Takayuki Masuko

Dedifferentiated fat cells convert to cardiomyocyte phenotype and repair infarcted cardiac tissue in rats

Adipose tissue-derived stem cells have been demonstrated to differentiate into cardiomyocytes and vascular endothelial cells. Here we investigate whether mature adipocyte-derived dedifferentiated fat (DFAT) cells can differentiate to cardiomyocytes in vitro and in vivo by establishing DFAT cell lines via ceiling culture of mature adipocytes. DFAT cells were obtained by dedifferentiation of mature adipocytes from GFP-transgenic rats. We evaluated the differentiating ability of DFAT cells into cardiomyocytes by detection of the cardiac phenotype markers in immunocytochemical and RT-PCR analyses in vitro. We also examined effects of the transplantation of DFAT cells into the infarcted heart of rats on cardiomyocytes regeneration and angiogenesis. DFAT cells expressed cardiac phenotype markers when cocultured with cardiomyocytes and also when grown in MethoCult medium in the absence of cardiomyocytes, indicating that DFAT cells have the potential to differentiate to cardiomyocyte lineage. In a rat acute myocardial infarction model, transplanted DFAT cells were efficiently accumulated in infarcted myocardium and expressed cardiac sarcomeric actin at 8 weeks after the cell transplantation. The transplantation of DFAT cells significantly (p<0.05) increased capillary density in the infarcted area when compared with hearts from saline-injected control rats. We demonstrated that DFAT cells have the ability to differentiate to cardiomyocyte-like cells in vitro and in vivo. In addition, transplantation of DFAT cells led to neovascuralization in rats with myocardial infarction. We propose that DFAT cells represent a promising candidate cell source for cardiomyocyte regeneration in severe ischemic heart disease.

Single-incision laparoscopic-assisted appendectomy in children: exteriorization of the appendix is a key component of a simple and cost-effective surgical technique

PurposeSingle-incision laparoscopic appendectomy was recently introduced as a less-invasive surgical technique than existing methods, and is increasingly being performed worldwide. We have started to perform transumbilical laparoscopic-assisted appendectomy (TULAA) in children. In this study, we assessed its feasibility, efficacy, and cost-effectiveness compared with conventional multiport laparoscopic appendectomy (MLA).MethodsWe retrospectively reviewed all of the patients who underwent MLA or TULAA between August 2009 and December 2012. Patient characteristics, operative time, length of hospital stay, postoperative complications, cost-effectiveness, and cosmetic results were analyzed.ResultsA total of 88 patients underwent laparoscopic appendectomy, with MLA in 48 patients and TULAA in 40 patients. TULAA was successful in 36/40 patients. There were no significant differences in mean operative time or mean length of stay between the two groups of patients. There were no postoperative complications in the TULAA group. The cost of TULAA was much lower than that of MLA. TULAA also provided nearly scarless surgery.ConclusionTULAA, in which the appendix is exteriorized via a combination of open and laparoscopic techniques, is a safe and effective procedure for treating appendicitis in children. TULAA is a cost-effective treatment that provides excellent cosmetic results.

Long-term follow-up of nutritional status, pancreatic function, and morphological changes of the pancreatic remnant after pancreatic tumor resection in children.

Objectives The objectives of the present study were to determine nutritional status, pancreatic function, and morphological changes of the pancreatic remnant after pancreatic tumor resection in children. Methods The nutritional status was evaluated by the patterns of growth. Pancreatic function was evaluated by using a questionnaire, the Bristol stool form chart, the serum levels of fasting blood glucose, and hemoglobin A1c (HbA1c). Morphological changes of the pancreatic remnant were evaluated by computed tomography, magnetic resonance image, or magnetic resonance cholangiopancreatography. Results The present study consisted of 6 patients with pancreatic tumor (5 solid pseudopapillary tumors of the pancreas and 1 pancreatoblastoma) who underwent the following operations: tumor enucleation (3), distal pancreatectomy with splenectomy (1), and pylorus-preserving pancreatoduodenectomy (PPPD [2]). The serum levels of HbA1c have been gradually elevated in 2 patients with PPPD. A significant decrease in pancreatic parenchymal thickness and dilatation of the main pancreatic duct were observed in 2 patients with PPPD. Conclusion Endocrine pancreatic insufficiency after PPPD may be explainable by obstructive pancreatitis after operation. Taking together the results of pancreatic endocrine function and morphological changes of pancreatic remnant after PPPD, tumor enucleation should be considered as surgical approach in children with pancreas head tumor whenever possible.

Mouse embryonic stem cells give rise to gut-like morphogenesis, including intestinal stem cells, in the embryoid body model

Embryonic stem (ES) cells have been proposed as candidates for cell replacement therapy in patients with intestinal failure because these cells can be expanded indefinitely without losing their pluripotent phenotype. We investigated the differentiation capacity of mouse ES cells into gut-like structures, including intestinal stem cells, and defined culture conditions for efficient induction of formation of these structures. ES cell-derived gut-like structures (ES-guts) were reproducibly induced in developing embryoid bodies (EBs) by day 21 of differentiation culture. ES-guts contained an endodermal epithelium, a smooth muscle layer, interstitial cells of Cajal, and enteric neurons and showed spontaneous contraction. Transplantation of ES-guts under the kidney capsules of immunodeficient mice induced formation of highly differentiated epithelium composed of absorptive cells and goblet cells in the grafts. Immunoreactivity for Musashi-1 (Msi-1), a marker of intestinal stem cells, was detected in 1.9% of the columnar epithelial cells in the graft. Culture with 0.1% dimethyl sulfoxide increased the numbers of ES-guts in EBs, and serum-replacement (SR) culture, in comparison to standard ES culture containing 15% serum, increased the area ratio of ES-guts to EBs. SR culture also promoted maturation of epithelium to form a single layer of columnar epithelial cells, including absorptive cells and goblet cells. Expression of Msi-1 mRNA and protein was significantly enhanced when EBs were cultured under SR conditions. In conclusion, SR conditions efficiently induce formation of ES-guts and promote differentiation of epithelium, including intestinal stem cells. These results suggest the feasibility of cell-based therapy for intestinal failure based on ES cell culture systems.

Complete Thoracoscopic Versus Video-Assisted Thoracoscopic Resection of Congenital Lung Lesions

BACKGROUND Minimally invasive surgery is recognized as a safe and feasible technique for resecting congenital lung lesions. In our hospital, video-assisted thoracoscopic surgery (VATS) was initially performed through a 5-6-cm incision with several trocars under differential lung ventilation (assisted-VATS). Complete thoracoscopic surgery (complete-VATS) with artificial pneumothorax was introduced in 2009 and allowed surgery in smaller infants. The aim of this study was to compare the outcomes of complete-VATS and assisted-VATS for congenital lung lesions. PATIENTS AND METHODS Between January 2004 and October 2012, 22 children underwent pulmonary lobectomy by complete-VATS or assisted-VATS at our hospital. We retrospectively reviewed the intraoperative and early postoperative results of these patients. RESULTS Of the 22 children, 10 underwent assisted-VATS, and 12 underwent complete-VATS to treat various diseases, including congenital cystic adenomatous malformation, sequestration, and bronchial atresia. The age, body weight, and mean operative time were not significantly different between the two groups. Four patients in the complete-VATS group, but none in the assisted-VATS group, weighed <10 kg. Intraoperative bleeding was significantly less, and hospital stay was significantly shorter, in the complete-VATS group. One patient in the assisted-VATS group had intraoperative bleeding and required conversion to open surgery. Postoperative complications included transient paralysis of the affected arm and transient atelectasis in 1 patient each in the complete-VATS group. CONCLUSIONS Complete-VATS can be safely performed with less bleeding and shorter hospital stay than assisted-VATS. As differential lung ventilation is not essential during complete-VATS, complete-VATS can be performed in small infants.

Non-promoter DNA hypermethylation of Zygote Arrest 1 (ZAR1) in neuroblastomas

BACKGROUND The comprehensive methylation analysis of tumor-specific differently methylated regions in malignant melanomas and brain tumors has led to the identification of non-promoter hypermethylation of zygote arrest 1 (ZAR1). To search the non-promoter ZAR1 hypermethylation in neuroblastomas, we analyzed the levels of the methylation and transcript expression of ZAR1. METHODS The MassARRAY® EpiTYPER (Sequenom Inc., San Diego, CA, USA) system was optimized to determine the quantitative methylation levels of ZAR1 for 12 neuroblastoma cell lines, 23 neuroblastoma samples and four adrenal samples. ZAR1 expression levels were evaluated through a quantitative, real-time reverse transcription-polymerase chain reaction. The quantitative methylation levels of ZAR1 were subjected to correlation studies with the established markers of progressive disease and outcome. RESULTS Strikingly, the hypermethylation of ZAR1 regions and ZAR1 expression levels was observed in the neuroblastoma cell lines and neuroblastoma samples, compared to the adrenal samples. Somatic changes in ZAR1 methylation and ZAR1 expression were found in all three neuroblastoma patients. In the ZAR1 regions, poor-outcome tumors that were MYCN-amplified and/or Stage 3 or 4 and/or the age at diagnosis was≥18months, and/or showed an unfavorable histology were frequently hypermethylated. CONCLUSION Our results indicate that the hypermethylation of ZAR1 regions is extremely frequent in neuroblastomas and correlates with established markers of progressive disease and outcome.

Identification of aberrant methylation regions in neuroblastoma by screening of tissue-specific differentially methylated regions†‡§

The identification of tissue‐specific differentially methylated regions (tDMRs) is key to our understanding of mammalian development. Research has indicated that tDMRs are aberrantly methylated in cancer and may affect the oncogenic process.

Urethroscopic holmium: YAG laser ablation for acquired posterior urethral diverticulum after repair of anorectal malformations

Two patients with acquired posterior urethral diverticulum that is a complication of laparoscopic assisted anorectoplasty underwent urethroscopic holmium: YAG laser ablation. After the ablation therapies, the size of the diverticulum markedly decreased in both patients. Holmium: YAG laser is safe and easy to handle in the small pediatric urethra.

Diagnostic laparoscopy for neonatal perforated Meckel's diverticulum.

Pneumoperitoneum in a neonate is a serious condition for which bowel perforation is indicated in most cases. Because the transdiaphragmatic air dissection could occur in some ventilated neonates without peritonitis, making the right diagnosis is sometimes difficult, and exploratory laparotomy is often necessary. Here, we report the first case of neonatal pneumoperitoneum caused by a perforated Meckel′s diverticulum in which diagnostic laparoscopy was useful in achieving minimal access surgery. Using a multiuse single-site port on the umbilicus could enable the extraction of a certain length of a small intestine with good cosmetic results. This method would decrease the hurdles of early surgical interventions for the suspected perforation of unknown aetiology and shorten the fasting period, which is beneficial for the neurodevelopment of small neonates.

Influence of surgical intervention on neurodevelopmental outcome in infants with focal intestinal perforation.

Intestinal perforation is known to correlate with neurodevelopmental outcome in very low‐birthweight (VLBW) infants, and its two major causes are necrotizing enterocolitis (NEC) and focal intestinal perforation (FIP). Infants with FIP are reported to have better neurodevelopmental outcome than infants with NEC, but outcome has not been compared with that in infants without diseases that require surgery. The aim of this study was to compare neurodevelopmental outcomes between FIP survivors and infants without diseases that require surgery.