Dual-color FISH analyses of xenogeneic human fibroblast sheets transplanted to repair lung pleural defects in an immunocompromised rat model.

BACKGROUND: Pulmonary air leaks (PALs) due to visceral pleura injury during surgery is frequently observed after pulmonary resections and the complication is difficult to avoid in thoracic surgery. The development of postoperative PALs is the most common cause of prolonged hospitalization. Previously, we reported that PALs sealants using autologous dermal fibroblast sheets (DFSs) harvested from temperature-responsive culture dishes successfully closed intraoperative PALs during lung resection. OBJECTIVE: In this study, we investigated the fate of human DFSs xenogenetically transplanted onto lung surfaces to seal PALs of immunocompromised rat. Dual-color FISH analyses of human fibroblast was employed to detect transplantation human cells on the lung surface. RESULTS: One month after transplantation, FISH analyses revealed that transplanted human fibroblasts still composed a sheet-structure, and histology also showed that beneath the sheet's angiogenesis migrating into the sheets was observed from the recipient tissues. FISH analyses revealed that even at 3 months after transplantation, the transplanted human fibroblasts still remained in the sheet. Dual-color FISH analyses of the transplanted human fibroblasts were sparsely present as a result of the cells reaching the end of their lifespan, the cells producing extracellular matrix, and remained inside the cell sheet and did not invade the lungs of the host. CONCLUSIONS: DFS-transplanted human fibroblasts showed that they are retained within cell sheets and do not invade the lungs of the host.

Variation in the rate of detection of minute and small early gastric cancers at diagnostic endoscopy may reflect the performance of individual endoscopists.

OBJECTIVE: The documented variation in gastric cancer (GC) detection among endoscopists has often been dismissed as a coincidental artefact of the low incidence of gastric neoplasms; it is not considered associated with differences in physicians' performance of the esophagogastroduodenoscopy procedure. This study is to confirm whether significant variations among endoscopists in early GC detection suggest the individual performance of the upper endoscopy. DESIGN: A retrospective observational study at a single centre in Japan assessed the results of 218 early GCs detected during 25 688 routine esophagogastroduodenoscopies by 12 endoscopists. The main outcome was the rate of early GC detection for each endoscopist under the same circumstances. Other measures included the major diameters and locations of the lesions, Helicobacter pylori infection status, and baseline patient characteristics that could affect the prevalence of GC. RESULTS: The early GC detection rates exhibited wide variation among endoscopists (0.09%-2.87%) despite performing routine esophagogastroduodenoscopies in a population with a similar background. Endoscopists were assigned to a low-detection group (n=6; detection rate: 0.47% (range: 0.09%-0.55%)) and a high-detection group (n=5; detection rate: 0.83% (range: 0.63%-1.12%)), with the single highest detector analysed separately due to his distinct detection rate (2.87%). Endoscopists in the high-detection group had better detection rates for minute (major diameter ≤5 mm) and small (major diameter 6-10 mm) GCs than the low-detection group (0.19%/0.23% vs 0.085%/0.098%). These differences were significant (p<0.01), although there were no significant differences in detection of larger tumours (major diameter ≥11 mm; 0.40% vs 0.28%; p=0.13). The tumour location and H. pylori status were similar in the low-detection group, high-detection group and for the highest detector. CONCLUSION: Significant variation in the detection of hard-to-find, smaller GCs may reflect individual performance of the examination.

Polydactyly-derived allogeneic chondrocyte cell-sheet transplantation with high tibial osteotomy as regenerative therapy for knee osteoarthritis.

Allogeneic cell therapies are not fully effective in treating osteoarthritis of the knee (OAK). We recently reported that transplantation of autologous chondrocyte cell-sheets along with open-wedge high tibial osteotomy promoted hyaline cartilage repair in humans. Here we describe our regenerative therapy for OAK using polydactyly-derived allogeneic chondrocyte cell-sheets (PD sheets) and temperature-responsive culture inserts. Ten patients with OAK and cartilage defects categorized arthroscopically as Outerbridge grade III or IV received the therapy. Cartilage viscoelasticity and thickness were assessed before and after transplantation. Arthroscopic biopsies obtained 12 months after transplantation were analyzed histologically. Gene expression was analyzed to evaluate the PD sheets. In this small initial longitudinal series, PD sheet transplantation was effective in treating OAK, as indicated by changes in cartilage properties. Gene marker sets in PD sheets may predict outcomes after therapy and provide markers for the selection of donor cells. This combined surgery may be an ideal regenerative therapy with disease-modifying effects in OAK patients.

In utero transplantation of myoblasts and adipose-derived mesenchymal stem cells to murine models of Duchenne muscular dystrophy does not lead to engraftment and frequently results in fetal death.

Introduction: Duchenne muscular dystrophy (DMD) is a progressive disease that leads to damage of muscle and myocardium due to genetic abnormalities in the dystrophin gene. In utero cell transplantation that might facilitate allogenic transplantation is worth considering to treat this disease. Methods: We performed allogeneic in utero transplantation of GFP-positive myoblasts and adipose-derived mesenchymal stem cells into murine DMD model animals. The transplantation route in this study was fetal intraperitoneal transplantation and transplacental transplantation. Transplanted animals were examined at 4-weeks old by immunofluorescence staining and RT-qPCR. Results: No GFP-positive cells were found by immunofluorescence staining of skeletal muscle and no GFP mRNA was detected by RT-qPCR in any animal, transplantation method and cell type. Compared with previous reports, myoblast transplantation exhibited an equivalent mortality rate, but adipose-derived stem cell (ASC) transplantation produced a higher mortality rate. Conclusions: In utero transplantation of myoblasts or ASCs to murine models of DMD does not lead to engraftment and, in ASC transplantation primarily, frequently results in fetal death.

Allogeneic transplantation of epidermal cell sheets followed by endoscopic submucosal dissection to prevent severe esophageal stricture in a porcine model.

Introduction: Endoscopic submucosal dissection (ESD) is a minimally invasive treatment for early esophageal cancer. However, large mucosal defects after esophageal ESD result in refractory strictures. In the present study, we histologically evaluated the endoscopic transplantation of allogeneic epidermal cell sheets (ECSs) as a feasible therapy for preventing esophageal stricture after circumferential ESD in a porcine model. Methods: Epidermal cells were isolated from the skin tissue of allogeneic pigs and cultured on temperature-responsive cell culture inserts for 2 weeks. Transplantable ECSs were harvested by reducing the temperature and endoscopically transplanting the sheets to ulcer sites immediately after esophageal ESD. The engraftment of transplanted ECSs was then evaluated in two pigs at 7 days after transplantation. Next, ten pigs were divided into two groups to evaluate the endoscopic transplantation of allogeneic ECSs for the prevention of esophageal strictures after ESD. Allogeneic ECSs were transplanted immediately after esophageal ESD in the transplantation group (n = 5), whereas the control group (n = 5) did not undergo transplantation. Results: Most of the transplanted allogeneic ECSs were successfully engrafted at the ulcer sites in the early phase. Fluorescence in situ hybridization analysis revealed that several allogeneic cells were present in the transplanted area at 7 days after ESD. At 14 days after ESD, significant differences in body weight loss, dysphagia scores, and mucosal strictures were observed between the control and transplantation groups. Transplanting allogeneic ECSs after esophageal ESD promotes mucosal healing and angiogenesis and prevents excessive inflammation and granulation tissue formation. Conclusions: Endoscopic and histological analyses revealed that allogeneic ECSs promoted artificial ulcer healing after ESD, preventing esophageal strictures after ESD.

Tissue-engineered nerve guides with mesenchymal stem cells in the facial nerve regeneration.

Nerve guides with mesenchymal stem cells have been investigated in the rat facial nerve defect model to promote peripheral nerve regeneration and shorten recovery time to improve patients' quality of life. A 7-mm facial nerve gap experimental rat model is frequently employed in facial nerve regeneration studies. Facial nerve regeneration with nerve guides is evaluated by (1) assessing myelinated fiber counts using toluidine blue staining, (2) immunohistological analysis, (3) determining the g-ratio (axon diameter/total outer diameter) of regenerated nerve on transmission electron microscopic images, (4) retrograde nerve tracing in the facial nucleus, (5) electrophysiological evaluations using compound muscle action potential, and (6) functional evaluations using rat facial palsy scores. Dental pulp and adipose-derived stem cells, easily harvested using a minimally invasive procedure, possess characteristics of mesenchymal tissue lineages and can differentiate into Schwann-like cells. Cultured dental pulp-derived cells can produce neurotrophic factors, including nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor. These neurotrophic factors promote peripheral nerve regeneration and afford protection against facial motor neuron death. Moreover, artificial nerve guides can maneuver axonal regrowth, and dental pulp-derived cells and adipose-derived Schwann cells may supply neurotrophic factors, promoting axonal regeneration. In the present review, the authors discuss facial nerve regeneration using nerve guides with mesenchymal stem cells.

Cytomegalovirus-associated esophagitis on early esophageal cancer in immunocompetent host: a case report.

BACKGROUND: Cytomegalovirus (CMV)-associated gastrointestinal diseases usually occur in immunocompromised patients; however, few cases has also been described in healthy hosts despite still unclear pathological mechanisms. CMV esophagitis causes various lesions, such as erythematous mucosa, erosions, and ulcers, although such inflammatory changes can appear in superficial esophageal cancers or in surrounding areas. CMV-associated esophagitis has been also reported in cancer patients, but typically in those with advanced and/or terminal stage cancers secondary to chemoradiotherapy-induced immunosuppression or the physiologic demands of the malignancy itself. To our best knowledge, we firstly report on an immunocompetent patient subject to endoscopic submucosal dissection (ESD) for early esophageal cancer complicated with CMV infection. CASE PRESENTATION: A 77-year-old man underwent esophagogastroduodenoscopy (EGD) at a local clinic. EGD revealed a lugol-unstained reddish lesion with whitish exudates in the middle-distal esophagus. Histological evaluation of lesion biopsy revealed atypical squamous epithelium with CMV-positive granulation tissue and aggregates of macrophages, prompting referral for further examination and treatment. Magnifying endoscopy with narrow-band imaging showed an erosive lesion with white moss in a well-demarcated brownish area with irregular mesh-like microvessels. ESD was performed for diagnosis and treatment. Histopathological examination of the resected specimen revealed superficial, moderately differentiated squamous cell carcinoma (SCC) with multiple lymphatic infiltration, and few CMV-positive cells were found in the erosive part of the SCC. Interestingly, he had no underlying conditions to predispose to CMV infection and no risk factors for esophageal cancer, other than gender and age. He received neither steroids for stricture prevention nor antiviral agents post-EGD and 4-month follow-up was negative for esophagitis. CONCLUSIONS: This is the first report of a case of CMV esophagitis superimposed on early esophageal cancer in an immunocompetent host and might provide valuable information for possible adverse effects of steroid administration during ESD procedures, despite their common use for prevention of post-ESD stricture.

Preclinical assessment of transplantable human nasal mucosal epithelial cell sheets.

INTRODUCTION: We previously reported a new cell transplantation therapy for patients with intractable otitis media using autologous nasal mucosal epithelial cell sheets, manufactured using temperature-responsive cell culture inserts. The current study aimed to verify whether the transplantable cell sheets could be manufactured for application in clinical trials, according to standard operational procedures (SOP), in a cell processing facility (CPF). METHODS: Human nasal mucosal epithelial cells from four volunteer donors were aseptically cultured and transplantable cell sheets successfully manufactured, with reproducibility, using temperature-responsive cell culture inserts in the CPF. During the manufacture of cell sheets, the CPF environment was confirmed to be aseptic by sterilization tests. Purity of the cell sheets was confirmed by histological analysis and flow cytometry. Both safety and quality of the human nasal mucosal epithelial cell sheets were validated. RESULTS: The cultured and manipulated human nasal mucosal epithelial cells showed no evidence of malignant transformation in vitro. The study confirmed the safety and suitability of the manufactured human nasal mucosal epithelial cell sheets for use in clinical trials. CONCLUSIONS: The results led to the establishment of a coherent system in which transplantation could be achieved smoothly.

Bio-artificial pleura using autologous dermal fibroblast sheets to mitigate air leaks during thoracoscopic lung resection.

Lung air leaks (LALs) due to visceral pleura injury during surgery are a difficult-to-avoid complication in thoracic surgery (TS). Reliable LAL closure is an important patient management issue after TS. We demonstrated both safeties of transplantation of a cultured human autologous dermal fibroblast sheet (DFS) to LALs. From May 2016 to March 2018, five patients who underwent thoracoscopic lung resection met all the inclusion criteria. Skin biopsies were acquired from each patient to source autologous dermal cells for DFS fabrication. During the primary culture, fibroblasts migrated from the dermal tissue pieces and proliferated to form cell monolayers. These fibroblasts were subcultured to confluence. Transplantable DFSs were fabricated from these subcultured fibroblasts that were trypsinized and seeded onto temperature-responsive culture dishes. After 10 days of fabrication culture, intact patient-specific DFS were harvested. DFSs were analyzed for fibroblast cell content and tissue contaminants prior to application. For closing intraoperative LAL, mean number of transplanted autologous DFS per patient was 6 ± 2 sheets. Mean chest drainage duration was 5.0 ± 4.8 days. The two patients with major LAL had a drainage duration of more than 7 days. All patients currently have no LAL recurrence after discharge. DFSs effectively maintain LAL closure via remodeling of the deposited extracellular matrix. The use of autologous DFSs to permanently close air leaks using a patient-derived source is expected to reduce surgical complications during high-risk lung resections.

Development of a nasal mucosa-removal model for evaluating cell therapy.

INTRODUCTION: Endoscopic sinus surgery is an effective surgical procedure for treating chronic sinusitis; however, extensive exposure of the bone in the nasal cavity can result in permanent disability postoperatively. Particularly, closure of the sinus drainage pathway due to bone hyperplasia associated with bone exposure can trigger the recurrence of sinusitis. It is essential to regenerate the nasal mucosa after surgery to avoid bone hyperplasia. Regenerative medicine, including cell therapy, could be one of the leading options for nasal mucosa regeneration. To date, there is a lack of effective models for evaluating treatments for prevention of bone hyperplasia that occurs after sinus surgery. The purpose of this study was to develop a model of nasal mucosal removal to evaluate cellular therapies. METHODS: The model was created in rabbits, a species with a wide nasal structure, and was generated by approaching the maxillary sinus from the nasal bone side and solely removing the maxillary sinus mucosa without destroying the structures in the nasal cavity. Adipose-derived mesenchymal stromal cell sheets prepared in temperature-responsive cell culture dishes were examined for the effect of transplantation in the animal model. Intranasal evaluation was assessed by micro-computed tomography and tissue staining. RESULTS: Significant bone hyperplasia in the maxillary sinus occurred on the side of mucosal removal, and no bone hyperplasia occurred in the control sham side in the same rabbits on postoperative day 28. Bone hyperplasia was observed over a short time period, with the presence of bone hyperplasia in the maxillary sinus on day 14 and calcification of the bone on day 28. The adipose-derived mesenchymal stromal cell (ADSC) sheet was transplantable in a nasal mucosa-removal model. No significant differences in bone hyperplasia were found between the transplantation side and the sham side in terms of the effect of transplantation of the ADSC sheet; however, bone hyperplasia tended to be suppressed on the transplantation side. CONCLUSIONS: This animal model is simple, highly reproducible, and does not require special equipment or drugs. In addition, this model can be used for various therapeutic interventions, including cell therapy. The presence or absence of the nasal mucosa affects bone remodeling, which highlights the importance of regeneration of the nasal mucosa. In the nasal mucosal regeneration therapy, the ADSC sheet had an inhibitory effect on bone hyperplasia. The nasal mucosa-removal model allows observation of conditions associated with nasal mucosa removal and evaluation of the effectiveness of cell therapy.

[Cancer Therapy with Cell Sheets].

We have developed cell sheet-based regenerative medicine, in which cell sheets are fabricated with temperature-responsive culture surfaces. We succeeded in clinical translation and large animal model experiments of cell sheet-based regenerative medicine to treat various complications of cancer therapy including esophageal stricture after esophageal early cancer endoscopic submucosal dissection(ESD)and lung air leaks. We would like to continue development of cell sheet-based regenerative medicine to treat frail, sarcopenia, and cancer cachexia after surgery, chemotherapy, and radio therapy by supplying stem cells and paracrine effects.

Transplantation of autologous oral mucosal epithelial cell sheets inhibits the development of acquired external auditory canal atresia in a rabbit model.

Acquired external auditory canal atresia is characterized by fibrous tissue formation in the ear canal, hearing loss and chronic otorrhea. Although the disease can be treated surgically, the recurrence rate is high. This study explored whether autologous oral mucosal epithelial cell sheets could be used as a novel therapy for ear canal atresia. We succeeded in generating a rabbit model of acquired external auditory canal atresia by dissecting the skin of the ear canal. Endoscopic and histological findings in this model indicated that atresia developed over a 4-week period and was not inhibited by the placement of polyglycolic acid sheets immediately after skin dissection. By contrast, transplantation of autologous oral mucosal epithelial cell sheets, which had been fabricated by culture on temperature-responsive inserts without a feeder layer, prevented the development of atresia during the 4-week period after skin dissection. Transplantation of autologous epithelial cell sheets after surgical treatment of acquired external auditory canal atresia could be a promising new method to reduce the risk of disease recurrence. STATEMENT OF SIGNIFICANCE: Acquired external auditory canal atresia is characterized by fibrous tissue formation in the ear canal, which leads to hearing loss and chronic otorrhea. Although surgical treatments are available, the recurrence rate is high. In this study, we successfully generated a rabbit model of acquired external auditory canal atresia by dissecting the skin of the ear canal. Furthermore, we utilized this new animal model to investigate whether the transplantation of autologous oral mucosal epithelial cell sheets could be used as a novel therapy for ear canal atresia. Our results raise the possibility that the transplantation of autologous epithelial cell sheets after surgical treatment of ear canal atresia could be a promising new method to reduce the risk of disease recurrence.

Evaluation of human keratinocyte sheets transplanted onto porcine excised esophagus after submucosal dissection in an ex vivo model.

BACKGROUND: The utility of endoscopic transplantation of epithelial cell sheets to ulcer sites after endoscopic submucosal dissection (ESD) has been shown to prevent scar stenosis after ESD of early esophageal cancer. Previously, our group reported use of an endoscopic transplantation device fabricated with a 3-dimensional printer. Cell sheets are transplanted to the esophageal wound site with the following procedure: first, a cell sheet harvested from temperature-responsive culture dishes is placed on the device's deflated balloon surface and transported to the wound site with endoscopic forceps; second, by applying pressure from inflating the balloon locally at the wound site, the cell sheet is successfully transferred and adhered to the wound tissue; third, the balloon is deflated, and the device is removed. By repeating the procedure, several cell sheets can be safely transplanted to a wider ESD area. Nonetheless, possible damage to cell sheets using this procedure has not yet been assessed. OBJECTIVE: Effects of endoscopic transplantation balloon inflation on cell viability and damage of normal human epidermal keratinocyte sheets resident on the device's balloon surface were evaluated by histology after sheet placement onto lumenal surfaces in the ex vivo porcine submucosal dissection esophagus model. Endoscopic transplantation of these same cell sheets with conventional methods using a polyvinylidene fluoride (PVDF) cell sheet support membrane, balloon device transfer, and also using a novel modified balloon transfer procedure was also examined. Cell sheet transfer results obtained with these three procedures were compared. METHOD: Normal human epidermal keratinocyte sheets were fabricated on temperature-responsive culture inserts. By temperature reduction to 20 °C, all cells were harvested as a single contiguous cell sheet. Freshly excised porcine esophagi purchased in a slaughter house were turned inside-out, and the exposed lumenal mucosa and submucosal layers were removed by Cooper scissors. This luminal surface was then utilized as a transplantation bed in ex vivo cell sheet experiments. Cell sheets were adhered to the endoscopic transfer device balloon, expanded by balloon inflation and resulting cell viability was evaluated by trypan blue exclusion test after cell sheet trypsinization and dispersion. Cell sheets were transferred onto the esophagus lumen ex vivo using forceps and the balloon device, and also using a modified balloon transfer method. The obtained results were compared with those without balloon expansion, and evaluated for sheet thickness and lumenal histology. Finally, TUNEL staining was performed to examine cell apoptosis. RESULT: Cell sheets thinned after cell sheet balloon expansion, but no apoptosis was observed after these procedures. CONCLUSION: Expanding keratinocyte cell sheets on a balloon endoscopic transfer device did not damage the cell sheets. This sheet transplantation method using the endoscopic balloon transfer device may be considered as a future standard cell sheet endoscopic transplantation procedure for repairing the esophagus.

Fabrication of tissue-engineered cell sheets by automated cell culture equipment.

Most cells for regenerative medicine are currently cultured manually. In order to promote the widespread use of regenerative medicine, it will be necessary to develop automated culture techniques so that cells can be produced in greater quantities at lower cost and with more stable quality. In the field of regenerative medicine technology, cell sheet therapy is an effective tissue engineering technique whereby cells can be grafted by attaching them to a target site. We have developed automated cell culture equipment to promote the use of this cell sheet regenerative treatment. This equipment features a fully closed culture vessel and circuit system that avoids contamination with bacteria and the like from the external environment, and it was designed to allow 10 cell sheets to be simultaneously cultured in parallel. We used this equipment to fabricate 50 sheets of human oral mucosal epithelial cells in five automated culture tests in this trial. By analyzing these sheets, we confirmed that 49 of the 50 sheets satisfied the quality standards of clinical research. To compare the characteristics of automatically fabricated cell sheets with those of manually fabricated cell sheets, we performed histological analyses using immunostaining and transmission electron microscopy. The results confirmed that cell sheets fabricated with the automated cell culture are differentiated in the same way as cultures fabricated manually.

Analysis of human nasal mucosal cell sheets fabricated using transported tissue and blood specimens.

Previously, we succeeded in transplanting autologous nasal mucosal cell sheets in the middle ears of 5 patients, who underwent cholesteatoma resection, which prevents recurrence of cholesteatoma in clinical settings. Current good manufacturing practice (GMP) standards for human cell cultivation requires the establishment of cell processing centers (CPC) which act as germ-free facilities. However, due to practical difficulties involved in establishing and maintaining such facilities at each individual hospital, a functional transport system is felt to be needed for the continuation of effective regenerative therapy. In the current study, nasal mucosal tissue and autologous blood obtained from 3 human volunteers were transported for over 3 h. Disinfected nasal tissues were cultured using keratinocyte culture medium, which included autologous serum prepared from blood. After 24 d, cultured nasal mucosal cells were transported for over 3 h and subsequently assessed for cell number, viability and purity. Moreover, CK4, CK8, and CK18 were analyzed the suitability of these nasal mucosal cell sheets for middle ear regenerative therapy. Overall, we confirmed that nasal mucosal cell sheets can be fabricated using transported nasal mucosal tissue and blood. This study would be contribute to establish a new regenerative therapy for clinical application, accompanied with transportation between companies and hospitals.