Koichiro Yoshimaru

In vivo hepatogenic capacity and therapeutic potential of stem cells from human exfoliated deciduous teeth in liver fibrosis in mice.

IntroductionLiver transplantation is a gold standard treatment for intractable liver diseases. Because of the shortage of donor organs, alternative therapies have been required. Due to their potential to differentiate into a variety of mature cells, stem cells are considered feasible cell sources for liver regeneration. Stem cells from human exfoliated deciduous teeth (SHED) exhibit hepatogenic capability in vitro. In this study, we investigated their in vivo capabilities of homing and hepatocyte differentiation and therapeutic efficacy for liver disorders in carbon tetrachloride (CCl4)-induced liver fibrosis model mice.MethodsWe transplanted SHED into CCl4-induced liver fibrosis model mice through the spleen, and analyzed the in vivo homing and therapeutic effects by optical, biochemical, histological, immunological and molecular biological assays. We then sorted human leukocyte antigen-ABC (HLA-ABC)-positive cells from primary CCl4-damaged recipient livers, and analyzed their fusogenicity and hepatic characteristics by flow cytometric, genomic DNA, hepatocyte-specific gene assays. Furthermore, we examined the treatment effects of HLA-positive cells to a hepatic dysfunction by a secondary transplantation into CCl4-treated mice.ResultsTransplanted SHED homed to recipient livers, and expressed HLA-ABC, human hepatocyte specific antigen hepatocyte paraffin 1 and human albumin. SHED transplantation markedly recovered liver dysfunction and led to anti-fibrotic and anti-inflammatory effects in the recipient livers. SHED-derived HLA-ABC-positive cells that were sorted from the primary recipient liver tissues with CCl4 damage did not fuse with the host mouse liver cells. Sorted HLA-positive cells not only expressed human hepatocyte-specific genes including albumin, cytochrome P450 1A1, fumarylacetoacetase, tyrosine aminotransferase, uridine 5′-diphospho-glucuronosyltransferase, transferrin and transthyretin, but also secreted human albumin, urea and blood urea nitrogen. Furthermore, SHED-derived HLA-ABC-positive cells were secondary transplanted into CCl4-treated mice. The donor cells homed into secondary recipient livers, and expressed hepatocyte paraffin 1 and human albumin, as well as HLA-ABC. The secondary transplantation recovered a liver dysfunction in secondary recipients.ConclusionsThis study indicates that transplanted SHED improve hepatic dysfunction and directly transform into hepatocytes without cell fusion in CCl4-treated mice, suggesting that SHED may provide a feasible cell source for liver regeneration.

Reevaluation of acetylcholinesterase staining for the diagnosis of Hirschsprung disease and allied disorders

Objectives: Acetylcholinesterase (AChE) staining has become the gold standard for definitively diagnosing Hirschsprung disease (HD), although some pitfalls have been reported. We reevaluated a large series at our institute in order to validate the accuracy of AChE staining for detecting HD. Methods: A retrospective study of the rectal mucosal specimens of all of the children with suspected HD during a 13-year period was performed. The specimens were stained according to the modified Karnovsky-Roots method for AChE staining. The final diagnosis, prognosis, and management after the histopathological diagnosis were analyzed with a questionnaire sent to the patients original hospital. Results: Three hundred and fifty-eight specimens were collected. One hundred twenty-two (34%) specimens were diagnosed as HD, 198 (55%) as nonHD, 25 (7%) as “undetermined,” and 13 (4%) as “inappropriate.” The non-HD group contained 190 (96%) specimens with a normal appearance and 8 (4%) specimens with suspected intestinal neuronal dysplasia (IND). Three hundred and six of 358 questionnaires were returned. The final diagnosis showed that no specimens first diagnosed as HD were identified as non-HD and vice versa, for a sensitivity and specificity of 100%. Four cases were finally diagnosed as chronic idiopathic intestinal pseudo-obstruction (CIIP) in the non-HD group. All of the patients with HD underwent radical surgery. Most non-HD patients were managed conservatively, although some continued to have constipation. Conclusions: AChE staining is an accurate tool for differentiating between HD and non-HD with high sensitivity and specificity. CIIP can be included in cases of non-HD; therefore, careful follow-up is mandatory.

In vivo and ex vivo methods of growing a liver bud through tissue connection

Cell-based therapy has been proposed as an alternative to orthotopic liver transplantation. The novel transplantation of an in vitro-generated liver bud might have therapeutic potential. In vivo and ex vivo methods for growing a liver bud are essential for paving the way for the clinical translation of liver bud transplantation. We herein report a novel transplantation method for liver buds that are grown in vivo involving orthotopic transplantation on the transected parenchyma of the liver, which showed long engraftment and marked growth in comparison to heterotopic transplantation. Furthermore, this study demonstrates a method for rapidly fabricating scalable liver-like tissue by fusing hundreds of liver bud-like spheroids using a 3D bioprinter. Its system to fix the shape of the 3D tissue with the needle-array system enabled the fabrication of elaborate geometry and the immediate execution of culture circulation after 3D printing—thereby avoiding an ischemic environment ex vivo. The ex vivo-fabricated human liver-like tissue exhibited self-tissue organization ex vivo and engraftment on the liver of nude rats. These achievements conclusively show both in vivo and ex vivo methods for growing in vitro-generated liver buds. These methods provide a new approach for in vitro-generated liver organoids transplantation.

The Safety and Efficacy of Laparoscopic Percutaneous Extraperitoneal Closure for Inguinal Hernia in Neonates and Infants Younger than 1 Year of Age in Comparison to Older Patients

PURPOSE Laparoscopic percutaneous extraperitoneal closure (LPEC) has been performed in Japan for the repair of the pediatric inguinal hernias for over a decade. However, the safety and efficacy of LPEC in neonates and infants under 1 year of age remain unknown. The aim of the present study is to elucidate the safety and efficacy of LPEC in the treatment of inguinal hernia in patients who are younger than 1 year of age. PATIENTS AND METHODS The medical records of the patients who underwent LPEC at Saga-Ken Medical Center Koseikan (Saga, Japan) between August 2007 and November 2012 were collected. The intraoperative findings and postoperative outcomes were retrospectively investigated. The data of the patients who were younger than 1 year of age (Group A) were compared with the data of patients who were older than 1 year of age (Group B). RESULTS During the study period, 150 LPEC procedures were performed in 112 Group A patients, whereas 607 LPEC procedures were performed in 456 Group B patients. There were no serious complications in either group. After a mean follow-up period of 50.4 ± 15.6 months (range, 28-91 months), there were no significant differences between the two groups in the operating time or the incidence of intraoperative or postoperative complications. Postoperative testicular ascent and recurrence were observed in some cases of each group. CONCLUSIONS LPEC is a safe and effective procedure for the repair of an inguinal hernia, even in neonatal and infant patients who are younger than 1 year of age.

Transient hyperphosphatasemia after pediatric liver transplantation: TH after liver transplantation

Transient hyperphosphatasemia (TH), the incidence of which in healthy children is 1.5–2.8%, is associated with a temporary elevation of serum alkaline phosphatase (ALP) without any other liver function test (LFT) abnormalities. Fast α2 band, detected on agarose gel electrophoresis, is known to be a highly sensitive phenomenon in TH. The aim of this study was to elucidate the characteristics of TH after liver transplantation (LT).

Bowel obstruction without history of laparotomy: Clinical analysis of 70 patients

Determining the cause of bowel obstruction without a history of laparotomy (BO without HL) is difficult and can result in delay of treatment and development of a potentially life‐threatening situation. We herein investigated the clinical characteristics of pediatric patients who underwent laparotomy due to BO without HL.

The role of splenectomy before liver transplantation in biliary atresia patients

BACKGROUND/PURPOSE There is currently no unified view regarding whether liver transplantation or splenectomy should be performed for hypersplenism before liver transplantation in biliary atresia (BA) patients. We herein describe the efficacy of splenectomy before liver transplantation. METHODS Splenectomy was performed in ten patients with hypersplenism associated with BA. We retrospectively reviewed their perioperative and postoperative courses, the number of leukocytes and thrombocytes, and the MELD score. RESULTS The mean age was 17.5±7.0years (range 11-31years), and the male-to-female ratio was 1:1. The platelet and leukocyte levels increased after splenectomy and returned to normal levels one month postoperatively. The mean MELD score after splenectomy was significantly decreased after splenectomy: 10±2.1 vs 7.6±1.8. In particular, PT-INR improved. Five patients underwent liver transplantation because of hepatopulmonary syndrome and repeated bouts of cholangitis, whereas the remaining five patients did not undergo liver transplantation because of improvements in the liver function (the mean follow-up period was 56months). The postoperative complications included portal vein thrombosis and intestinal perforation, but the patient survival rates remained at 100%. CONCLUSION After splenectomy, both pancytopenia and the liver function clearly improved. Splenectomy should therefore be a treatment option for patients with hypersplenism before liver transplantation. LEVEL OF EVIDENCE Retrospective Comparative Study - Level III.

Surgical strategy according to the anatomical types of congenital portosystemic shunts in children

BACKGROUND Congenital portosystemic shunts (CPSS) with intrahepatic portal vein (IHPV) hypoplasia or absence cause encephalopathy or pulmonary hypertension (PH). Acute shunt closure may result in postoperative portal hypertension. The aim of this study was to propose a surgical strategy according to the anatomical types of CPSS and IHPV. METHODS Twenty-three CPSS patients were diagnosed from1990 to 2015. All patients were evaluated by computed tomography, angiography, and PV pressure monitoring under a shunt occlusion test. CPSS were categorized into 5 types according to the anatomical shunt location. RESULTS The median age at diagnosis was 34months. Three of 23 total patients, who had an extrahepatic portosystemic shunt with a hypoplastic IHPV, died before treatment initiation because of severe PH. Fourteen cases received surgical or interventional treatment at the median age of 5years. A total of 6 cases received surgical therapy, including liver transplants for 2 absent IHPV cases. The remaining 8 cases received interventional coiling. All shunt ligations were successfully accomplished in 1-stage ligation without any complications. After the treatment, the hypoplastic IHPV gradually enlarged with an efficient portal inflow. CONCLUSION A precise pretreatment anatomical evaluation of CPSS and IHPV types is mandatory for the selection of surgical treatment. LEVEL OF EVIDENCE Diagnostic study - level II and treatment study - level III.

Liver graft-to-spleen volume ratio as a useful predictive factor of the early graft function in children and young adults transplanted for biliary atresia: a retrospective study

A graft volume/standard liver volume ratio (GV/SLV) > 35% or graft/recipient weight ratio (GRWR) > 0.8% has been considered as a standard criteria of graft selection. Even if the graft size meets these selection criteria, small‐for‐size syndrome can still occur depending on the portal venous flow (PVF). The aim of this study was to identify other factors contributing to portal hyperperfusion and the post‐transplant course, focusing on the graft volume‐to‐spleen volume ratio (GV/SV). Thirty‐seven BA patients who underwent living donor liver transplantation were reviewed retrospectively. First, we evaluated the preoperative factors contributing to portal hyperperfusion. Second, we evaluated the factors contributing to post‐transplant complications, such as thrombocytopenia, hyperbilirubinemia, and coagulopathy. The GV/SLV was >35% in all cases; however, portal hyperperfusion (≥250 ml/min/100 g graft) was found in 12 recipients (35.3%). Furthermore, although the GRWR was >0.8% in over 90% of cases, portal hyperperfusion was found in 10 recipients (32.3%). In contrast, the GV/SV showed a significant correlation with the PVF after reperfusion. If the GV/SV was <0.88, about 80% of recipients developed portal hyperperfusion. Furthermore, the GV/SV also showed a significant correlation with post‐transplant persistent thrombocytopenia and hyperbilirubinemia. The GV/SV < 0.88 predicts portal hyperperfusion, post‐transplant persistent thrombocytopenia, and hyperbilirubinemia.

Insufficient Portal Vein Inflow in Children without Major Shunt Vessels During Living Donor Liver Transplantation

BACKGROUND Liver cirrhosis is frequently accompanied by insufficient portal vein inflow (IPVF) with large portosystemic shunts. However, pediatric cases often manifested IPVF without any apparent major portosystemic shunts. Although IPVF is a very critical issue, the intraoperative assessment has not been well established. In this study, we reviewed the intraoperative approach and the outcome of the IPVF cases at our department. MATERIAL AND METHODS Eighty-three living donor liver transplantations (LDLT) were performed from 1996 to 2014. The IPVF occurred in 5 cases and necessitated some additional assessments and intraoperative PV flow modulations. We retrospectively reviewed the operative records and analyzed the risk factors and the outcome of the IPVF. RESULTS All 5 IPVF cases were biliary atresia and the mean age at LDLT was 0.74±0.19 years old. The mean recipient PV diameter was 4.3±0.8 mm and the donor IMV patch grafts were applied. To increase the PV inflow, the collaterals around the spleen were ligated in all cases. Intraoperative portal venography was performed in 1 case for selective shunt vessel ligation. In 1 case, the graft was removed and returned to the back table to prevent graft loss during the IPVF. As a result, the final PVF/GV increased to 66.4±20.0 ml/min/100 g. CONCLUSIONS IPVF is a very critical problem. Intraoperative portal venography is helpful and collateral veins ligation is crucial. In some cases, returning the graft to the back table during the PV inflow modulation can prevent graft loss.