Younan Liu

Bone marrow-derived cells rescue salivary gland function in mice with head and neck irradiation

Treatment for most patients with head and neck cancers includes ionizing radiation. A consequence of this treatment is irreversible damage to salivary glands (SGs), which is accompanied by a loss of fluid-secreting acinar-cells and a considerable decrease of saliva output. While there are currently no adequate conventional treatments for this condition, cell-based therapies are receiving increasing attention to regenerate SGs. In this study, we investigated whether bone marrow-derived cells (BMDCs) can differentiate into salivary epithelial cells and restore SG function in head and neck irradiated mice. BMDCs from male mice were transplanted into the tail-vein of 18Gy-irradiated female mice. Salivary output was increased in mice that received BMDCs transplantation at week 8 and 24 post-irradiation. At 24 weeks after irradiation (IR), harvested SGs (submandibular and parotid glands) of BMDC-treated mice had greater weights than those of non-treated mice. Histological analysis shows that SGs of treated mice demonstrated an increased level of tissue regenerative activity such as blood vessel formation and cell proliferation, while apoptotic activity was increased in non-transplanted mice. The expression of stem cell markers (Sca-1 or c-kit) was detected in BMDC-treated SGs. Finally, we detected an increased ratio of acinar-cell area and approximately 9% of Y-chromosome-positive (donor-derived) salivary epithelial cells in BMDC-treated mice. We propose here that cell therapy using BMDCs can rescue the functional damage of irradiated SGs by direct differentiation of donor BMDCs into salivary epithelial cells.

Mesenchymal Stromal Cells Improve Salivary Function and Reduce Lymphocytic Infiltrates in Mice with Sjogren's-Like Disease

Background Non-obese diabetic (NOD) mice develop Sjögrens-like disease (SS-like) with loss of saliva flow and increased lymphocytic infiltrates in salivary glands (SGs). There are recent reports using multipotent mesenchymal stromal cells (MSCs) as a therapeutic strategy for autoimmune diseases due to their anti-inflammatory and immunomodulatory capabilities. This paper proposed a combined immuno- and cell-based therapy consisting of: A) an injection of complete Freunds adjuvant (CFA) to eradicate autoreactive T lymphocytes, and B) transplantations of MSCs to reselect lymphocytes. The objective of this was to test the effectiveness of CD45−/TER119− cells (MSCs) in re-establishing salivary function and in reducing the number of lymphocytic infiltrates (foci) in SGs. The second objective was to study if the mechanisms underlying a decrease in inflammation (focus score) was due to CFA, MSCs, or CFA+MSCs combined. Methodology/Principal Findings Donor MSCs were isolated from bones of male transgenic eGFP mice. Eight week-old female NOD mice received one of the following treatments: insulin, CFA, MSC, or CFA+MSC (combined therapy). Mice were followed for 14 weeks post-therapy. CD45−/TER119− cells demonstrated characteristics of MSCs as they were positive for Sca-1, CD106, CD105, CD73, CD29, CD44, negative for CD45, TER119, CD11b, had high number of CFU-F, and differentiated into osteocytes, chondrocytes and adipocytes. Both MSC and MSC+CFA groups prevented loss of saliva flow and reduced lymphocytic infiltrations in SGs. Moreover, the influx of T and B cells decreased in all foci in MSC and MSC+CFA groups, while the frequency of Foxp3+ (Treg) cell was increased. MSC-therapy alone reduced inflammation (TNF-α, TGF-β), but the combination of MSC+CFA reduced inflammation and increased the regenerative potential of SGs (FGF-2, EGF). Conclusions/Significance The combined use of MSC+CFA was effective in both preventing saliva secretion loss and reducing lymphocytic influx in salivary glands.

Matrigel improves functional properties of human submandibular salivary gland cell line.

Sjogrens syndrome and radiotherapy for head and neck cancers result in irreversible damage to functional salivary tissue, for which no adequate treatment is available. The microenvironment for salivary gland cell cytodifferentiation is critical for the future development of salivary gland regeneration, repair and tissue engineering treatments. Results from this study indicate that human submandibular cell line (HSG) cultured on Matrigel (2mg/ml) could be induced to differentiate into polarized secretory acinar-like cells. The HSG cells grown on Matrigel were evaluated by physiological functional assays, molecular and immunohistochemistry, immunofluorescence, and morphological assessments. The results showed (1) a decrease in cell proliferation; (2) an increase in cell apoptosis; (3) cellular polarization evident by transepithelial electrical resistance (TER), expressions of tight junction proteins (claudin-1, -2, -3, -4, occludin, JAM-A, and ZO-1) and transmission electron microscopy (TEM); (4) an increase in the production and/or secretion of acinar cell proteins, i.e., alpha-amylase, aquaporin-5, cytokeratins, and mucin-1, that were not associated with increases in mRNA transcription; (5) a decrease in vimentin expression; and (6) expression of potential stem cell biomarkers CD44 and CD166. The data indicated that Matrigel provided a suitable microenvironment for morphological and functional differentiation of HSG cells into 3D acinar like cells. This study provides an in vitro model and baseline data on future developments of new strategies for salivary gland regeneration and replacement.

Paracrine Effects of Bone Marrow Soup Restore Organ Function, Regeneration, and Repair in Salivary Glands Damaged by Irradiation

Background There are reports that bone marrow cell (BM) transplants repaired irradiated salivary glands (SGs) and re-established saliva secretion. However, the mechanisms of action behind these reports have not been elucidated. Methods To test if a paracrine mechanism was the main effect behind this reported improvement in salivary organ function, whole BM cells were lysed and its soluble intracellular contents (termed as “BM Soup”) injected into mice with irradiation-injured SGs. The hypothesis was that BM Soup would protect salivary cells, increase tissue neovascularization, function, and regeneration. Two minor aims were also tested a) comparing two routes of delivering BM Soup, intravenous (I.V.) versus intra-glandular injections, and b) comparing the age of the BM Soup’s donors. The treatment-comparison group consisted of irradiated mice receiving injections of living whole BM cells. Control mice received irradiation and injections of saline or sham-irradiation. All mice were followed for 8 weeks post-irradiation. Results BM Soup restored salivary flow rates to normal levels, protected salivary acinar, ductal, myoepithelial, and progenitor cells, increased cell proliferation and blood vessels, and up-regulated expression of tissue remodeling/repair/regenerative genes (MMP2, CyclinD1, BMP7, EGF, NGF). BM Soup was as an efficient therapeutic agent as injections of live BM cells. Both intra-glandular or I.V. injections of BM Soup, and from both young and older mouse donors were as effective in repairing irradiated SGs. The intra-glandular route reduced injection frequency/dosage by four-fold. Conclusion BM Soup, which contains only the cell by-products, can be advantageously used to repair irradiation-damaged SGs rather than transplanting whole live BM cells which carry the risk of differentiating into unwanted/tumorigenic cell types in SGs.

Microchimerism in Salivary Glands after Blood- and Marrow-Derived Stem Cell Transplantation

Blood- and marrow-derived stem cells (BMDSCs) provide disease-ameliorating effects for cardiovascular and autoimmune diseases. Microchimerism from donor BMDSCs has been reported in several recipient tissues. We hypothesized that this finding suggests a potential use of BMDSCs in the treatment of salivary dysfunctions. We investigated the presence of Y chromosome-positive cells in salivary gland biopsies of 5 females who had received a marrow or blood stem cell transplant from male donors. One to 16 years after transplantation, all recipients exhibited scattered Y chromosome-positive cells in the acini, ducts, and stroma of their salivary glands (mean of 1.01%). Potentially, these cells can be markers of transplantation tolerance, contribute to neoplastic epithelial tissues, or engraft at sites of injury. In addition, transplantation of BMDSCs could be used for treatment of Sjögrens syndrome and salivary glands damaged by therapeutic irradiation for cancers of the head and neck.

Identification of the active components in Bone Marrow Soup: a mitigator against irradiation-injury to salivary glands

In separate studies, an extract of soluble intracellular contents from whole bone marrow cells, named “Bone Marrow (BM) Soup”, was reported to either improve cardiac or salivary functions post-myocardial infarction or irradiation (IR), respectively. However, the active components in BM Soup are unknown. To demonstrate that proteins were the active ingredients, we devised a method using proteinase K followed by heating to deactivate proteins and for safe injections into mice. BM Soup and “deactivated BM Soup” were injected into mice that had their salivary glands injured with 15Gy IR. Control mice received either injections of saline or were not IR. Results at week 8 post-IR showed the ‘deactivated BM Soup’ was no better than injections of saline, while injections of native BM Soup restored saliva flow, protected salivary cells and blood vessels from IR-damage. Protein arrays detected several angiogenesis-related factors (CD26, FGF, HGF, MMP-8, MMP-9, OPN, PF4, SDF-1) and cytokines (IL-1ra, IL-16) in BM Soup. In conclusion, the native proteins (but not the nucleic acids, lipids or carbohydrates) were the therapeutic ingredients in BM Soup for functional salivary restoration following IR. This molecular therapy approach has clinical potential because it is theoretically less tumorigenic and immunogenic than cell therapies.

Quantitative Analysis of Protein and Gene Expression in Salivary Glands of Sjogren’s-Like Disease NOD Mice Treated by Bone Marrow Soup

Background Bone marrow cell extract (termed as BM Soup) has been demonstrated to repair irradiated salivary glands (SGs) and restore saliva secretion in our previous study. In the present study, we aim to investigate if the function of damaged SGs in non-obese diabetic (NOD) mice can be restored by BM Soup treatment and the molecular alterations associated with the treatment. Methods Whole BM cells were lysed and soluble intracellular contents (“BM Soup”) were injected I.V. into NOD mice. Tandem mass tagging with 2-D liquid chromatography-mass spectrometry was used to quantify proteins in the submandibular glands (SMGs) between untreated and BM Soup-treated mice. Quantitative PCR was used to identify genes with altered expression in the treated mice. Results BM Soup restored salivary flow rates to normal levels and significantly reduced the focus scores of SMGs in NOD mice. More than 1800 proteins in SMG cells were quantified by the proteomic approach. Many SMG proteins involved in inflammation and apoptosis were found to be down-regulated whereas those involved in salivary gland biology and development/regeneration were up-regulated in the BM Soup-treated mice. qPCR analysis also revealed expression changes of growth factors and cytokines in the SMGs of the treated NOD mice. Conclusion BM Soup treatment is effective to restore the function of damaged SGs in NOD mice. Through gene/protein expression analysis, we have found that BM Soup treatment might effectuate via inhibiting apoptosis, focal adhesion and inflammation whereas promoting development, regeneration and differentiation of the SG cells in NOD mice. These findings provide important insights on the potential mechanisms underlying the BM Soup treatment for functional restoration of damaged SGs in NOD mice. Additional studies are needed to further confirm the identified target genes and their related signaling pathways that are responsible for the BM Soup treatment.

Treatment for salivary gland hypofunction at both initial and advanced stages of Sjögren-like disease: a comparative study of bone marrow therapy versus spleen cell therapy with a 1-year monitoring period

BACKGROUND AIMS Non-obese diabetic mice (NOD) exhibit autoimmune Sjögren-like disease (SS-like). We reported previously that a combined-therapy consisting of immuno- and cell-based therapy rescued NOD from SS-like. However, therapies tested to date on NOD mice were aimed at the initial phase of SS-like. It is unknown whether therapies are effective in restoring salivary function when given at an advanced phase of SS-like. METHODS The efficacy of two therapies (bone marrow versus spleen cells) was compared head-to-head for halting/reversing salivary hypofunction at two critical time points of SS-like (7-week-old NOD with normal saliva output and 20-week-old NOD with minimal saliva). NOD mice were divided into four groups: (i) control, (ii) complete Freunds adjuvant (CFA), (iii) bone marrow transplants with CFA or (iv) spleen cell transplants with CFA. Mice were monitored 8-12 months after therapy. RESULTS Both cell therapies were effective during the initial phase of SS-like; salivary flow rates were maintained between 80-100% of pre-symptomatic levels. Spleen cell therapy was better than bone marrow when administered in the initial phase of SS-like. When cell therapies were given at an advanced phase of SS-like (20 weeks and older), salivary flow rates improved but were at best 50% of pre-symptomatic levels. Both cell therapies decreased tumor necrosis factor-α, transforming growth factor-β1 levels and T and B cells while increasing epidermal growth factor and regulatory T cells. Elevated serum epidermal growth factor levels were measured in spleen-treated mice. CONCLUSIONS A therapeutic effect in advanced phase disease, albeit in mice, holds promise for humans in which Sjögren syndrome is generally not diagnosed until a late stage.

GDFs promote tenogenic characteristics on human periodontal ligament-derived cells in culture at late passages.

Abstract Tendon/ligament injures are leading disabilities worldwide. The periodontal ligament (PDL) connects teeth to bone, and is comparable to a tendon/ligament-to-bone insertion. PDL-derived cells (PDLCs) express both osteo/cementogenesis and teno/ligamentogenesis genes. However, an efficient method to induce a tenogenic differentiation of PDLCs has not been thoroughly examined. Therefore, this study tested if growth/differentiation factors (GDFs) enhanced tenogenic characteristics of human PDLCs, as a potential cell source for tendon/ligament engineering. Results demonstrated recombinant GDF-5/GDF-7 inhibited alkaline phosphatase (ALP) activity of PDLCs from passage 3 to 6, while GDF-5 enhanced ALP in dental pulp-derived cells and mesenchymal stem cells. GDF-5 (particularly at 10 ng/ml concentration) induced high expression of both early (scleraxis) and mature (tenomodulin, aggrecan, collagen3) tenogenic genes in P4-6 PDLCs, while inhibiting expression of specific transcription-factors for osteogenic, chondrogenic and adipogenic differentiation. Exogenous GDFs might lead PDLCs being expanded in culture during several passages to highly useful cell source for tendon/ligament engineering.

Cannulation of the Mouse Submandibular Salivary Gland via the Wharton's Duct

Severe salivary gland hypofunction is frequently found in patients with Sjögrens syndrome and those who receiving therapeutic irradiation in their head and neck regions for cancer treatment. Both groups of patients experience symptoms such as xerostomia (dry mouth), dysphagia (impaired chewing and swallowing), severe dental caries, altered taste, oro-pharyngeal infections (candidiasis), mucositis, pain and discomfort. One innovative approach of regenerative medicine for the treatment of salivary gland hypo-function is speculated in RS Redman, E Mezey et al. 2009: stem cells can be directly deposited by cannulation into the gland as a potent method in reviving the functions of the impaired organ. Presumably, the migrated foreign stem cells will differentiate into glandular cells to function as part of the host salivary gland. Also, this cannulation technique is an expedient and effective delivery method for clinical gene transfer application. Here we illustrate the steps involved in performing the cannulation procedure on the mouse submandibular salivary gland via the Whartons duct (Fig 1). C3H mice (Charles River, Montreal, QC, Canada) are used for this experiment, which have been kept under clean conventional conditions at the McGill University animal resource center. All experiments have been approved by the University Animal Care Committee and were in accordance with the guidelines of the Canadian Council on Animal Care. For this experiment, a trypan blue solution is infused into the gland through the opening of the Whartons duct using a insulin syringe with a 29-gauge needle encased inside a polyethylene tube. Subsequently, the mouse is dissected to show that the infusions migrated into the gland successfully.