The Mayo Clinic Salivary Tissue-Organoid Biobanking: A Resource for Salivary Regeneration Research.

The salivary gland (SG) is an essential organ that secretes saliva, which supports versatile oral function throughout life, and is maintained by elusive epithelial stem and progenitor cells (SGSPC). Unfortunately, aging, drugs, autoimmune disorders, and cancer treatments can lead to salivary dysfunction and associated health consequences. Despite many ongoing therapeutic efforts to mediate those conditions, investigating human SGSPC is challenging due to lack of standardized tissue collection, limited tissue access, and inadequate purification methods. Herein, we established a diverse and clinically annotated salivary regenerative biobanking at the Mayo Clinic, optimizing viable salivary cell isolation and clonal assays in both 2D and 3D-matrigel growth environments. Our analysis identified ductal epithelial cells in vitro enriched with SGSPC expressing the CD24/EpCAM/CD49f+ and PSMA- phenotype. We identified PSMA expression as a reliable SGSPC differentiation marker. Moreover, we identified progenitor cell types with shared phenotypes exhibiting three distinct clonal patterns of salivary differentiation in a 2D environment. Leveraging innovative label-free unbiased LC-MS/MS-based single-cell proteomics, we identified 819 proteins across 71 single cell proteome datasets from purified progenitor-enriched parotid gland (PG) and sub-mandibular gland (SMG) cultures. We identified distinctive co-expression of proteins, such as KRT1/5/13/14/15/17/23/76 and 79, exclusively observed in rare, scattered salivary ductal basal cells, indicating the potential de novo source of SGSPC. We also identified an entire class of peroxiredoxin peroxidases, enriched in PG than SMG, and attendant H2O2-dependent cell proliferation in vitro suggesting a potential role for PRDX-dependent floodgate oxidative signaling in salivary homeostasis. The distinctive clinical resources and research insights presented here offer a foundation for exploring personalized regenerative medicine.

Neurotrophin signaling is a central mechanism of salivary dysfunction after irradiation that disrupts myoepithelial cells.

The mechanisms that prevent regeneration of irradiated (IR) salivary glands remain elusive. Bulk RNAseq of IR versus non-IR human salivary glands showed that neurotrophin signaling is highly disrupted post-radiation. Neurotrophin receptors (NTRs) were significantly upregulated in myoepithelial cells (MECs) post-IR, and single cell RNAseq revealed that MECs pericytes, and duct cells are the main sources of neurotrophin ligands. Using two ex vivo models, we show that nerve growth factor (NGF) induces expression of MEC genes during development, and upregulation of NTRs in adult MECs is associated with stress-induced plasticity and morphological abnormalities in IR human glands. As MECs are epithelial progenitors after gland damage and are required for proper acinar cell contraction and secretion, we propose that MEC-specific upregulation of NTRs post-IR disrupts MEC differentiation and potentially impedes the ability of the gland to regenerate.

Characterization of Transgenic NSG-SGM3 Mouse Model of Precision Radiation-Induced Chronic Hyposalivation.

Regenerative medicine holds promise to cure radiation-induced salivary hypofunction, a chronic side effect in patients with head and neck cancers, therefore reliable preclinical models for salivary regenerative outcome will promote progress towards therapies. In this study, our objective was to develop a cone beam computed tomography-guided precision ionizing radiation-induced preclinical model of chronic hyposalivation using immunodeficient NSGSGM3 mice. Using a Schirmer's test based sialagogue-stimulated saliva flow kinetic measurement method, we demonstrated significant differences in hyposalivation specific to age, sex, precision-radiation dose over a chronic (6 months) timeline. NSG-SMG3 mice tolerated doses from 2.5 Gy up to 7.5 Gy. Interestingly, 5-7.5 Gy had similar effects on stimulated-saliva flow (∼50% reduction in young female at 6 months after precision irradiation over sham-treated controls), however, >5 Gy led to chronic alopecia. Different groups demonstrated characteristic saliva fluctuations early on, but after 5 months all groups nearly stabilized stimulated-saliva flow with low-inter-mouse variation within each group. Further characterization revealed precision-radiation-induced glandular shrinkage, hypocellularization, gland-specific loss of functional acinar and glandular cells in all major salivary glands replicating features of human salivary hypofunction. This model will aid investigation of human cell-based salivary regenerative therapies.

Changes in salivary biomarkers associated with periodontitis and diabetic neuropathy in individuals with type 1 diabetes.

The objective of this pilot clinical study was to identify salivary biomarkers that are associated with periodontal disease and measures of diabetic autonomic dysfunction. Saliva samples from 32 participants were obtained from 3 groups: healthy (H), type 1 diabetes mellitus (DM), and type 1 diabetes mellitus with neuropathy (DMN). Based on the periodontal examination, individuals' mean Periodontal Screening and Recording scores were categorized into two groups (periodontally healthy and gingivitis), and correlated to specific salivary inflammatory biomarkers assessed by a customized protein array and enzyme assay. The mean salivary IgA level in DM was 9211.5 ± 4776.4 pg/ml, which was significantly lower than H (17,182.2 ± 8899.3 pg/ml). IgA in DMN with healthy periodontium was significantly lower (5905.5 ± 3124.8 pg/ml) compared to H, although IgA levels in DMN patients with gingivitis (16,894. 6 ± 7084.3) were not. According to the result of a logistic regression model, IgA and periodontal condition were the indicators of the binary response given by H versus DM, and H versus DMN, respectively. These data suggest that selected salivary biomarkers, such as IgA, combined with a periodontal examination prior to obtaining salivary samples can offer a non-invasive method to assess risk for developing diabetic neuropathy.

Neuronal-epithelial cross-talk drives acinar specification via NRG1-ERBB3-mTORC2 signaling.

Acinar cells are the principal secretory units of multiple exocrine organs. A single-cell, layered, lumenized acinus forms from a large cohort of epithelial progenitors that must initiate and coordinate three cellular programs of acinar specification, namely, lineage progression, secretion, and polarization. Despite this well-known outcome, the mechanism(s) that regulate these complex programs are unknown. Here, we demonstrate that neuronal-epithelial cross-talk drives acinar specification through neuregulin (NRG1)-ERBB3-mTORC2 signaling. Using single-cell and global RNA sequencing of developing murine salivary glands, we identified NRG1-ERBB3 to precisely overlap with acinar specification during gland development. Genetic deletion of Erbb3 prevented cell lineage progression and the establishment of lumenized, secretory acini. Conversely, NRG1 treatment of isolated epithelia was sufficient to recapitulate the development of secretory acini. Mechanistically, we found that NRG1-ERBB3 regulates each developmental program through an mTORC2 signaling pathway. Thus, we reveal that a neuronal-epithelial (NRG1/ERBB3/mTORC2) mechanism orchestrates the creation of functional acini.

Long-term functional regeneration of radiation-damaged salivary glands through delivery of a neurogenic hydrogel.

Salivary gland acinar cells are severely depleted after radiotherapy for head and neck cancer, leading to loss of saliva and extensive oro-digestive complications. With no regenerative therapies available, organ dysfunction is irreversible. Here, using the adult murine system, we demonstrate that radiation-damaged salivary glands can be functionally regenerated via sustained delivery of the neurogenic muscarinic receptor agonist cevimeline. We show that endogenous gland repair coincides with increased nerve activity and acinar cell division that is limited to the first week after radiation, with extensive acinar cell degeneration, dysfunction, and cholinergic denervation occurring thereafter. However, we found that mimicking cholinergic muscarinic input via sustained local delivery of a cevimeline-alginate hydrogel was sufficient to regenerate innervated acini and retain physiological saliva secretion at nonirradiated levels over the long term (>3 months). Thus, we reveal a previously unknown regenerative approach for restoring epithelial organ structure and function that has extensive implications for human patients.

Immunosuppressed Miniswine as a Model for Testing Cell Therapy Success: Experience With Implants of Human Salivary Stem/Progenitor Cell Constructs.

An urgent need exists to develop large animal models for preclinical testing of new cell therapies designed to replace lost or damaged tissues. Patients receiving irradiation for treatment of head and neck cancers frequently develop xerostomia/dry mouth, a condition that could one day be treated by cell therapy to repopulate functional saliva-producing cells. Using immunosuppression protocols developed for patients receiving whole face transplants, we successfully used immunosuppressed miniswine as a suitable host animal to evaluate the long-term stability, biocompatibility, and fate of matrix-modified hyaluronate (HA) hydrogel/bioscaffold materials containing encapsulated salivary human stem/progenitor cells (hS/PCs). An initial biocompatibility test was conducted in parotids of untreated miniswine. Subsequent experiments using hS/PC-laden hydrogels were performed in animals, beginning an immunosuppression regimen on the day of surgery. Implant sites included the kidney capsule for viability testing and the parotid gland for biointegration time periods up to eight weeks. No transplant rejection was seen in any animal assessed by analysis of the tissues near the site of the implants. First-generation implants containing only cells in hydrogel proved difficult to handle in the surgical suite and were modified to adhere to a porcine small intestinal submucosa (SIS) membrane for improved handling and could be delivered through the da Vinci surgical system. Several different surgical techniques were assessed using the second-generation 3D-salivary tissue (3D-ST) for ease and stability both on the kidney capsule and in the capsule-less parotid gland. For the kidney, sliding the implant under the capsule membrane and quick stitching proved superior to other methods. For the parotid gland, creation of a tissue "pocket" for placement and immediate multilayer tissue closure were well tolerated with minimal tissue damage. Surgical clips were placed as fiduciary markers for tissue harvest. Some implant experiments were conducted with miniswine 90 days post-irradiation when salivation decreased significantly. Sufficient parotid tissue remained to allow implant placement, and animals tolerated immunosuppression. In all experiments, viability of implanted hS/PCs was high with clear signs of both vascular and nervous system integration in the parotid implants. We thus conclude that the immunosuppressed miniswine is a high-value emerging model for testing human implants prior to first-in-human trials.

Hybrid Three-Dimensional-Printed Ear Tissue Scaffold With Autologous Cartilage Mitigates Soft Tissue Complications.

OBJECTIVES/HYPOTHESIS: To analyze the use of highly translatable three-dimensional (3D)-printed auricular scaffolds with and without novel cartilage tissue inserts in a rodent model. STUDY DESIGN: Preclinical rodent animal model. METHODS: This prospective study assessed a single-stage 3D-printed auricular bioscaffold with or without porcine cartilage tissue inserts in an athymic rodent model. Digital Imaging and Communications in Medicine computed tomography images of a human auricle were segmented to create an external anatomic envelope filled with orthogonally interconnected spherical pores. Scaffolds with and without tissue inset sites were 3D printed by laser sintering bioresorbable polycaprolactone, then implanted subcutaneously in five rats for each group. RESULTS: Ten athymic rats were studied to a goal of 24 weeks postoperatively. Precise anatomic similarity and scaffold integrity were maintained in both scaffold conditions throughout experimentation with grossly visible tissue ingrowth and angiogenesis upon explantation. Cartilage-seeded scaffolds had relatively lower rates of nonsurgical site complications compared to unseeded scaffolds with relatively increased surgical site ulceration, though neither met statistical significance. Histology revealed robust soft tissue infiltration and vascularization in both seeded and unseeded scaffolds, and demonstrated impressive maintenance of viable cartilage in cartilage-seeded scaffolds. Radiology confirmed soft tissue infiltration in all scaffolds, and biomechanical modeling suggested amelioration of stress in scaffolds implanted with cartilage. CONCLUSIONS: A hybrid approach incorporating cartilage insets into 3D-printed bioscaffolds suggests enhanced clinical and histological outcomes. These data demonstrate the potential to integrate point-of-care tissue engineering techniques into 3D printing to generate alternatives to current reconstructive surgery techniques and avoid the demands of traditional tissue engineering. LEVEL OF EVIDENCE: NA Laryngoscope, 131:1008-1015, 2021.

Sestrins regulate muscle stem cell metabolic homeostasis.

The health and homeostasis of skeletal muscle are preserved by a population of tissue-resident muscle stem cells (MuSCs) that maintain a state of mitotic and metabolic quiescence in adult tissues. The capacity of MuSCs to preserve the quiescent state declines with aging and metabolic insults, promoting premature activation and stem cell exhaustion. Sestrins are a class of stress-inducible proteins that act as antioxidants and inhibit the activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling complex. Despite these pivotal roles, the role of Sestrins has not been explored in adult stem cells. We show that SESTRIN1,2 loss results in hyperactivation of the mTORC1 complex, increased propensity to enter the cell cycle, and shifts in metabolic flux. Aged SESTRIN1,2 knockout mice exhibited loss of MuSCs and a reduced ability to regenerate injured muscle. These findings demonstrate that Sestrins help maintain metabolic pathways in MuSCs that protect quiescence against aging.

Microengineered 3D pulmonary interstitial mimetics highlight a critical role for matrix degradation in myofibroblast differentiation.

Fibrosis, characterized by aberrant tissue scarring from activated myofibroblasts, is often untreatable. Although the extracellular matrix becomes increasingly stiff and fibrous during disease progression, how these physical cues affect myofibroblast differentiation in 3D is poorly understood. Here, we describe a multicomponent hydrogel that recapitulates the 3D fibrous structure of interstitial tissue regions where idiopathic pulmonary fibrosis (IPF) initiates. In contrast to findings on 2D hydrogels, myofibroblast differentiation in 3D was inversely correlated with hydrogel stiffness but positively correlated with matrix fibers. Using a multistep bioinformatics analysis of IPF patient transcriptomes and in vitro pharmacologic screening, we identify matrix metalloproteinase activity to be essential for 3D but not 2D myofibroblast differentiation. Given our observation that compliant degradable 3D matrices amply support fibrogenesis, these studies demonstrate a departure from the established relationship between stiffness and myofibroblast differentiation in 2D, and provide a new 3D model for studying fibrosis and identifying antifibrotic therapeutics.

CERE-120 Prevents Irradiation-Induced Hypofunction and Restores Immune Homeostasis in Porcine Salivary Glands.

Salivary gland hypofunction causes significant morbidity and loss of quality of life for head and neck cancer patients treated with radiotherapy. Preventing hypofunction is an unmet therapeutic need. We used an adeno-associated virus serotype 2 (AAV2) vector expressing the human neurotrophic factor neurturin (CERE-120) to treat murine submandibular glands either pre- or post-irradiation (IR). Treatment with CERE-120 pre-IR, not post-IR, prevented hypofunction. RNA sequencing (RNA-seq) analysis showed reduced gene expression associated with fibrosis and the innate and humoral immune responses. We then used a minipig model with CERE-120 treatment pre-IR and also compared outcomes of the contralateral non-IR gland. Analysis of gene expression, morphology, and immunostaining showed reduced IR-related immune responses and improved secretory mechanisms. CERE-120 prevented IR-induced hypofunction and restored immune homeostasis, and there was a coordinated contralateral gland response to either damage or treatment. CERE-120 gene therapy is a potential treatment for head and neck cancer patients to influence communication among neuronal, immune, and epithelial cells to prevent IR-induced salivary hypofunction and restore immune homeostasis.

Keratinocyte growth factor prevents radiation damage to salivary glands by expansion of the stem/progenitor pool.

Irradiation of salivary glands during radiotherapy treatment of patients with head and neck cancer evokes persistent hyposalivation. This results from depletion of stem cells, which renders the gland incapable of replenishing saliva to produce acinar cells. The aim of this study was to investigate whether it is possible to expand the salivary gland stem/progenitor cell population, thereby preventing acinar cell depletion and subsequent gland dysfunction after irradiation. To induce cell proliferation, keratinocyte growth factor (DeltaN23-KGF, palifermin) was administered to C57BL/6 mice for 4 days before and/or after local irradiation of salivary glands. Salivary gland vitality was quantified by in vivo saliva flow rates, morphological measurements, and a newly developed in vitro salisphere progenitor/stem cell assay. Irradiation of salivary glands led to a pronounced reduction in the stem cells of the tissues, resulting in severe hyposalivation and a reduced number of acinar cells. DeltaN23-KGF treatment for 4 days before irradiation indeed induced salivary gland stem/progenitor cell proliferation, increasing the stem and progenitor cell pool. This did not change the relative radiation sensitivity of the stem/progenitor cells, but, as a consequence, an absolute higher number of stem/progenitor cells and acinar cells survived after radiation. Postirradiation treatment with DeltaN23-KGF also improved gland function, and this effect was much more pronounced in DeltaN23-KGF pretreated animals. Post-treatment with DeltaN23-KGF seemed to act through accelerated expansion of the pool of progenitor/stem cells that survived the irradiation treatment. Overall, our data indicate that DeltaN23-KGF is a promising drug to enhance the number of salivary gland progenitor/stem cells and consequently prevent radiation-induced hyposalivation. Disclosure of potential conflicts of interest is found at the end of this article.

Cytokine treatment improves parenchymal and vascular damage of salivary glands after irradiation.

PURPOSE: During radiotherapy for head and neck cancer, co-irradiation (IR) of salivary glands results in acute and often lifelong hyposalivation. Recently, we showed that bone marrow-derived cells (BMC) can partially facilitate postradiation regeneration of the mouse submandibular gland. In this study, we investigate whether optimized mobilization of BMCs can further facilitate regeneration of radiation-damaged salivary glands. EXPERIMENTAL DESIGN: Salivary glands of mice reconstituted with eGFP+ bone marrow cells were irradiated with a single dose of 15 Gy. One month later, BMCs were mobilized using granulocyte colony-stimulating factor (G-CSF) or the combination of FMS-like tyrosine kinase-3 ligand, stem cell factor, and G-CSF (termed F/S/G) as mobilizing agents. Salivary gland function and morphology were evaluated at 90 days post-IR by measuring the saliva flow rate, the number of acinar cells, and the functionality of the vasculature. RESULTS: Compared with G-CSF alone, the combined F/S/G treatment mobilized a 10-fold higher number and different types of BMCs to the bloodstream and increased the number of eGFP+ cells in the irradiated submandibular gland from 49% to 65%. Both treatments reduced radiation-induced hyposalivation from almost nothing in the untreated group to approximately 20% of normal amount. Surprisingly, however, F/S/G treatment resulted in significant less damage to submandibular blood vessels and induced BMC-derived neovascularization. CONCLUSIONS: Post-IR F/S/G treatment facilitates regeneration of the submandibular gland and ameliorates vascular damage. The latter is partly due to BMCs differentiating in vascular cells but is likely to also result from direct stimulation of existing blood vessel cells.

3D Organoid Formation from the Murine Salivary Gland Cell Line SIMS.

Salivary glands consist of multiple phenotypically and functionally unique cell populations, such as the acinar, ductal, and myoepithelial cells that help produce, modify, and secrete saliva (Lombaert et al., 2011). Identification of mechanisms and factors that regulate these populations has been of key interest, as salivary gland-related diseases have detrimental effects on these cell populations. A variety of approaches have been used to understand the roles different signaling mechanisms and transcription factors play in regulating salivary gland development and homeostasis. Differentiation assays have been performed with primary salivary cells in the past (Maimets et al., 2016), however this approach may sometimes be limiting due to tissue availability, labor intensity of processing the tissue samples, and/or inability to long-term passage the cells. Here we describe in detail a 3D differentiation assay to analyze the differentiation potential of a salivary gland cell line, SIMS, which was immortalized from an adult mouse submandibular salivary gland (Laoide et al., 1996). SIMS cells express cytokeratin 7 and 19, which is characteristic for a ductal cell type. Although adult acinar and myoepithelial cells were found in vivo to preserve their own cell population through self-duplication (Aure et al., 2015; Song et al. 2018), in some cases duct cells can differentiate into acinar cells in vivo, such as after radiation injury (Lombaert et al., 2008; Weng et al., 2018). Thus, utilization of SIMS cells allows us to target and analyze the self-renewal and differentiation effects of ductal cells under specific in vitro controlled conditions.

Sox10 Regulates Plasticity of Epithelial Progenitors toward Secretory Units of Exocrine Glands.

Understanding how epithelial progenitors within exocrine glands establish specific cell lineages and form complex functional secretory units is vital for organ regeneration. Here we identify the transcription factor Sox10 as essential for both the maintenance and differentiation of epithelial KIT+FGFR2b+ progenitors into secretory units, containing acinar, myoepithelial, and intercalated duct cells. The KIT/FGFR2b-Sox10 axis marks the earliest multi-potent and tissue-specific progenitors of exocrine glands. Genetic deletion of epithelial Sox10 leads to loss of secretory units, which reduces organ size and function, but the ductal tree is retained. Intriguingly, the remaining duct progenitors do not compensate for loss of Sox10 and lack plasticity to properly form secretory units. However, overexpression of Sox10 in these ductal progenitors enhances their plasticity toward KIT+ progenitors and induces differentiation into secretory units. Therefore, Sox10 controls plasticity and multi-potency of epithelial KIT+ cells in secretory organs, such as mammary, lacrimal, and salivary glands.

Serial patient-derived orthotopic xenografting of adenoid cystic carcinomas recapitulates stable expression of phenotypic alterations and innervation.

BACKGROUND: Patient-derived xenograft (PDX) models have significantly enhanced cancer research, and often serve as a robust model. However, enhanced growth rate and altered pathological phenotype with serial passages have repeatedly been shown in adenoid cystic carcinoma (ACC) PDX tumors, which is a major concern. METHODS: We evaluated the fidelity of ACCs in their natural habitat by performing ACC orthotopic xenotransplantation (PDOX) in salivary glands. FINDINGS: Our PDOX model enabled solid tumors to integrate within the local epithelial, stromal and neuronal environment. Over serial passages, PDOX tumors maintained their stereotypic MYB-NFIB translocation, and FGFR2 and ATM point mutations. Tumor growth rate and histopathology were retained, including ACCs hallmark presentations of cribriform, tubular, solid areas and innervation. We also demonstrate that the PDOX model retains its capacity as a tool for drug testing. INTERPRETATION: Unlike the precedent PDX model, our data shows that the PDOX is a superior model for future cancer biology and therapy research. FUND: This work was supported by the National Institutes of Health (NIH)/National Institute of Dental and Craniofacial Research (NIDCR) grants DE022557, DE027034, and DE027551.

Neurturin Gene Therapy Protects Parasympathetic Function to Prevent Irradiation-Induced Murine Salivary Gland Hypofunction.

Head and neck cancer patients treated with irradiation often present irreversible salivary gland hypofunction for which no conventional treatment exists. We recently showed that recombinant neurturin, a neurotrophic factor, improves epithelial regeneration of mouse salivary glands in ex vivo culture after irradiation by reducing apoptosis of parasympathetic neurons. Parasympathetic innervation is essential to maintain progenitor cells during gland development and for regeneration of adult glands. Here, we investigated whether a neurturin-expressing adenovirus could be used for gene therapy in vivo to protect parasympathetic neurons and prevent gland hypofunction after irradiation. First, ex vivo fetal salivary gland culture was used to compare the neurturin adenovirus with recombinant neurturin, showing they both improve growth after irradiation by reducing neuronal apoptosis and increasing innervation. Then, the neurturin adenovirus was delivered to mouse salivary glands in vivo, 24 hr before irradiation, and compared with a control adenovirus. The control-treated glands have ∼50% reduction in salivary flow 60 days post-irradiation, whereas neurturin-treated glands have similar flow to nonirradiated glands. Further, markers of parasympathetic function, including vesicular acetylcholine transporter, decreased with irradiation, but not with neurturin treatment. Our findings suggest that in vivo neurturin gene therapy prior to irradiation protects parasympathetic function and prevents irradiation-induced hypofunction.

The Future of Radiobiology.

Innovation and progress in radiation oncology depend on discovery and insights realized through research in radiation biology. Radiobiology research has led to fundamental scientific insights, from the discovery of stem/progenitor cells to the definition of signal transduction pathways activated by ionizing radiation that are now recognized as integral to the DNA damage response (DDR). Radiobiological discoveries are guiding clinical trials that test radiation therapy combined with inhibitors of the DDR kinases DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM), ataxia telangiectasia related (ATR), and immune or cell cycle checkpoint inhibitors. To maintain scientific and clinical relevance, the field of radiation biology must overcome challenges in research workforce, training, and funding. The National Cancer Institute convened a workshop to discuss the role of radiobiology research and radiation biologists in the future scientific enterprise. Here, we review the discussions of current radiation oncology research approaches and areas of scientific focus considered important for rapid progress in radiation sciences and the continued contribution of radiobiology to radiation oncology and the broader biomedical research community.

Mobilization of bone marrow stem cells by granulocyte colony-stimulating factor ameliorates radiation-induced damage to salivary glands.

PURPOSE: One of the major reasons for failure of radiotherapeutic cancer treatment is the limitation in dose that can be applied to the tumor because of coirradiation of the normal healthy tissue. Late radiation-induced damage reduces the quality of life of the patient and may even be life threatening. Replacement of the radiation-sterilized stem cells with unirradiated autologous stem cells may restore the tissue function. Here, we assessed the potential of granulocyte colony-stimulating factor (G-CSF)-mobilized bone marrow-derived cells (BMC) to regenerate and functionally restore irradiated salivary glands used as a model for normal tissue damage. EXPERIMENTAL DESIGN: Male-eGFP+ bone marrow chimeric female C57BL/6 mice were treated with G-CSF, 10 to 60 days after local salivary gland irradiation. Four months after irradiation, salivary gland morphology and flow rate were assessed. RESULTS: G-CSF treatment induced homing of large number of labeled BMCs to the submandibular glands after irradiation. These animals showed significant increased gland weight, number of acinar cells, and salivary flow rates. Donor cells expressed surface markers specific for hematopoietic or endothelial/mesenchymal cells. However, salivary gland acinar cells neither express the G-CSF receptor nor contained the GFP/Y chromosome donor cell label. CONCLUSIONS: The results show that BMCs home to damaged salivary glands after mobilization and induce repair processes, which improve function and morphology. This process does not involve transdifferentiation of BMCs to salivary gland cells. Mobilization of BMCs could become a promising modality to ameliorate radiation-induced complications after radiotherapy.

Exosomal MicroRNA Transport from Salivary Mesenchyme Regulates Epithelial Progenitor Expansion during Organogenesis.

Epithelial-mesenchymal interactions involve fundamental communication between tissues during organogenesis and are primarily regulated by growth factors and extracellular matrix. It is unclear whether RNA-containing exosomes are mobile genetic signals regulating epithelial-mesenchymal interactions. Here we identify that exosomes loaded with mesenchyme-specific mature microRNA contribute mobile genetic signals from mesenchyme to epithelium. The mature mesenchymal miR-133b-3p, loaded into exosomes, was transported from mesenchyme to the salivary epithelium, which did not express primary miR-133b-3p. Knockdown of miR-133b-3p in culture decreased endbud morphogenesis, reduced proliferation of epithelial KIT+ progenitors, and increased expression of a target gene, Disco-interacting protein 2 homolog B (Dip2b). DIP2B, which is involved in DNA methylation, was localized with 5-methylcytosine in the prophase nucleus of a subset of KIT+ progenitors during mitosis. In summary, exosomal transport of miR-133b-3p from mesenchyme to epithelium decreases DIP2B, which may function as an epigenetic regulator of genes responsible for KIT+ progenitor expansion during organogenesis.