– mice have fewer acinar structures than wildtype mice. ALDH3A1 deletion or pharmacological inhibition in SSPC leads to a decrease in mitochondrial DNA copy number, lower expression of mitochondrial specific genes and proteins, structural abnormalities, lower membrane potential, and reduced cellular respiration. Loss or inhibition of ALDH3A1 also elevates ROS levels, depletes glutathione pool, and accumulates ALDH3A1 substrate 4-hydroxynonenal (4-HNE, a lipid peroxidation product), leading to decreased survival of murine SSPC that can be rescued by treatment with 4-HNE specific carbonyl scavengers. Our data indicate that ALDH3A1 activity protects mitochondrial function and is important for the regeneration activity of SSPC. This knowledge will help to guide our translational strategy of applying ALDH3A1 activators in the clinic to prevent radiation-related hyposalivation in head and neck cancer patients.

Emerging organoid-based platforms to study salivary gland hypofunction

Xerostomia is a pathologic condition of hyposalivation due to salivary gland (SG) dysfunction. Although xerostomia significantly affects the quality of patients’ life, there is no satisfactory treatment for this disease. Importantly, the senior population is more susceptible to xerostomia than younger individuals and the prevalence of the disease is much higher in elderly women than in men. However, the mechanisms underlying these clinical correlations have not yet been elucidated and further studies are required. Given that cell lines exhibiting saliva-producing abilities are not available, the generation and maturation of salivary gland organoids (SGOs) have been spotlighted as a modeling system to investigate the homeostasis of SG stem cells, as well as the pathophysiology of SGs in disease. In this review article, we will review the latest reports dealing with the generation and maturation of SGOs by defining the stem cells in SGs. We will also discuss the recent literature proposing strategies to model disease and regenerate damaged tissues.

Marrow Derived Autologous Stromal Cells for the Restoration of Salivary Hypofunction (MARSH): Study Protocol for a Phase I Dose Escalation Trial of Patients with Xerostomia after Radiation Therapy for Head and Neck Cancer

<h3>Purpose/Objective(s)</h3> Xerostomia, or dry mouth, is a common side effect of head neck radiation. Current treatment options for radiation-induced xerostomia are generally supportive in nature. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are a viable cell-based therapy for xerostomia. Adult stem cells are the ultimate source for replenishment of salivary gland tissue and the secretome of BM-MSCs may allow for the regeneration of salivary gland stem cells after radiation. We have undertaken FDA IND enabling studies demonstrating the phenotype, functionality, and secretome of interferon gamma (IFNγ) stimulated BM-MSCs taken from patients with head and neck cancer who have undergone radiation. Previous clinical trials with MSCs often required injection of MSCs rapidly after expansion, without cryopreservation. However this is unrealistic for patient care where patients are traveling for therapy or may require multiple injections of MSCs. Cryopreservation allows for a more inclusive therapy but can result in cellular injury or immune dysfunction. Stimulating the BM-MSCs with IFNγ prior to cryopreservation has been shown to prevent this. Here we present the protocol of MARSH, a first-in-human clinical trial of bone marrow derived, interferon gamma activated BM-MSCs for the treatment of radiation-induced xerostomia. <h3>Materials/Methods</h3> This single-center phase I dose-escalation with expansion cohort, study will assess the safety and tolerability of BM-MSCs for treatment of radiation-induced xerostomia in patients who had head and neck cancer. This is a 2-part trial. The first is a phase I trial of unilateral injection of MSCs into the submandibular gland of 6 patients to demonstrate safety. The second part is a phase I dose escalation study with a 3+3 design with staggered enrollment. A total of 21-30 subjects (9-18 in phase I study, 12 in expansion cohort) will be enrolled. The primary endpoint is determining the recommended phase 2 dose (RP2D) of interferon gamma stimulated BM-MSCs in order to conduct further studies on the efficacy of BM-MSCs. Patients will have their bone marrow aspirated, BM-MSCs expanded and stimulated with IFNγ, and then injected into the submandibular gland. The RP2D will be determined by dose limiting toxicities occurring within one month of BM-MSC injection. Secondary outcomes of salivary amounts, composition, ultrasound of salivary glands, and quality of life surveys will be taken at 3, 6, 12, and 24 month visits. <h3>Results</h3> Recruiting will start January 3, 2022. Subject accrual will occur over 2.5 years with the total duration of the trial expected to be approximately 4.5 years. <h3>Conclusion</h3> Autotransplantation of IFNγ-stimulated BM-MSCs in salivary glands post-radiation may provide an innovative remedy to treat xerostomia and restore quality of life.

Thermoresponsive fiber-based microwells capable of formation and retrieval of salivary gland stem cell spheroids for the regeneration of irradiation-damaged salivary glands

Three-dimensional spheroid culture enhances cell-to-cell interactions among stem cells and promotes the expression of stem cell properties; however, subsequent retrieval and delivery of these cells remain a challenge. We fabricated a thermoresponsive fiber-based microwell scaffold by combining electrospinning and hydrogel micropatterning. The resultant scaffold appeared to facilitate the formation of cellular spheroids of uniform size and enabled the expression of more stem cell-secreting growth factor genes (EGF, IGF-1, FGF1, FGF2, and HGF), pluripotent stem cell-related genes (SOX2 and NANOG), and adult epithelial stem cell-related genes (LGR4, LGR5, and LGR6) than salivary gland stem cells in a monolayer culture (SGSCmonolayer). The spheroids could be retrieved efficiently by decreasing temperature. SGSC-derived spheroid (SGSCspheroid) cells were then implanted into the submandibular glands of mice at 2 weeks after fractionated X-ray irradiation at a dose of 7.5 Gy/day. At 16 weeks post-irradiation, restoration of salivary function was detected only in SGSCspheroid-implanted mice. The production of submandibular acini specific mucin increased in SGSCspheroid-implanted mice, compared with PBS control. More MIST1+ mature acinar cells were preserved in the SGSCspheroid-implanted group than in the PBS control group. Intriguingly, SGSCspheroid-implanted mice exhibited greater amelioration of tissue damage and preservation of KRT7+ terminally differentiated luminal ductal cells than SGSCmonolayer-implanted mice. The SGSCspheroid-implanted mice also showed less DNA damage and apoptotic cell death than the SGSCmonolayer-implanted mice at 2 weeks post-implantation. Additionally, a significant increase in Ki67+AQP5+ proliferative acinar cells was noted only in SGSCspheroid-implanted mice. Our results suggest that a thermoresponsive fiber-based scaffold could be of use to facilitate the production of function-enhanced SGSCspheroid cells and their subsequent retrieval and delivery to damaged salivary glands to alleviate radiation-induced apoptotic cell death and promote salivary gland regeneration.

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

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.

Induction of salivary gland-like cells from epithelial tissues transdifferentiated from mouse embryonic fibroblasts

Salivary gland hypofunction due to radiation therapy for head and neck cancer or Sjögren syndrome may cause various oral diseases, which can lead to a decline in the quality of life. Cell therapy using salivary gland stem cells is a promising method for restoring hypofunction. Herein, we show that salivary gland-like cells can be induced from epithelial tissues that were transdifferentiated from mouse embryonic fibroblasts (MEFs). We introduced four genes, Dnp63a, Tfap2a, Grhl2, and Myc (PTMG) that are known to transdifferentiate fibroblasts into oral mucosa-like epithelium in vivo into MEFs. MEFs overexpressing these genes showed epithelial cell characteristics, such as cobblestone appearance and E-cadherin positivity, and formed oral epithelial-like tissue under air–liquid interface culture conditions. The epithelial sheet detached from the culture dish was infected with adenoviruses encoding Sox9 and Foxc1, which we previously identified as essential factors to induce salivary gland formation. The cells detached from the cell sheet formed spheres 10 days after infection and showed a branching morphology. The spheres expressed genes encoding basal/myoepithelial markers, cytokeratin 5, cytokeratin 14, acinar cell marker, aquaporin 5, and the myoepithelial marker α-smooth muscle actin. The dissociated cells of these primary spheres had the ability to form secondary spheres. Taken together, our results provide a new strategy for cell therapy of salivary glands and hold implications in treating patients with dry mouth.