Participation of Somatic Stem Cells, Recognized by a Unique A3 Antibody, in Mucosal Epithelial Regeneration in Dextran Sulfate Sodium (DSS)-Induced Rat Colonic Lesions.

A3, generated as a monoclonal antibody against rat malignant fibrous histiocytoma cells, recognizes somatic stem cells in rats. We analyzed the distribution of A3-positive cells in dextran sulfate sodium (DSS)-induced colonic lesions consisting of regenerating mucosa and fibrosis. Male 6-week-old F344 rats were administered 5% DSS in drinking water for 5 to 7 days, and lesions at recovery stage were also examined. In untreated control adult colons, A3-positive cells are localized around the crypts where stem cell niche is formed. Histopathologically, in colons of DSS-administered rats, mucosal atrophy, inflammatory cell infiltration, and fibrosis were observed in the lamina propria; thereafter, mucosal epithelia were desquamated, and crypts were decreased gradually with decrease in surrounding A3-positive cells. At the early recovery stage, crypts showed regeneration with reappearance of A3-positive cells. Interestingly, A3-positive cells aggregated in desquamated mucosa surface of fibrosis. Aggregated A3-positive cells coexpressed with vimentin, Thy-1, and partly CK19 but did not react simultaneously with α-SMA. Likely, aggregated A3-positive cells may be rescue cells with nature of both mesenchymal and epithelial cells to maintain self-renewal after injury in the colon. A3 antibody would become a useful tool to investigate the participation of stem cells in rat colonic lesions.

Concise Review: Cellular Therapies: The Potential to Regenerate and Restore Tolerance in Immune-Mediated Intestinal Diseases.

Chronic inflammatory enteropathies, including celiac disease, Crohn's disease, and ulcerative colitis, are lifelong disabling conditions whose cure is still an unmet need, despite the great strides made in understanding their complex pathogenesis. The advent of cellular therapies, mainly based on the use of stem cells, represents a great step forward thanks to their multitarget strategy. Both hematopoietic stem cells (HSC) and mesenchymal stem/stromal cells (MSC) have been employed in the treatment of refractory cases with promising results. The lack of immunogenicity makes MSC more suitable for therapeutic purposes as their infusion may be performed across histocompatibility locus antigen barriers without risk of rejection. The best outcome has been obtained when treating fistulizing Crohn's disease with local injections of MSC. In addition, both HSC and MSC proved successful in promoting regeneration of intestinal mucosa, and favoring the expansion of a T-cell regulatory subset. By virtue of the ability to favor mucosal homeostasis, this last cell population has been exploited in clinical trials, with inconsistent results. Finally, the recent identification of the epithelial stem cell marker has opened up the possibility of tissue engineering, with an array of potential applications for intestinal diseases. However, the underlying mechanisms of action of these interconnected therapeutic strategies are still poorly understood. It is conceivable that over the next few years their role will become clearer as the biological interactions with injured tissues and the hierarchy by which they deliver their action are unraveled through a continuous moving from bench to bedside and vice versa. Stem Cells 2016;34:1474-1486.

Therapeutic effects of mouse bone marrow-derived clonal mesenchymal stem cells in a mouse model of inflammatory bowel disease.

Mouse bone marrow-derived clonal mesenchymal stem cells (mcMSCs), which were originated from a single cell by a subfractionation culturing method, are recognized as new paradigm for stem cell therapy featured with its homogenous cell population. Next to proven therapeutic effects against pancreatitis, in the current study we demonstrated that mcMSCs showed significant therapeutic effects in dextran sulfate sodium (DSS)-induced experimental colitis model supported with anti-inflammatory and restorative activities. mcMSCs significantly reduced the disease activity index (DAI) score, including weight loss, stool consistency, and intestinal bleeding and significantly increased survival rates. The pathological scores were also significantly improved with mcMSC. We have demonstrated that especial mucosal regeneration activity accompanied with significantly lowered level of apoptosis as beneficiary actions of mcMSCs in UC models. The levels of inflammatory cytokines including TNF-α, IFN-γ, IL-1ß, IL-6, and IL-17 were all significantly concurrent with significantly repressed NF-κB activation compared to the control group and significantly decreased infiltrations of responsible macrophage and neutrophil. Conclusively, our findings provide the rationale that mcMSCs are applicable as a potential source of cell-based therapy in inflammatory bowel diseases, especially contributing either to prevent relapse or to accelerate healing as solution to unmet medical needs in IBD therapy.

General Semi-Solid Freeze Casting for Uniform Large-Scale Isotropic Porous Scaffolds: An Application for Extensive Oral Mucosal Reconstruction.

Ice-templated porous biomaterials possess transformative potential in regenerative medicine; yet, scaling up ice-templating processes for broader applications-owing to inconsistent pore formation-remains challenging. This study reports an innovative semi-solid freeze-casting technique that draws inspiration from semi-solid metal processing (SSMP) combined with ice cream-production routines. This versatile approach allows for the large-scale assembly of various materials, from polymers to inorganic particles, into isotropic 3D scaffolds featuring uniformly equiaxed pores throughout the centimeter scale. Through (cryo-)electron microscopy, X-ray tomography, and finite element modeling, the structural evolution of ice grains/pores is elucidated, demonstrating how the method increases the initial ice nucleus density by pre-fabricating a semi-frozen slurry, which facilitates a transition from columnar to equiaxed grain structures. For a practical demonstration, as-prepared scaffolds are integrated into a bilayer tissue patch using biodegradable waterborne polyurethane (WPU) for large-scale oral mucosal reconstruction in minipigs. Systematic analyses, including histology and RNA sequencing, prove that the patch modulates the healing process toward near-scarless mucosal remodeling via innate and adaptive immunomodulation and activation of pro-healing genes converging on matrix synthesis and epithelialization. This study not only advances the field of ice-templating fabrication but sets a promising precedent for scaffold-based large-scale tissue regeneration.

Adrenomedullin alleviates mucosal injury in experimental colitis and increases claudin-4 expression in the colonic epithelium.

Adrenomedullin (AM) is a peptide with pleiotropic physiological functions that attenuates intestinal mucosal inflammation. However, the mechanism underpinning mucosal protection by AM is not fully understood, and its effect on intestinal epithelial cells remains unclear. Here, we investigated the effects of AM on junctional molecules in primary-cultured murine intestinal epithelial cells and discovered that AM upregulates claudin-4 expression. In a mouse model of dextran sulfate sodium-induced colitis, AM administration also enhanced claudin-4 expression and accelerated mucosal regeneration. Furthermore, AM reversed TNFα-mediated downregulation of claudin-4 and loss of cell-cell adhesion of the HCT116 human intestinal epithelial cell line in vitro. These results indicate that AM may enhance intestinal epithelial integrity by upregulating claudin-4 expression.

Cell sheet transplantation prevents inflammatory adhesions: A new treatment for adhesive otitis media.

INTRODUCTION: We developed a new treatment method that combines tympanoplasty with transplantation of autologous cultured nasal mucosal epithelial cell sheets to regenerate the mucosa of patients with adhesive otitis media, which has been difficult to treat effectively. We verified whether this procedure could be performed safely and measured its therapeutic efficacy. METHODS: Autologous nasal mucosal epithelial cell sheets were manufactured at a good manufacturing practice-compliant cell processing facility using autologous nasal mucosal tissue. We performed tympanoplasty and transplanted the cell sheets into the middle ear cavity in six patients with adhesive otitis media. RESULTS: The manufactured autologous cultured epithelial cell sheets met the predetermined quality standards and were successfully transplanted safely in all cases. Computed tomography findings after cell sheet transplantation showed that aeration in the tympanic cavity was maintained or restored in five of the six patients (83.3%). Four of the six (66.7%) patients had postoperative air-bone gap within 20 dB, which is considered a postoperative success in tympanoplasty for chronic middle ear disease. CONCLUSIONS: The results of this clinical study suggest that tympanoplasty with cell sheet transplantation can be used to treat adhesive otitis media by reliably preventing re-adhesion of the tympanic membrane.

Lessons learned from conventional animals: Encouragement to use specific-pathogen-free animals.

Experimental rabbits provide evidence for translational research regarding the pathogenies or treatment of human diseases. We developed a novel method for regenerating the middle ear mucosa using autologous cultured nasal mucosal epithelial cell sheets, and evaluated the wound healing process in the middle ear mucosa of experimental rabbits. Nonetheless, vigilant microbiological monitoring of experimental animals is essential to effectively prevent a decline in their health conditions, which may affect the research results. We experimented with contamination of Pasteurella multocida in non-specific-pathogen-free (SPF) rabbits (without microbiological monitoring). Most non-SPF rabbits had otitis media, whereas SPF rabbits did not, which affected their results during the mucosal regeneration study. The contamination was resolved by changing the experimental design from using non-SPF rabbits to that using SPF rabbits. It is crucial to use the SPF animals for any surgical intervention studies.

Switching to a Healthy Diet Prevents the Detrimental Effects of Western Diet in a Colitis-Associated Colorectal Cancer Model.

Inflammatory bowel disease increases the odds of developing colitis-associated cancer. We hypothesized that Western-style diet (WD) aggravates azoxymethane (AOM)/dextran sulfate sodium salt (DSS)-induced colitis-associated tumorigenesis and that switching to the standard AIN93G diet will ameliorate disease symptoms even after cancer initiation. Female BALB/c mice received either WD (WD group) or standard AIN93G diet (AIN group) for the whole experimental period. After five weeks, the mice received 12.5 mg/kg AOM intraperitoneally, followed by three DSS cycles. In one group of mice, the WD was switched to AIN93G the day before starting the first DSS cycle (WD/AIN group). Feeding the WD during the whole experimental period aggravated colitis symptoms, shortened the colon (p < 0.05), changed microbiota composition and increased tumor promotion. On molecular level, the WD reduced proliferation (p < 0.05) and increased expression of the vitamin D catabolizing enzyme Cyp24a1 (p < 0.001). The switch to the AIN93G diet ameliorated this effect, reflected by longer colons, fewer (p < 0.05) and smaller (p < 0.01) aberrant colonic crypt foci, comparable with the AIN group. Our results show that switching to a healthy diet, even after cancer initiation is able to revert the deleterious effect of the WD and could be an effective preventive strategy to reduce colitis symptoms and prevent tumorigenesis.

Middle ear mucosal regeneration with three-dimensionally tissue-engineered autologous middle ear cell sheets in rabbit model.

The likelihood of recurrent retraction and adhesion of newly formed tympanic membrane is high when middle ear mucosa is extensively lost during cholesteatoma and adhesive otitis media surgery. If rapid postoperative regeneration of the mucosa on the exposed bone surface can be achieved, prevention of recurrent eardrum adhesion and cholesteatoma formation, for which there has been no definitive treatment, can be expected. Suture-less transplantation of tissue-engineered mucosal cell sheets was examined immediately after the operation of otitis media surgery in order to quickly regenerate middle ear mucosa lost during surgery in a rabbit model. Transplantable middle ear mucosal cell sheets with a three-dimensional tissue architecture very similar to native middle ear mucosa were fabricated from middle ear mucosal tissue fragments obtained in an autologous manner from middle ear bulla on temperature-responsive culture surfaces. Immediately after the mucosa was resected from middle ear bone bulla inner cavity, mucosal cell sheets were grafted at the resected site. Both bone hyperplasia and granulation tissue formation were inhibited and early mucosal regeneration was observed in the cell sheet-grafted group, compared with the control group in which only mucosal removal was carried out and the bone surface exposed. This result indicates that tissue engineered mucosal cell sheets would be useful to minimize complications after the surgical operation on otitis media and future clinical application is expected.

Synergistic effect of laminin and mesenchymal stem cells on tracheal mucosal regeneration.

Although several studies have been successfully undertaken of tracheal reconstruction in terms of the maintaining the framework of the graft, most cases of reconstruction failure have resulted from delayed mucosal regeneration. The purposes of this study were to evaluate whether laminin-coated asymmetrically porous membrane (APM) scaffold enhances mucosal regeneration, to compare the mucosalization capability with mesenchymal stem cell (MSC) seeded APM, and to determine whether laminin coating and MSC seeding has a synergistic effect on mucosal regeneration. We reconstructed the full-thickness anterior tracheal defect of 36 New Zealand White rabbits with the APM scaffold. MSCs were isolated from the rabbit's inguinal fat. The animals were divided into 4 groups by the presence of laminin coating on APM and application of MSC [Group I, -/- (laminin/MSC); Group II, -/+; Group III, +/-; Group IV, +/+]. Endoscopy and histologic evaluation were performed and the results were compared among the groups. The results showed that ciliated columnar epithelium was regenerated earlier in groups II and III than in group I. Furthermore, the application of laminin and MSC had synergistic effects on tracheal epithelial regeneration. These results demonstrate that tracheal reconstruction by laminin-coated APM seeded with MSCs is most effective in enhancing tracheal mucosalization, and appears to be promising strategy in the regenerative treatment of tracheal defects.

The effect of transplantation of nasal mucosal epithelial cell sheets after middle ear surgery in a rabbit model.

Postoperative regeneration of the middle ear mucosa and pneumatization of the middle ear cavity are of great importance after middle ear surgery. This study developed a new method to transplant autologous nasal mucosal epithelial cell sheets into the damaged middle ear cavity. The aim of this study was to evaluate postoperative healing after the transplantation of the cell sheets. Rabbit nasal mucosal epithelial cell sheets were fabricated on a temperature-responsive culture dish, and transplanted into the damaged middle ear of rabbit, which was surgically created. The healing of middle ears was evaluated by histology and X-ray computed tomography after transplantation. Functional evaluation was performed by measuring the maximum middle ear total pressure reflecting a trans-mucosal gas exchange function. Two control groups were used: the normal control group and the mucosa-eliminated control group. Transplantation of cell sheets suppressed the bone hyperplasia and the narrowing of pneumatic space in the middle ear cavity compared with the mucosa-eliminated control group. The mucosal gas exchange function was also better in the cell sheet-transplanted group. Nasal mucosal epithelial cell sheet was confirmed to be useful as an effective graft material after middle ear surgery and hopefully become a novel therapy in the future.

Bone Marrow-derived Cell Therapy for Oral Mucosal Repair after Irradiation.

Oral mucositis (ulcer) is a serious and painful side effect for patients with head and neck cancer following radiation therapy. However, current clinical strategies cannot efficiently prevent the occurrence of oral mucositis. In this study, we investigated whether bone marrow-derived cells (BMDCs) prevented the occurrence and/or decreased the severity of radiation-induced oral mucositis. Fresh concentrated BMDCs from male C3H mice were transplanted intravenously into female mice after tongue irradiation. For 14 days postirradiation, the changes of body weight and the time courses of ulceration were observed. Until the ulcer reached maximum size (7 days postirradiation), macroscopic and histologic analyses of harvested tongues were performed to detect the behavior of donor BMDCs. Between 2 and 5 days postirradiation, BMDCs-transplanted mice showed more expression of stem cell markers (c-Kit, Sca-1) and EGFR and fewer apoptotic cells when compared with nontransplanted control mice (irradiation group). On day 7, there were fewer and smaller ulcers observed in the BMDCs-transplanted group. Tongues of these mice had preserved their epithelial thickness, and regenerative activities (blood vessels formation, cell proliferation) were higher than they were in the irradiation group. Fluorescently labeled BMDCs were not detected in tongue epithelium but rather in connective tissue (dermis) just below the basal cell layer. These findings suggest that exogenous BMDCs behave to reduce radiogenic oral mucositis in a paracrine manner.