Salivary Gland Stem Cells Research Articles

Functional Parotid Sparing: Does Sparing the Parotid Ducts Yield Improvements in Patient Reported Xerostomia?

Literature suggests that salivary gland stem cells, capable of regenerating salivary tissue, may preferentially reside in the large ducts of salivary glands. We conducted a prospective trial in patients receiving definitive chemoradiotherapy (CRT) for oropharyngeal cancer where we preferentially spared the major parotid ducts (localized via MRI sialography) during treatment planning. We hypothesized that (1) this technique would yield improvements in patient reported xerostomia (PRX) at 6- and 12-months post-CRT compared to a prospectively accrued cohort of similar patients, and (2) that parotid duct doses would be predictive for PRX.A total of 38 Patients were prospectively enrolled and underwent MRI sialography guided ductal sparing. 28/38 patients received de-escalated CRT (60Gy). PRX was classified as none/mild versus moderate to very severe. (1) PRX in the 28 de-escalated patients were compared using a fisher exact test to a cohort of 232 patients on the same clinical trials but who did not undergo ductal sparing. (2) Competing nested multivariate logistic models (+/- parotid duct doses) were assessed using dosimetric data from all 38 patients for their ability to predict PRX. Models included contralateral submandibular mean dose, mean parotid gland dose (both) +/- parotid duct mean dose (both). The two nested models were compared using a likelihood ratio test to determine if incorporating mean parotid duct doses significantly improved model fit.(1) Patients reporting moderate to very severe xerostomia was lower in patients with vs without parotid-ductal sparing at 6 months post-CRT (46% vs 67%, P = 0.03), and trended lower at 12 months post-CRT (32% vs 49%, P = 0.11). At 6 months post CRT, the mean parotid duct dose was the only covariate significantly associated with PRX (P = 0.047). At 12 months post CRT, no covariate was significantly associated with PRX but both mean contralateral submandibular dose and mean parotid duct dose approached significance (P = 0.06 and 0.07, respectively). (2) The models +/- parotid duct doses were compared and the addition of parotid ductal mean dose significantly improved model fit at 6- and 12-months post CRT (P = 0.030 and 0.047, respectively).The absolute rates of moderate to very severe patient reported xerostomia were approximately 20% lower for those undergoing MRI sialography guided parotid ductal sparing versus those without ductal sparing at 6- and 12-months post CRT. The significant improvement of model fit upon adding mean parotid ductal dose to the mean major salivary gland doses at 6 and 12 months suggests that the dose received by the parotid ducts relates to patient reported xerostomia severity even when considering traditional metrics such as mean parotid and contralateral submandibular dose.

Aging-associated stem/progenitor cell dysfunction in the salivary glands of mice

Although stem cell aging leads to a decline in tissue homeostasis and regenerative capacity, it remains unclear whether salivary gland stem cell function changes during this process. However, the salivary glands are gradually replaced by connective tissue during aging. Here, we show a decline in the stem cell ability of CD133-positive stem/progenitor cells in the salivary glands of aged mice. The CD133-positive cells were isolated from young, adult, and aged mice. The number of CD133-positive cells was significantly decreased in aged mice. They also showed a lower sphere formation capacity compared to young and adult mice. RNA sequencing revealed that CD133-positive cells in aged mice exhibited lower gene expression of several aging-related genes, including FoxO3a, than those in young and adult mice. Salivary gland cells infected with a recombinant lentivirus encoding the FoxO3a gene showed a reduction in oxidative stress induced by hydrogen peroxide compared with those infected with a control virus. Thus, FoxO3a may inhibit stem cell aging via oxidative stress.

OC-0063 Role of microenvironment on the post-irradiation regenerative potential of salivary gland stem cells

OC-0063 Role of microenvironment on the post-irradiation regenerative potential of salivary gland stem cells

A Rho Kinase (ROCK) Inhibitor, Y-27632, Inhibits the Dissociation-Induced Cell Death of Salivary Gland Stem Cells.

Salivary gland stem cells (SGSCs) are potential cell sources for the treatment of salivary gland diseases. The control of cell survival is an essential factor for applying stem cells to regenerative medicine or stem cell-based research. The purpose of this study was to investigate the effects of the ROCK inhibitor Y-27632 on the survival of SGSCs and its underlying mechanisms. SGSCs were isolated from mouse submandibular glands and cultured in suspension. Treatment with Y-27632 restored the viability of SGSCs that was significantly decreased during isolation and the subsequent culture. Y-27632 upregulated the expression of anti-apoptotic protein BCL-2 in SGSCs and, in the apoptosis assay, significantly reduced apoptotic and necrotic cell populations. Matrigel was used to mimic the extracellular environment of an intact salivary gland. The expression of genes regulating apoptosis and the ROCK signaling pathway was significantly reduced when SGSCs were embedded in Matrigel. SGSCs cultured in Matrigel and treated with Y-27632 showed no difference in the total numbers of spheroids and expression levels of apoptosis-regulating genes. Matrigel-embedded SGSCs treated with Y-27632 increased the number of spheroids with budding structures and the expression of acinar cell-specific marker AQP5. We demonstrate the protective effects of Y-27632 against dissociation-induced apoptosis of SGSCs during their culture in vitro.

Abstract IA-020: Optimizing stem cell niche for post-irradiation regeneration

Abstract Radiotherapy involves unavoidable co-irradiation of the normal tissue, often resulting in side effects that limit the dose to the tumor and/or reduce quality of life of cancer survivors. The response of normal tissues to irradiation is mainly determined by the survival and regenerative potential of the tissue stem cells, and modulated by inflammatory processes, vasculature damage, altered neuronal innervation and fibrosis. Indeed, transplantation of tissue specific stem cells has been shown to restores tissue homeostasis and prevent late radiation effects. However, the radiation-induced senescence, fibrosis, and chronic inflammation may chance the stem cell niche engender an unfavorable environment for the regenerative potential of the tissue’s stem cells. Here changes in the salivary gland stem cell niche will be discussed using irradiated murine submandibular salivary gland tissue and derived organoids. Recently, we have developed methods to culture murine and patient specific tissue resembling salivary gland organoids (SGO). SGO contain all the glandular lineages, are able to extensively self-renew and up on transplantation rescue salivary gland function, allowing the study of radiation responses. The role of stemness factors, senescence and inflammatory processes in stem cell self-renewal and differentiation and post-irradiation regeneration will be discussed. Using SGO formation efficiency (OFE) as a measure of regenerative potential it is shown that stem cell potency after in vivo irradiation, is time dependent reduced. Interestingly, the conditioned medium of irradiated SGO reduced the OFE of unirradiated SGO. The potential role of radiation-induced senescence-associated secretory phenotype, hippo-signaling and inflammation through activation of cytosolic DNA sensing pathways on stem cells function will be addressed. Radiation-induced environmental changes in the stem cells niche have a severe impact on stem cell function. Modulation of these effect may enhance regenerative potential of surviving stem cells and improve engraftment and regeneration after stem cell transplantation. Supported by the Dutch Cancer Society (KWF, grant No. 10650 and 12092) and The Netherlands Organisation for Health Research and Development (ZonMw, Grant nrs. 11.600.1023 and 40-43600-98-14003). Citation Format: Rob P. Coppes. Optimizing stem cell niche for post-irradiation regeneration [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-020.

Oral squamous cell carcinoma may originate from bone marrow-derived stem cells.

Molecules that demonstrate a clear association with the aggressiveness of oral squamous cell carcinoma (OSCC) have not yet been identified. The current study hypothesized that tumor cells in OSCC have three different origins: Epithelial stem cells, oral tissue stem cells from the salivary gland and bone marrow (BM) stem cells. It was also hypothesized that carcinomas derived from less-differentiated stem cells have a greater malignancy. In the present study, sex chromosome analysis by fluorescence in situ hybridization and/or microdissection PCR was performed in patients with OSCC that developed after hematopoietic stem cell transplantation (HSCT) from the opposite sex. OSCC from 3 male patients among the 6 total transplanted patients were considered to originate from donor-derived BM cells. A total of 2/3 patients had distant metastasis, resulting in a poor prognosis. In a female patient with oral potentially malignant disorder who underwent HSCT, there were 10.7% Y-containing cells in epithelial cells, suggesting that some epithelial cells were from the donor. Subsequently, gene expression patterns in patients with possible BM stem cell-derived OSCC were compared with those in patients with normally developed OSCC by microarray analysis. A total of 3 patients with BM stem cell-derived OSCC exhibited a specific pattern of gene expression. Following cluster analysis by the probes identified on BM stem cell-derived OSCC, 2 patients with normally developed OSCC were included in the cluster of BM stem cell-derived OSCC. If the genes that could discriminate the origin of OSCC were identified, OSCCs were classified into the three aforementioned categories. If diagnosis can be performed based on the origin of the cancer cells, a more specific therapeutic strategy may be implemented to improve prognosis. This would be a paradigm shift in diagnostic and therapeutic strategies for OSCC.

Mouse parotid salivary gland organoids for the in vitro study of stem cell radiation response.

ObjectiveHyposalivation‐related xerostomia is an irreversible, untreatable, and frequent condition after radiotherapy for head and neck cancer. Stem cell therapy is an attractive option of treatment, but demands knowledge of stem cell functioning. Therefore, we aimed to develop a murine parotid gland organoid model to explore radiation response of stem cells in vitro.Materials and MethodsSingle cells derived from murine parotid gland organoids were passaged in Matrigel with defined medium to assess self‐renewal and differentiation potential. Single cells were irradiated and plated in a 3D clonogenic stem cell survival assay to assess submandibular and parotid gland radiation response.ResultsSingle cells derived from parotid gland organoids were able to extensively self‐renew and differentiate into all major tissue cell types, indicating the presence of potential stem cells. FACS selection for known salivary gland stem cell markers CD24/CD29 did not further enrich for stem cells. The parotid gland organoid‐derived stem cells displayed radiation dose–response curves similar to the submandibular gland.ConclusionsMurine parotid gland organoids harbor stem cells with long‐term expansion and differentiation potential. This model is useful for mechanistic studies of stem cell radiation response and suggests similar radiosensitivity for the parotid and submandibular gland organoids.

The secretory senescence in otorhinolaryngology: principles of treatment

Atrophy or hypofunction of the salivary gland because of aging, radiotherapy or disease causes hyposalivation and impairs the quality of life of patients by compromising mastication, swallowing and speech and by leading to a loss of taste. Moreover, hyposalivation exacerbates dental caries and induces periodontal disease, and oral candidiasis. Currently, no satisfactory therapies have been established to solve salivary hypofunction. Current treatment options for atrophy or hypofunction of the salivary glands in clinical practice are only symptomatic and include saliva substitutes and parasympathetic agonists, such as pilocarpine, to stimulate salivary flow. However, parasympathomimetics have systemic side effects, so different treatment options are necessary, and research has recently focused on this. The main strategies that have been proposed to restore salivary gland atrophy and hypofunction are gene therapy by gene activation/silencing during stem cell differentiation and by the use of viral vectors, such as adenoviruses; cell-based therapy with salivary gland cells, stem cells and non-salivary gland and/ or non-epithelial cells to regenerate damaged salivary gland cells; replacement with tissue bioengineering in which organoids from pluripotent stem cells are used in the development of organ replacement regenerative therapy. Remarkable progression in this research field has been made in the last decade, but a definitive therapy for salivary gland hypofunction has not been developed due to intrinsic challenges that come with each approach. However, with research efforts in the future, a range of precision medicine therapies may become available individualized to each patient.

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.

Labial Stem Cell Extract Mitigates Injury to Irradiated Salivary Glands

Stem cell–based therapies could provide a permanent treatment for salivary gland (SG) hypofunction caused by ionizing radiation (IR) injury. However, current challenges for SG stem cells to reach the clinic include surgical invasiveness, amount of tissue needed, cell delivery, and storage methods. The objective of this study was to develop a clinically less invasive method to isolate and expand human SG stem cells and then to obtain a cell-free extract to be used as a therapy for IR-injured SGs. Human labial glands were biopsied, and labial stem cells (LSCs) were expanded by explant culture. The LSC extract (LSCE) was obtained by releasing the cellular components after 3 freeze-thaw cycles and 17,000g force centrifugation. LSCE was injected intravenously into mice that had their SGs injured with 13-Gy IR. Positive (non-IR) and negative (IR) control mice received injections of saline (vehicle control). Three pieces of labial glands (0.1 g weight) could expand 1 to 2 million cells. LSCs had a doubling time of 18.8 h; could differentiate into osteocytes, adipocytes, and chondrocytes; and were positive for mesenchymal stem cell markers. Both angiogenic (FGF-1, FGF-2, KGF, angiopoietin, uPA, VEGF) and antiangiogenic factors (PAI-1, TIMP-1, TSP-1, CD26) were detected in LSCE. In addition, some angiogenic factors (PEDF, PTX3, VEGF) possessed neurotrophic functions. Mice treated with LSCE had 50% to 60% higher salivary flow rate than saline-treated mice at 8 and 12 wk post-IR. Saliva lag time measurements also confirmed that LSCE restored SG function. Histologic analyses of parotids and submandibular glands reported comparable numbers of acinar cells, blood vessels, and parasympathetic nerves and cell proliferation rates in sham IR and LSCE-treated mice, though significantly lower in saline-treated mice. An explant culture method can harvest a large number of LSCs from small pieces of labial glands. LSCE showed clinical potential to mitigate IR-injured SGs.

Concise Review: A Critical Evaluation of Criteria Used to Define Salivary Gland Stem Cells.

In the effort to develop cell-based therapies to treat salivary gland dysfunction, many different populations of cells in the adult salivary glands have been proposed as stem cells. These cell populations vary, depending on the assay used, and are often nonoverlapping, leading to the conclusion that salivary glands harbor multiple stem cells. The goal of this review is to critically appraise the assays and properties used to identify stem cells in the adult salivary gland, and to consider the caveats of each. Re-evaluation of the defining criteria may help to reconcile the many potential stem cell populations described in the salivary gland, in order to increase comparability between studies and build consensus in the field. Stem Cells 2019;37:1144-1150.

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.

Three-Dimensional Culture of Salivary Gland Stem Cell in Orthotropic Decellularized Extracellular Matrix Hydrogels.

Radiotherapy in patients with cancer can kill cancer cells but also damage normal cells or tissues. During the treatment of patients with head and neck cancer or thyroid cancer, hyposalivation is a representative chronic side effect of radio-damaged salivary glands (SGs). The major symptom of hyposalivation is mouth dryness, resulting in several subsequent long-term complications. No effective therapeutic approaches have been developed to manage this symptom. In this study, we developed the first rat SG tissue-derived decellularized extracellular matrix hydrogel (DSGM-hydrogel) as a functional orthotropic bioscaffold for future efficient SG stem cell therapy. DSGM-hydrogels were characterized by rheological or biochemical analyses, and rat SG stem/progenitor cells (rSGSCs) were then subjected to three-dimensional culture in the DSGM-hydrogels. Interestingly, DSGM-hydrogel-embedded rSGSCs survived and expressed SG functional differentiation marker of amylase IA and increased enzyme activity of α-amylase in protein level, whereas they showed reduced levels of adult ductal stem/progenitor markers, including c-Kit, c-Met, and CD44. Furthermore, the expression levels of basic epithelial tight junction markers were recovered to levels similar to those naked SG tissues after culture in DSGM-hydrogels in transcription level. Therefore, our findings suggested that the DSGM-hydrogels could provide an appropriate microenvironment for stem/progenitor cell survival and a source of SG cytodifferentiation. This approach could be an applicable method to SG stem cell research as a potential source for an organoid and for clinical regenerative reagents to manage radio-damaged SGs in vivo. Impact Statement In this study, we established the first rat salivary gland (SG) tissue-derived decellularized extracellular matrix hydrogel (DSGM-hydrogel) and assessed the role of this hydrogel as a functional orthotropic bioscaffold. Our findings provide important insights into the applications of the DSGM-hydrogel as a biocompatible matrix for regenerative therapy of radio-damaged SGs.

Salivary Gland Stem Cells and Tissue Regeneration: An Update on Possible Therapeutic Application

The aim of this review is to combine literature and experimental data concerning the impact of salivary gland (SG) stem cells (SCs) and their therapeutic prospects in tissue regeneration. So far, SCs were isolated from human and rodent major and minor SGs that enabled their regeneration. Several scaffolds were also combined with "SCs" and different "proteins" to achieve guided differentiation, although none have been proven as ideal. A new aspect of SC therapy aims to establish a vice versa relationship between SG and other ecto- or endodermal organs such as the pancreas, liver, kidneys, and thyroid. SC therapy could be a cheap and simple, non-traumatic, and individualized therapy for medically challenging cases like xerostomia and major organ failures. Functional improvement has been achieved in these organs, but till date, the whole organ in vivo regeneration was not achieved. Concerns about malignant formations and possible failures are yet to be resolved. In this review article, we highlight the basic embryology of SGs, existence of SG SCs with a detailed exploration of various cellular markers, scaffolds for tissue engineering, and, in the later part, cover potential therapeutic applications with a special focus on the pancreas and liver. Keywords: Salivary gland stem cells, Stem cell therapy, Tissue regeneration.

The effect of salivary gland-derived stem cell transplantation on the regeneration of gamma-irradiated rat submandibular salivary glands: an immunohistochemical study

Background Hyposalivation could be a sequela of radiation impairment in patients with head and neck cancer. Regenerative approaches based on the reactivation of endogenous stem cells or the transplant of exogenous stem cells hold substantial promise in restoring the structure and function of these organs to improve patient quality of life. Recently, tissue-specific stem cell therapy has attracted public attention as a next-generation therapeutic reagent. The aim of this work is to assess the regenerative potential of salivary gland-derived stem cells transplantation in gamma-irradiated rat submandibular salivary glands (SMSGs). Materials and methods Forty-six adult male albino rats were used in this study. Both SMSGs were harvested from five healthy donor rats and used as a source of stem cells. Cells were cultured for 3 and 10 days. Characterization and assessment of stemness after isolation by flow cytometry was carried out using CD24 stem cell marker by fluorescent analysis cell sorting. The rats were grouped as follows: group I (normal untreated control), group II (irradiation group), and group III (irradiation and transplantation group). They were subjected to whole-body gamma radiation with a single dose of 6 Gy. Results Stem cells were successfully isolated from rat SMSGs with positively expressing CD24 and C-kit markers. The statistical analysis revealed significant increase in cell proliferation (proliferating cell nuclear antigen) in both groups II and III compared with the control group I (P˂0.05). Regarding caspase 3 results, the statistical analysis revealed highly significant increase in the mean values of groups II and subgroups III compared with the control group I (P˂0.05). Concerning C-kit expression, group III showed the highest statistically significant mean C-kit expression, followed by group II and group I (P˃0.05). At 2 weeks after transplantation, group II showed the highest statistically significant mean C-kit expression followed by III and control group I (P˃0.05). Conclusion Transplantation of these C-kit+ submandibular salivary gland stem cell (SMSGSCs) could result in amelioration of the severely reduced quality of life of surviving patients with cancer.

Salivary Gland Stem Cells Age Prematurely in Primary Sjögren's Syndrome.

ObjectiveA major characteristic of the autoimmune disease primary Sjögren's syndrome (SS) is salivary gland (SG) hypofunction. The inability of resident SG stem cells (SGSCs) to maintain homeostasis and saliva production has never been explained and limits our comprehension of mechanisms underlying primary SS. The present study was undertaken to investigate the role of salivary gland stem cells in hyposalivation in primary SS.Methods SGSCs were isolated from parotid biopsy samples from controls and patients classified as having primary SS or incomplete primary SS, according to the American College of Rheumatology/European League Against Rheumatism criteria. Self‐renewal and differentiation assays were used to determine SGSC regenerative potential, RNA was extracted for sequencing analysis, single telomere length analysis was conducted to determine telomere length, and frozen tissue samples were used for immunohistochemical analysis.Results SGSCs isolated from primary SS parotid gland biopsy samples were regeneratively inferior to healthy control specimens. We demonstrated that SGSCs from samples from patients with primary SS are not only lower in number and less able to differentiate, but are likely to be senescent, as revealed by telomere length analysis, RNA sequencing, and immunostaining. We further found that SGSCs exposed to primary SS–associated proinflammatory cytokines we induced to proliferate, express senescence‐associated genes, and subsequently differentiate into intercalated duct cells. We also localized p16+ senescent cells to the intercalated ducts in primary SS SG tissue, suggesting a block in SGSC differentiation into acinar cells.ConclusionThis study represents the first characterization of SGSCs in primary SS, and also the first demonstration of a linkage between an autoimmune disease and a parenchymal premature‐aging phenotype. The knowledge garnered in this study indicates that disease‐modifying antirheumatic drugs used to treat primary SS are not likely to restore saliva production, and should be supplemented with fresh SGSCs to recover saliva production.

Salivary gland cell aggregates are derived from self-organization of acinar lineage cells

Objective: The objective of this study was to characterize the mechanism by which salivary gland cells (SGC) aggregate in vitro.Design: Timelapse microscopy was utilized to analyze the process of salivary gland aggregate formation using both primary murine and human salivary gland cells. The role of cell density, proliferation, extracellular calcium, and secretory acinar cells in aggregate formation was investigated. Finally, the ability of cells isolated from irradiated glands to form aggregates was also evaluated.Results: Salivary gland cell self-organization rather than proliferation was the predominant mechanism of aggregate formation in both primary mouse and human salivary gland cultures. Aggregation was found to require extracellular calcium while acinar lineage cells account for ∼80% of the total aggregate cell population. Finally, aggregation was not impaired by irradiation.Conclusions: The data reveal that aggregation occurs as a result of heterogeneous salivary gland cell self-organization rather than from stem cell proliferation and differentiation, contradicting previous dogma. These results suggest a re-evaluation of aggregate formation as a criterion defining salivary gland stem cells.

Organotypic 3D Culture in Nanoscaffold Microwells Supports Salivary Gland Stem-Cell-Based Organization.

The self-organizing properties of stem cells have been exploited to generate organoids, organ-specific, cell-containing, three-dimensional (3D) structures. The present study aimed to introduce a novel bioengineering technique for driving the effective organization of adult tissue stem cells via niche-independent 3D microwell culture. Microwells were fabricated by photopatterning poly(ethylene glycol) hydrogel in the presence of an electrospun polycaprolactone nanofibrous scaffold. Human single clonal salivary gland stem cells (SGSCs) were cultured in nanofibrous microwells through two simple steps, priming and differentiation. Before the induction of 3D organization, single clonal SGSCs were preconditioned to aggregate to form 3D spheroids in different matrices, such as Matrigel, floating dish, and microwells. Expression of salivary stem cell markers and pluripotency markers was greater in 3D spheroid cultures than in 2D plastic culture. Lobular structures were organized by changing media, and those in microwells exhibited higher salivary acinar, ductal, and tight junction marker levels and decreased stem-cell marker levels relative to other 3D cultures. Furthermore, higher α-amylase secretion and intracellular calcium levels were observed in the presence of adrenergic or cholinergic agonists, respectively, along with an increased intracellular pH, suggesting more functional salivary organoid formation. These microwell-driven organoids also engrafted successfully into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Our results showed that microwell-cultured SGSCs organize into salivary structures and that this biomimetic 3D culture technique can promote effective generation of niche-independent single stem-cell-based 3D organoids.

C-Kit+ Cells in Adult Salivary Glands do not Function as Tissue Stem Cells

A rare population of salivary gland cells isolated based on c-Kit immunoreactivity are thought to represent tissue stem cells since they exhibit the most robust proliferative and differentiation capacity ex vivo. Despite their high promise for cell-based therapies aimed at restoring salivary function, the precise location and in vivo function of c-Kit+ stem cells remain unclear. Here, by combining immunostaining with c-KitCreERT2-based genetic labeling and lineage tracing in the adult mouse salivary glands, we show that c-Kit is expressed in a relatively large and heterogeneous cell population that consists mostly of differentiated cells. Moreover, c-Kit does not mark ductal stem cells that are known to express cytokeratin K14. Tracking the fate of in vivo-labeled c-Kit+ or that of K14+ cells in spheroid cultures reveals a limited proliferative potential for c-Kit+ cells and identifies K14+ cells as the major source of salispheres in these cultures. Long-term in vivo lineage tracing studies indicate that although c-Kit marks at least two discrete ductal cell lineages, c-Kit+ cells do not contribute to the normal maintenance of any other cell lineages. Our results indicate that c-Kit is not a reliable marker for salivary gland stem cells, which has important implications for salivary gland regenerative therapies.

Salivary gland stem cells: A review of development, regeneration and cancer.

Salivary glands are responsible for maintaining the health of the oral cavity and are routinely damaged by therapeutic radiation for head and neck cancer as well as by autoimmune diseases such as Sjögren's syndrome. Regenerative approaches based on the reactivation of endogenous stem cells or the transplant of exogenous stem cells hold substantial promise in restoring the structure and function of these organs to improve patient quality of life. However, these approaches have been hampered by a lack of knowledge on the identity of salivary stem cell populations and their regulators. In this review we discuss our current knowledge on salivary stem cells and their regulators during organ development, homeostasis and regeneration. As increasing evidence in other systems suggests that progenitor cells may be a source of cancer, we also review whether these same salivary stem cells may also be cancer initiating cells.