Anorectal Transplantation: The First Long-term Success in a Canine Model.

OBJECTIVE: Anorectal transplantation is a challenging procedure but a promising option for patients with weakened or completely absent anorectal function. SUMMARY BACKGROUND DATA: We constructed a canine model of anorectal transplantation, evaluated the long-term outcomes, and controlled rejection and infection in allotransplantation. METHODS: In the pudendal nerve function study, 6 dogs were randomly divided into 2 groups, transection and anastomosis, and were compared with a control using anorectal manometry, electromyography, and histological examination. In the anorectal transplantation model, 4 dogs were assigned to 4 groups: autotransplant, allotransplant with immunosuppression, allotransplant without immunosuppression, and normal control. Long-term function was evaluated by defecography, videography, and histological examination. RESULTS: In the pudendal nerve function study, anorectal manometry indicated that the anastomosis group recovered partial function 6 months postoperatively. Microscopically, the pudendal nerve and the sphincter muscle regenerated in the anastomosis group. Anorectal transplantation was technically successful with a 3-stage operation: colostomy preparation, anorectal transplantation, and stoma closure. The dog who underwent allotransplantation and immunosuppression had 2 episodes of mild rejection, which were reversed with methylprednisolone and tacrolimus. The dog who underwent allotransplantation without immunosuppression had a severe acute rejection that resulted in graft necrosis. Successful dogs had full defecation control at the end of the study. CONCLUSIONS: We describe the critical role of the pudendal nerve in anorectal function and the first long-term success with anorectal transplantation in a canine model. This report is a proof-of-concept study for anorectal transplantation as a treatment for patients with an ostomy because of anorectal dysfunction.

Pathological changes within two weeks following spinal cord injury in a canine model.

PURPOSES: This study aimed to investigate the histopathological changes that occur within 2 weeks following spinal cord injury (SCI) in dogs. METHODS: Eight adult female Beagle dogs were included in this study, and SCI was induced using an epidural balloon catheter. Two dogs were killed at each of the following four time points: immediately after the procedure and 1 day, 1 week, and 2 weeks after the procedure. Neurological status was evaluated with five categories. Histopathological changes were visually observed for stained sections of formalin-fixed spinal cord to evaluate hemorrhage, spongiosis, necrosis, and gliosis morphologically. RESULTS: Along the 2 weeks post-injury, severe hemorrhage was observed at the primary injury site, the average diameter of which expanded quickly from 8 to 10 mm in 1 day and then decreased to 5 mm in 1 week. This indicates that the bleeding cavity expanded at the initial injury site to produce ascending and descending hemorrhage. The hemorrhage at the injury site resolved in 2 weeks. In contrast, spongiosis, parenchymal necrosis, and gliosis were first inconspicuous or mild and then became severe in 1 week or 2 weeks. Hemorrhage, hematoma, and other similar changes occurred at the regions approximately 20-mm rostral and caudal to the primary injury site. These changes were observed in both gray matter and white matter. CONCLUSIONS: This study is the first to assess the sequential histopathological changes in the acute and intermediate phases following SCI in dogs. Our findings enhance the usefulness of the canine intervertebral disk disease model in the assessment of secondary spinal cord histopathology in human SCI.

Regenerated nephrons in kidney cortices ameliorate exacerbated serum creatinine levels in rats with adriamycin nephropathy.

Kidney regeneration could be classified into 2 groups: kidney generation and kidney repair. We have attempted in vivo nephron generation for kidney repair, as a therapy for chronic renal failure (CRF), by exploiting cellular interactions via conditioned media. In the previous report, we demonstrated the generation of rich nephrons in rat intact kidney cortices through percapsular injection of mesenchymal stem cell (MSC)-differentiated tubular epithelial cells (TECs) after pretreatment of 3-dimensional culture using a small amount of gel complex and condensed medium. In this study, to verify the amelioration of serum creatinine (sCr) levels by regenerated nephrons in rats with CRF, we first created damaged kidneys through systemic administration of adriamycin, and implanted the pretreated MSC-differentiated TECs into unilateral kidney cortices 2 weeks after adriamycin administration (A-2W, that is I-0W). After recovery of acute kidney injury, the control rats without cell implantation showed re-exacerbation of sCr levels, resulting in death within A-12W. Alternatively, the cell-implanted rats had a formation of mature nephrons in I-3W, and showed significant amelioration of sCr levels in I-7W. As a result, these rats could live until euthanization in I-12W or I-16W, indicating the utility of cell injection therapy into a kidney (K-CIT) for CRF. We expect that our K-CIT or the refined methods will be applied to patients with CRF.

Nephron generation in kidney cortices through injection of pretreated mesenchymal stem cell-differentiated tubular epithelial cells.

Transplantation of artificially treated metanephroi or pluripotent stem cell-injected blastocyst-derived whole kidneys will be established in the near future as a useful therapeutic method for renal failure. We have attempted in vivo nephron generation for kidney repair by exploiting cellular interactions via conditioned media (CMs). In a previous report, we showed stimulative cross-talks between vascular endothelial cells (VECs) and tubular epithelial cells (TECs) on cell proliferation and morphological changes, the differentiation of mesenchymal stem cells (MSCs) into TECs by TEC-CM, and nephron generation from TECs or MSCs in rat subcutaneous spaces. In this study adding collecting duct cells (CDCs) and their CM, we demonstrate the suppressive actions of CDC-CM against VECs and TECs, in addition to stimulative cross-talks between VECs and TECs, during the above changes. Furthermore, CDC-CM, similar to TEC-CM, caused differentiation of MSCs into TECs. Thus, we injected CDC-CM-induced MSC-differentiated TECs into rat kidney cortices. The pretreatment of cells in 3-dimensional culture using a small amount of gel complex before implantation triggered the generation of much more nephron-like structures, compared to the implantation of non-pretreated cells. Our method of injecting pretreated TECs into kidney cortices might have applications for repairing dysfunctional kidney tissue.

Long-term preservation of planar cell polarity in reversed tracheal epithelium.

BACKGROUND: Planar cell polarity (PCP) coordinates the patterning and orientation of cells and their structures along tissue planes, and although its acquisition during the formation of airway epithelium has been described, the mechanisms for its maintenance and reconstruction are poorly understood. We aimed to clarify whether ambient environment change by orthotropic autologous transplantation affected PCP at the cellular level. METHODS: We performed orthotropic autologous transplantation by inverting tracheal segments in rats, and then performed morphological evaluation by microscopy. The PCP of the tracheal epithelium was assessed over time by analyzing the directions of mucociliary transport and ciliary beat, the positional relationship between the basal body and basal foot, and the bias of Vang-like protein 1 (Vangl1) at 2, 4, and 6 months postoperatively. RESULTS: After 2 months, the directions of mucociliary transport and ciliary beat were preserved toward the lung in the inverted tracheal segments. The positional relationship between the basal body and the basal foot, and the bias of Vangl1, also indicated preservation of PCP in the inverted tracheal segments. Similar results were obtained at 6 months. CONCLUSION: The PCP of ciliated epithelium was preserved in reversed trachea, even after long-term observation.

Artificial sensory organs: latest progress.

This study introduces the latest progress on the study of artificial sensory organs, with a special emphasis on the clinical results of artificial nerves and the concept of in situ tissue engineering. Peripheral nerves have a strong potential for regeneration. An artificial nerve uses this potential to recover a damaged peripheral nerve. The polyglycolic acid collagen tube (PGA-C tube) is a bio-absorbable tube stuffed with collagen of multi-chamber structure that consists of thin collagen films. The clinical application of the PGA-C tube began in 2002 in Japan. The number of PGA-C tubes used is now beyond 300, and satisfactory results have been reported on peripheral nerve repairs. This PGA-C tube is also effective for patients suffering from neuropathic pain.

Development of a novel lung-stabilizing device for VATS procedures.

BACKGROUND: The use of video-assisted thoracoscopic surgery (VATS) has substantially increased in recent years. These procedures involve the insertion of specialized devices into the thoracic cavity via access ports. However, conventional devices such as cotton-tipped applicators and graspers can limit the field of view and injure the fragile lung tissue. The aim of this study was to develop a novel lung-stabilizing device for VATS that provides a good surgical field of view without causing lung injury. METHODS: We developed a novel suction-based lung-stabilizing device equipped with three hemispheric 20-mm-diameter silicon suction cups. The utility and safety of the novel device were evaluated using a resected pig lung and canine models. In order to assess potential organ damage arising from the use of the novel device, canine lung parenchyma and pleura were macroscopically and microscopically examined after the device had been continuously applied under negative pressure conditions of -400 or -540 mmHg for 1 h. RESULTS: To assess the utility of the novel device, we performed lobectomies in the resected pig lung and VATS in canine models. The device demonstrated sufficient power to stabilize the lungs and provided a clear field of view during surgery, which enabled us to perform VATS lobectomies more easily than conventional stabilizing forceps. Assessment of the dogs' lungs immediately after detaching the suction-based device revealed no complications such as hemorrhage, air leaks, and bullae formation. Pathological examination after 7 days also showed no substantial damage, except for a small impression in the parenchyma and pleura of the surface layer where the device had contacted the lung tissue. CONCLUSIONS: Although further validation studies in clinical settings are required, our study indicates that the novel lung-stabilizing device has potentially useful applications in VATS procedures.

Localizing small lung lesions in video-assisted thoracoscopic surgery via radiofrequency identification marking.

BACKGROUND: To facilitate accurate localization of small lung lesions in thoracoscopic surgery, we employed a micro-radiofrequency identification tag designed to be delivered through the 2-mm working channel of a flexible bronchoscope. This report presents the results of preclinical studies of our novel localizing technique in a canine model. METHODS: To evaluate functional placement, three types of tags [Group A, tag alone (n = 18); Group B, tag + resin anchor (n = 15); and Group C, tag + NiTi coil anchor (n = 15)] were bronchoscopically placed in subpleural areas and subsegmental bronchi via our new delivery device; tags were examined radiographically on days 0-7 and day 14. In addition, eight tags, which were placed at a mean depth of 13.3 mm (range 9-15.7 mm) from visceral pleura in bronchi with a mean diameter of 1.46 mm (range 0.9-2.3 mm), were recovered by partial lung resection under video-assisted thoracoscopic surgery using a 13.56-MHz wand-shaped probe with a 30-mm communication range. RESULTS: Peripheral airway placement: Group C had a significantly higher retention rate than the other two groups (retention rate at day 14: Group A, 11.1 %; Group B, 26.7 %; Group C, 100.0 %; P < 0.0001). Central airway placement: Overall retention rate was 73.3 % in Group C, and placement was possible in bronchi of up to 3.3 mm in diameter. Outcomes of partial resection: Tag recovery rate was 100 %, mean time required for tag detection was 10.8 s (range 8-15 s), and mean surgical margin from the delivered tag was 9.13 mm (range 6-13 mm). CONCLUSION: Radiofrequency identification marking enabled accurate localization with depth, which could ensure effective deep resection margins.

Prognosis After Surgical Treatment of Trigeminal Neuropathy with a PGA-c Tube: Report of 10 Cases.

OBJECTIVE: Although surgery using a polyglycolic acid-collagen (PGA-c) tube is effective for peripheral nerve injury-induced chronic hand pain, it has not been applied to trigeminal nerve lesions because of the difficult approach. We used a PGA-c tube during surgery for trigeminal neuropathy and evaluated its prognosis based on the outcomes. DESIGN: Case report. SETTING AND PATIENTS: In the dental anesthesia division of a university hospital, 11 patients with severe dysesthesia underwent surgical repair of a damaged lingual nerve (LN) or inferior alveolar nerve (IAN). One patient was lost to follow-up. Changes in quantitative sensory testing (QST) and the presence of dysesthesia as a treatment outcome were compared preoperatively and postoperatively in 10 patients. Two surgical treatments, bridging or encircling peripheral nerves, were applied. Bridging of both stumps was selected when neurotmesis was detected or the nerve was lacerated during surgery (N = 4). Otherwise, a longitudinal PGA-c tube was used to encircle the lesion (N = 6). Outcomes were evaluated 2 months to 8 years postoperatively. RESULTS: Both methods improved the patients' condition based on QST results (brush stroke perception, mechanical touch threshold, sensitivity to cold/hot stimuli). Preoperative allodynia or dysesthesia was resolved in six patients and greatly reduced in four. Two patients (one with inflammation-induced pain, one with implant-related pain) developed prolonged postoperative allodynia requiring pain-relief medication. CONCLUSIONS: Use of a PGA-c tube for surgical treatment of intractable pain due to LN or IAN neuropathy helps alleviate sensory impairment. The possibility of new dysesthesias emerging postoperatively, however, should be noted.

Effect of Structural Differences in Collagen Sponge Scaffolds on Tracheal Epithelium Regeneration.

OBJECTIVE: We developed an in situ regeneration-inducible artificial trachea composed of a porcine collagen sponge and polypropylene framework and used it for tracheal reconstruction. In the present study, collagen sponges with different structures were prepared from various concentrations of collagen solutions, and their effect on the regeneration of tracheal epithelium was examined. METHODS: Collagen sponges were prepared from type I and III collagen solutions. The structures of the sponges were analyzed using scanning electron microscopy (SEM). Artificial tracheae, which were formed using the collagen sponges with different structures, were implanted into rabbits, and regeneration of the tracheal epithelium on the artificial tracheae was evaluated by SEM analysis and histological examination. RESULTS: The SEM analysis showed that collagen sponges prepared from 0.5% and 1.0% collagen solutions had a porous structure. However, the sponges prepared from a 1.5% collagen solution had a nonporous structure. After implantation of artificial tracheae prepared from 0.5% and 1.0% collagen solutions, their luminal surfaces were mostly covered with epithelium within 14 days. However, epithelial reorganization occurred later on artificial tracheae prepared from the 1.5% collagen solution. CONCLUSION: Collagen sponges with a porous structure are suitable for regeneration of the tracheal epithelium in our artificial trachea.

Biocompatibility and efficacy of collagen/gelatin sponge scaffold with sustained release of basic fibroblast growth factor on vocal fold fibroblasts in 3-dimensional culture.

OBJECTIVE: Treatment of vocal fold scarring remains challenging. We have previously reported the therapeutic effects of local injection of basic fibroblast growth factor (bFGF) in animal models and humans. A novel collagen/gelatin sponge (CGS) is capable of sustained release of bFGF, which compensates for its quick absorption in vivo, avoiding multiple injections. This study aimed to evaluate the biocompatibility and efficacy of the CGS in rat vocal fold fibroblasts prior to human trials. METHODS: Fibroblasts extracted from Sprague-Dawley rat vocal folds were seeded onto a CGS and then cultivated with bFGF at concentrations of 0, 10, and 100 ng/mL. Vocal fold fibroblast morphology, adhesion, proliferation, and gene expression were measured under these 3-dimensional conditions. RESULTS: Cells adhered to the CGS from day 1. Although no significant differences in cell morphology were detected, cell proliferation was accelerated by bFGF administration. Expression of endogenous bFGF and hepatocyte growth factor was significantly up-regulated at 10 ng/mL bFGF. The expression of procollagen I and procollagen III was significantly suppressed, whereas HAS-1 and HAS-2 were up-regulated at 10 and 100 ng/mL bFGF. CONCLUSION: The collagen/gelatin sponge is biocompatible with vocal fold fibroblasts and may be useful as a bFGF drug delivery system for the treatment of scarred vocal folds.

Effect of Intratracheal Administration of Adipose-derived Stromal Cells on Bleomycin-induced Lung Injury in a Rat Model.

BACKGROUND: Mesenchymal stromal cells (MSCs) have been intensively investigated in regenerative medicine. Among the different types of MSCs, adipose tissue-derived stromal cells (ASCs) can be obtained with relatively less invasive techniques. Since ASC administration is a candidate strategy for the treatment of refractory diseases including pulmonary fibrosis, we investigated whether intratracheal injection of ASCs had therapeutic potential against bleomycin (BLM)-induced lung injury in rats. METHODS: BLM was intratracheally administered to rats, and 1 week later ASCs were harvested. Two weeks after BLM treatment, ASCs or phosphate-buffered saline (PBS) were injected autologously into the rats via the trachea A semi-quantitative histological evaluation was conducted to assess the injured lungs, followed by cell tracing at 3 or 6 weeks after BLM instillation. RESULTS: ASC administration did not affect the severity of lung damage on the third week after BLM exposure, but prevented further aggravation of the lung injury, as apparent on the sixth week. A fluorescent cell tracer revealed that the majority of ASCs did not appear to have penetrated inside the lung region injured by BLM on the third week after BLM instillation, but some of these cells sprouted deep into the thick distorted architecture of the injured lung on the sixth week after the BLM instillation. CONCLUSIONS: The results of the present study suggest that ASCs may play a role in the prevention of ongoing aggravation of lung injury in the long term.

Development of a novel marking system for laparoscopic gastrectomy using endoclips with radio frequency identification tags: feasibility study in a canine model.

BACKGROUND: Intraoperative identification of early gastric cancer is difficult to conduct during laparoscopic procedures. In this study, we investigated the feasibility and accuracy of a newly developed marking system using endoclips with radio frequency identification (RFID) tags in a canine model. METHODS: RFID is a wireless near field communication technology. Among the open frequency bands available for medical use, 13.56 MHz is suitable for a surgical marking system because of the similar and linear signal decay both in air and in biological tissues. The proposed system consists of four parts: (a) endoclips with RFID tags, (b) endo-clip applier equipment, (c) laparoscopic locating probe, and (d) signal processing units with audio interface. In the experimental setting using canine models, RFID-tagged endoclips were applied to the mucosa of each dog's stomach. During the subsequent operation, the clips with RFID tags placed in five dogs were located by the detection of the RFID signal from the tag (RFID group), and the conventional clips in the other six dogs were located by finger palpation (FP group). The detected sites were marked by ablation on the serosal surface. Distance between the clips and the metal pin needles indicating ablated sites were measured with X-ray radiographs of the resected specimen. RESULTS: All clips were successfully detected by the marking system in the RFID group (10/10) and by finger palpation in the FP group (17/17). The medians of detection times were 31.5 and 25.0 s, respectively; the distances were 5.63 and 7.62 mm, respectively. The differences were not statistically significant. No adverse event related to the procedures was observed. CONCLUSIONS: Endoclips with RFID tags were located by our novel marking system in an experimental laparoscopic setting using canine stomachs with substantial accuracy comparable to conventional endoclips located by finger palpation through an open approach.

Cellular interactions via conditioned media induce in vivo nephron generation from tubular epithelial cells or mesenchymal stem cells.

There are some successful reports of kidney generation by utilizing the natural course of kidney development, namely, the use of an artificially treated metanephros, blastocyst or ureteric bud. Under a novel concept of cellular interactions via conditioned media (CMs), we have attempted in vivo nephron generation from tubular epithelial cells (TECs) or mesenchymal stem cells (MSCs). Here we used 10× CMs of vascular endothelial cells (VECs) and TECs, which is the first to introduce a CM into the field of organ regeneration. We first present stimulative cross-talks induced by these CMs between VECs and TECs on cell proliferation and morphological changes. In MSCs, TEC-CM suppressed these changes, however, induced cytokeratin expression, indicating the differentiation of MSCs into TECs. As a result, glomerular and tubular structures were created following the implantation of TECs or MSCs with both CMs. Our findings suggest that the cellular interactions via CMs might induce in vivo nephron generation from TECs or MSCs. As a promoting factor, CMs could also be applied to the regeneration of other organs and tissues.

Adipose-derived stem cells enhance tissue regeneration of gastrotomy closure.

BACKGROUND: The aim of this study was to examine whether transplantation of adipose-derived stem cells (ADSCs) improves healing of a gastrotomy closure in rats. In digestive surgery, anastomotic leakage is a serious postoperative complication and anastomotic stenosis may reduce quality of life. Recent studies have suggested that ADSCs play material roles in intestinal healing, acceleration of angiogenesis, and reduction of fibrosis, and treatment with ADSCs may improve healing. MATERIALS AND METHODS: ADSCs were isolated from intra-abdominal white adipose tissue of 40 male Wistar rats (weight 300 g) in four groups (n = 10 each). Gastrotomy closures were prepared surgically in all rats. Controls were treated with phosphate-buffered saline injection and sacrificed 7 d (group 1) or 28 d (group 3) after the surgery. Other animals were treated with locally autotransplanted ADSCs (labeled by CM-DiI) and sacrificed 7 d (group 2) or 28 d (group 4) after the surgery. Histopathologic features were evaluated in the four groups. RESULTS: Injection of ADSCs significantly enhanced angiogenesis and collagen deposition after 7 d, indicating improved healing of the gastrotomy closure. In contrast, ADSC transplantation significantly reduced collagen deposition after 28 d. The bursting pressure was higher in the transplant groups after 7 d. CONCLUSIONS: ADSCs enhance tissue regeneration in gastrotomy closures by accelerating angiogenesis and fibrosis in the early healing period. In the late period, ADSCs prevent excessive fibrosis and assist in regeneration of tissues that closely resemble the native structure. These results suggest that therapy with transplanted ADSCs might improve postoperative complications in digestive surgery.

Intrahepatic biliary ablation with pure ethanol: an experimental model of biliary atresia.

PURPOSE: We describe a new rat model of biliary atresia, induced by biliary ablation with pure ethanol. METHODS: A catheter was inserted and fixed in the common bile duct of male rats. Saline or pure ethanol was injected through the catheter and the animals were monitored for 8 weeks thereafter. We measured total bilirubin (T-Bil), aspartate aminotransferase (AST), alanine transaminase (ALT), and hyaluronic acid (HA) and examined liver biopsy specimens immunohistochemically for α-smooth muscle actin staining (α-SMA) and transforming growth factor-ß1 (TGF-ß1). RESULTS: The ethanol injection group animals were further divided into a temporary and a persistent liver dysfunction group. In the persistent group, T-Bil, AST, ALT and HA levels were significantly higher after 8 weeks in the persistent group than in the control group and the temporary group. In the ethanol injection group, α-SMA expression was prominent in the surrounding proliferative bile ducts and portal areas. The distribution of TGF-ß1 was found prominently in hepatocytes in the center of nodules and in ductular epithelial cells. CONCLUSIONS: This study characterizes the effects of ethanol-induced bile duct injury in rats, resulting in sclerosing cholangitis and its secondary effects. We believe that this experimental model will prove useful in the study of biliary atresia.

Transplantation of autologous bone marrow-derived mononuclear cells into cerebrospinal fluid in a canine model of spinal cord injury.

Introduction: Spinal cord injury (SCI) is associated with severe dysfunction of nervous tissue, and repair via the transplantation of bone marrow-derived mononuclear cells (BM-MNCs) into cerebrospinal fluid yields promising results. It is essential to understand the underlying mechanisms; therefore, this study aimed to evaluate the regenerative potential of autologous BM-MNC transplantation in a canine model of acute SCI. Methods: Six dogs were included in this study, and SCI was induced using an epidural balloon catheter between L2 and L3, particularly in the area of the anterior longitudinal ligament. BM-MNC transplantation was performed, and T2-weighted magnetic resonance imaging (MRI) was conducted at specific time points (i.e., immediately after inducing SCI and at 1, 2, and 4 weeks after inducing SCI); moreover, the expression of growth-associated protein 43 (GAP-43) was evaluated. Results: MRI revealed that the signal intensity reduced over time in both BM-MNC-treated and control groups. However, the BM-MNC-treated group exhibited a significantly faster reduction than the control group during the early stages of SCI induction (BM-MNC-treated group: 4.82 ± 0.135 cm [day 0], 1.71 ± 0.134 cm [1 week], 1.37 ± 0.036 cm [2 weeks], 1.21 cm [4 weeks]; control group: 4.96 ± 0.211 cm [day 0], 2.49 ± 0.570 cm [1 week], 1.56 ± 0.045 cm [2 weeks], 1.32 cm [4 weeks]). During the early stages of treatment, GAP-43 was significantly expressed at the proximal end of the injured spinal cord in the BM-MSC-treated group, whereas it was scarcely expressed in the control group. Conclusions: In SCI, transplanted BM-MNCs can activate the expression of GAP-43, which is involved in axonal elongation (an important process in spinal cord regeneration). Thus, cell therapy with BM-MNCs can provide favorable outcomes in terms of better regenerative capabilities compared with other therapies.

Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia.

Induced pluripotent stem (iPS) cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large amounts of disease-specific cells for biomedical research. Despite their great potential, the long reprogramming process (up to 1month) remains one of the most significant challenges facing standard virus-mediated methodology. In this study, we report the accelerated generation of human iPS cells from adipose-derived stem (ADS) cells, using a new combination of chemical inhibitors under a setting of physiological hypoxia in conjunction with retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Under optimized conditions, we observed human embryonic stem (ES)-like cells as early as 6 days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate, morphology, pluripotency-associated markers, global gene expression patterns, genome-wide DNA methylation states, and the ability to differentiate into all three of the germ layers, both in vitro and in vivo. Our method, when combined with chemical inhibitors under conditions of physiological hypoxia, offers a powerful tool for rapidly generating bona fide human iPS cells and facilitates the application of iPS cell technology to biomedical research.

Expression of reactive oxygen species during wound healing of vocal folds in a rat model.

OBJECTIVES: Previous studies have indicated that although normal wound healing requires low levels of reactive oxygen species (ROS), excessive amounts of ROS impair wound healing. In injured vocal folds, this excess may result in dysphonia due to scarring that is difficult to treat. However, the expression of ROS during vocal fold wound healing has yet to be investigated. In this study, we assessed the expression and localization of ROS in injured vocal folds by immunohistochemical analysis. METHODS: Vocal folds of Sprague-Dawley rats were unilaterally injured by stripping the mucosa under transoral endoscopy. The larynges were harvested at specific time points after injury and were immunohistochemically examined for 4-hydroxy-2-nonenal (4-HNE), an ROS marker, and for the presence of inflammatory cells. RESULTS: We found that 4-HNE-immunopositive cells were significantly increased in the lamina propria of the injured vocal folds as compared to the normal vocal folds on postinjury days 1 and 3. More than half of the 4-HNE-immunopositive cells were also immunopositive for a macrophage- and granulocyte-specific antibody. CONCLUSIONS: This study suggests that a large amount of ROS is produced during early-phase wound healing, until postinjury day 3, and that this period may be crucial for regulating ROS levels. The results also suggest that inflammatory cells may contribute to ROS generation.

Regeneration of tracheal epithelium using a collagen vitrigel-sponge scaffold containing basic fibroblast growth factor.

OBJECTIVES: Our group has had good results in tracheal mucosal regeneration using a collagen vitrigel-sponge scaffold in an animal model. In this study, the effectiveness of this scaffold with the application of basic fibroblast growth factor (b-FGF) was investigated. METHODS: A collagen vitrigel-sponge scaffold was fabricated with simultaneous addition of b-FGF. Three types of collagen vitrigel-sponge scaffolds were made: no b-FGF, 10 ng of b-FGF, and 100 ng of b-FGF. At 3, 5, 7, and 14 days after implantation in rats, the tracheas were removed and histologically evaluated. The regeneration of mucosal epithelium and the subepithelial layer was evaluated. RESULTS: Mucosal epithelium, including pseudostratified epithelium and ciliated cells, regenerated earlier in the scaffolds when b-FGF was applied than when b-FGF was not applied. Regeneration of the subepithelial layer, infiltration of inflammatory cells and fibroblasts, and angiogenesis were promoted earlier in the scaffolds with b-FGF application. CONCLUSIONS: Our technique for tracheal reconstruction using collagen vitrigel-sponge scaffolds with b-FGF application affords a feasible approach for accelerating the regeneration of the intraluminal surface and subepithelial layer of tracheal tissue.