E.Y. Yoshitoshi

Living Donor Liver Transplantation for Pediatric Patients with Inheritable Metabolic Disorders

Forty‐six pediatric patients who underwent living donor liver transplantation (LDLT) using parental liver grafts for inheritable metabolic disorders (IMD) were evaluated to determine the outcomes of the surgery, decisive factors for post‐transplant patient survival and the impact of using donors who were heterozygous for the particular disorder. Disorders included Wilson disease (WD, n = 21), ornithine transcarbamylase deficiency (OTCD, n = 6), tyrosinemia type I (TTI, n = 6), glycogen storage disease (GSD, n = 4), propionic acidemia (PPA, n = 3), methylmalonic acidemia (MMA, n = 2), Crigler‐Najjar syndrome type I (CNSI, n = 2), bile acid synthetic defect (BASD, n = 1) and erythropoietic protoporphyria (EPP, n = 1). The post‐transplant cumulative patient survival rates were 86.8 and 81.2% at 1 and 5 years, respectively. Post‐transplant patient survival and recovery of the growth retardation were significantly better in the liver‐oriented diseases (WD, OTCD, TTI, CNSI and BASD) than in the non‐liver‐oriented diseases (GSD, PPA, MMA and EPP) and pre‐transplant growth retardation disadvantageously affected post‐transplant outcomes. Although 40 of 46 donors were considered heterozygous for each disorder, neither mortality nor morbidity related to the heterozygosis has been observed. LDLT using parental donors can be recommended as an effective treatment for pediatric patients with IMD. In the non‐liver‐oriented diseases, however, satisfactory outcomes were not obtained by hepatic replacement alone.

Long-term outcomes for 32 cases of Wilson's disease after living-donor liver transplantation

Background. Long-term outcomes after living donor liver transplantation (LDLT) in Wilson’s disease (WD) with heterozygous donors for WD gene are unknown. Methods. LDLT was performed for 32 patients with WD (15 men, 17 women; mean age 16 years; range, 6–40 years) at Kyoto University Hospital from 1992 to 2006. The mean follow-up time from LDLT was 6.7±4.0 years (0.04–15.0 years). Donors were mainly parents (90.6%), who were obligatory carriers of the WD mutation. The present study examined retrospectively the copper metabolism, recurrence of WD, survival rate, and neurologic outcomes after LDLT. Results. Mean ceruloplasmin at the time of LDLT was 9.7±7.3 mg/dL and increased to 22.3±5.3 mg/dL (normal, 18–37 mg/dL). Urinary copper decreased from 2704±901 to 73.7±5.2 &mgr;g/day (normal, <50 &mgr;g/day). Serum copper improved from 72.9±33.9 to 81.0±14.9 mg/dL (normal, 78–131 mg/dL). Although six patients died and two received retransplantation, the remaining 24 remain alive without recurrence, with overall survival rates of 90.6%, 83.7%, and 79.9% at 1, 5, and 10 years, respectively. Patients with chronic liver failure had a poorer prognosis (P<0.05). Conclusion. Use of liver grafts from heterozygous donors has been considered safe. Good improvements in copper metabolism were obtained without evidence of recurrence in long-term follow-up. Neuropsychologic presentations of WD improved or remained unchanged. Indications for liver transplantation in patients with WD with neurologic symptoms must be considered carefully based on the stage of neurologic damage and its irreversibility.

Keratin 19, a Cancer Stem Cell Marker in Human Hepatocellular Carcinoma

Purpose: Keratin 19 (K19) is a known marker of poor prognosis and invasion in human hepatocellular carcinoma (HCC). However, the relationship between K19 and cancer stem cells (CSCs) is unclear. Here, we determined whether K19 can be used as a new CSC marker and therapeutic target in HCC. Experimental Design: HCC cell lines were transfected with a K19 promoter–driven enhanced green fluorescence protein gene. CSC characteristics, epithelial–mesenchymal transition (EMT), and TGFb/Smad signaling were examined in FACS-isolated K19+/K19− cells. K19 and TGFb receptor 1 (TGFbR1) expression in 166 consecutive human HCC surgical specimens was examined immunohistochemically. Results: FACS-isolated single K19+ cells showed self-renewal and differentiation into K19− cells, whereas single K19− cells did not produce K19+ cells. K19+ cells displayed high proliferation capacity and 5-fluorouracil resistance in vitro. Xenotransplantation into immunodeficient mice revealed that K19+ cells reproduced, differentiated into K19− cells, and generated large tumors at a high frequency in vivo. K19+ cells were found to be involved in EMT and the activation of TGFb/Smad signaling, and these properties were suppressed by K19 knockdown or treatment with a TGFbR1 inhibitor. The TGFbR1 inhibitor also showed high therapeutic effect against K19+ tumor in the mouse xenograft model. Immunohistochemistry of HCC specimens showed that compared with K19− patients, K19+ patients had significantly poorer recurrence-free survival and higher tumor TGFbR1 expression. Conclusions: K19 is a new CSC marker associated with EMT and TGFb/Smad signaling, and it would thus be a good therapeutic target for TGFbR1 inhibition. Clin Cancer Res; 21(13); 3081–91. ©2015 AACR.

SOX9 is a novel cancer stem cell marker surrogated by osteopontin in human hepatocellular carcinoma.

The current lack of cancer stem cell (CSC) markers that are easily evaluated by blood samples prevents the establishment of new therapeutic strategies in hepatocellular carcinoma (HCC). Herein, we examined whether sex determining region Y-box 9 (SOX9) represents a new CSC marker, and whether osteopontin (OPN) can be used as a surrogate marker of SOX9 in HCC. In HCC cell lines transfected with a SOX9 promoter-driven enhanced green fluorescence protein gene, FACS-isolated SOX9+ cells were capable of self-renewal and differentiation into SOX9− cells, and displayed high proliferation capacity in vitro. Xenotransplantation experiments revealed that SOX9+ cells reproduced, differentiated into SOX9− cells, and generated tumors at a high frequency in vivo. Moreover, SOX9+ cells were found to be involved in epithelial-mesenchymal transition (EMT) and activation of TGFb/Smad signaling. Gain/loss of function experiments showed that SOX9 regulates Wnt/beta-catenin signaling, including cyclin D1 and OPN. Immunohistochemistry of 166 HCC surgical specimens and serum OPN measurements showed that compared to SOX9− patients, SOX9+ patients had significantly poorer recurrence-free survival, stronger venous invasion, and higher serum OPN levels. In conclusion, SOX9 is a novel HCC-CSC marker regulating the Wnt/beta-catenin pathway and its downstream target, OPN. OPN is a useful surrogate marker of SOX9 in HCC.

Efficient recellularisation of decellularised whole-liver grafts using biliary tree and foetal hepatocytes

A whole-organ regeneration approach, using a decellularised xenogeneic liver as a scaffold for the construction of a transplantable liver was recently reported. Deriving suitable scaffolds was the first step towards clinical application; however, effective recellularisation remains to be achieved. This report presents a strategy for the improvement of the recellularisation process, using novel cell-seeding technique and cell source. We evaluated recellularised liver grafts repopulated through the portal vein or the biliary duct with mice adult hepatocytes or E14.5 foetal hepatocytes. More than 80% of the cells seeded through the biliary tree entered the parenchyma beyond the ductule-lining matrix barrier and distributed throughout the liver lobule. In contrast, about 20% of the cells seeded through the portal tree entered the parenchyma. The gene expression levels of foetal hepatocyte albumin, glucose 6-phosphatase, transferrin, cytokeratin 19, and gamma-glutamyl transpeptidase were increased in three-dimensional cultures in the native liver-derived scaffolds, and the activation of liver detoxification enzymes and formation of biliary duct-like structures were supported. The metabolic functions of liver grafts recellularised with different cell types were similar. These results suggest that biliary tree cell-seeding approach is promising, and that liver progenitor cells represent a good cell source candidate.

Histology of intestinal allografts: lymphocyte apoptosis and phagocytosis of lymphocytic apoptotic bodies are diagnostic findings of acute rejection in addition to crypt apoptosis.

Acute rejection of a small-bowel transplant is often difficult to diagnose due to complicated immune responses. The present study aimed to elucidate the specific immune responses involved in intestinal transplant rejection. We correlated immunohistologic findings with an increase in crypt apoptosis, which has been commonly accepted as a criterion for the diagnosis of acute cellular rejection (ACR). Of 8 patients who received an intestinal allograft at Kyoto University Hospital, biopsy specimens from 7 patients were assessed immunohistologically with antibodies against 20 types of lymphocytic antigens including CD3, CD4, CD8, CD79a, CD20, IgG, and T-cell receptor, along with assessment of the patients’ clinical courses. It was revealed that, in addition to apoptotic crypts, T-lymphocyte apoptosis and phagocytosis of apoptotic bodies in the lamina propria of villi were findings of ACR; both were observed in all cases. Immunostaining of the Fas ligand, one of the apoptosis-inducing molecules, was useful for the identification of the apoptotic bodies in the lamina propria of villi. Apoptotic body phagocytosis may be a surrogate diagnostic finding of grafts undergoing ACR.

Identification of keratin19-positive cancer stem cells associating human hepatocellular carcinoma using 18F-fluorodeoxyglucose positron emission tomography.

Purpose: The current lack of tools for easy assessment of cancer stem cells (CSC) prevents the development of therapeutic strategies for hepatocellular carcinoma (HCC). We previously reported that keratin 19 (K19) is a novel HCC-CSC marker and that PET with 18F-fluorodeoxyglucose (18F-FDG) is an effective method for predicting postoperative outcome in hepatocellular carcinoma. Herein, we examined whether K19+ HCC-CSCs can be tracked using 18F-FDG-PET. Experimental Design: K19 and glucose transporter-1 (GLUT1) expression was evaluated by IHC in 98 hepatocellular carcinoma patients who underwent 18F-FDG-PET scans before primary tumor resection. Standardized uptake values (SUV) for primary tumors and tumor-to-nontumor SUV ratios (TNR) were calculated using FDG accumulation levels, and values were compared among K19+/K19− patients. Using hepatocellular carcinoma cell lines encoding with a K19 promoter–driven enhanced GFP, 18F-FDG uptake and GLUT1 expression were examined in FACS-isolated K19+/K19− cells. Results: In hepatocellular carcinoma patients, K19 expression was significantly correlated with GLUT1 expression and FDG accumulation. ROC analyses revealed that among preoperative clinical factors, TNR was the most sensitive indicator of K19 expression in hepatocellular carcinoma tumors. In hepatocellular carcinoma cells, FACS-isolated K19+ cells displayed significantly higher 18F-FDG uptake than K19− cells. Moreover, gain/loss-of-function experiments confirmed that K19 regulates 18F-FDG uptake through TGFβ/Smad signaling, including Sp1 and its downstream target GLUT1. Conclusions:18F-FDG-PET can be used to predict K19 expression in hepatocellular carcinoma and should thereby aid in the development of novel therapeutic strategies targeting K19+ HCC-CSCs. Clin Cancer Res; 23(6); 1450–60. ©2016 AACR.

The Protective Effect of Transplanting Liver Cells Into the Mesentery on the Rescue of Acute Liver Failure After Massive Hepatectomy.

Postoperative liver failure is one of the most critical complications following extensive hepatectomy. Although transplantation of allogeneic hepatocytes is an attractive therapy for posthepatectomy liver failure, transplanting cells via the portal veins typically causes portal vein embolization. The embolization by transplanted cells would be lethal in patients who have undergone massive hepatectomy. Thus, transplant surgeons need to select extrahepatic sites as transplant sites to prevent portal vein embolization. We aimed to investigate the mechanism of how liver cells transplanted into the mesentery protect recipient rats from acute liver failure after massive hepatectomy. We induced posthepatectomy liver failure by 90% hepatectomy in rats. Liver cells harvested from rat livers were transplanted into the mesenteries of hepatectomized rats. Twenty percent of the harvested cells, which consisted of hepatocytes and nonparenchymal cells, were transplanted into each recipient. The survival rate improved significantly in the liver cell transplantation group compared to the control group 7 days after hepatectomy (69 vs. 7%). Histological findings of the transplantation site, in vivo imaging system study findings, quantitative polymerase chain reaction assays of the transplanted cells, and serum albumin measurements of transplanted Nagase analbuminemic rats showed rapid deterioration of viable transplanted cells. Although viable transplanted cells deteriorated in the transplanted site, histological findings and an adenosine-5′-triphosphate (ATP) assay showed that the transplanted cells had a protective effect on the remaining livers. These results indicated that the paracrine effects of transplanted liver cells had therapeutic effects. The same protective effects were observed in the hepatocyte transplantation group, but not in the liver nonparenchymal cell transplantation group. Therefore, this effect on the remnant liver was mainly due to the hepatocytes among the transplanted liver cells. We demonstrated that transplanted liver cells protect the remnant liver from severe damage after massive hepatectomy.

Identification of keratin 19-positive cancer stem cells associating human hepatocellular carcinoma using CYFRA 21-1

The current lack of an easily measurable surrogate marker of cancer stem cells (CSCs) prevents the clinical application of CSCs for hepatocellular carcinoma (HCC). We previously reported that keratin 19 (K19) is a novel HCC‐CSC marker associated with transforming growth factor beta (TGFβ)/Smad signaling, and that K19+ HCC‐CSCs could be a new therapeutic target of TGFβ receptor 1 inhibitor LY2157299. In this study, we examined whether K19+ HCC‐CSCs can be tracked using cytokeratin 19 fragment CYFRA 21‐1. In 147 HCC patients who underwent curative resection and evaluated K19 expression by immunohistochemistry, preoperative serum CYFRA 21‐1 levels were significantly higher in K19+ patients than in K19− patients (P < 0.01). Receiver operating characteristic analyses revealed that serum CYFRA 21‐1 was the statistically significant and the most sensitive predictor of tumor K19 expression among preoperative laboratory test values (P < 0.001). In HCC cells encoding with a K19 promoter‐driven enhanced green fluorescent protein, fluorescence‐activated cell sorting (FACS)‐isolated K19+ cells displayed significantly higher levels of supernatant CYFRA 21‐1 than K19− cells (P < 0.01). Gain/loss of K19 function experiments confirmed that CYFRA 21‐1 levels were regulated by K19 function in HCC cells. Furthermore, CYFRA 21‐1 levels reflected the treatment efficacy of LY2157299 in K19+ cells. In conclusion, CYFRA 21‐1 can be used to predict K19 expression in HCC, and should thereby aid in the development of novel therapeutic strategies targeting K19+ HCC‐CSCs.

Generation of non-viral, transgene-free hepatocyte like cells with piggyBac transposon

Somatic cells can be reprogrammed to induced hepatocyte-like cells (iHeps) by overexpressing certain defined factors in direct reprogramming techniques. Of the various methods to deliver genes into cells, typically used genome-integrating viral vectors are associated with integration-related adverse events such as mutagenesis, whereas non-integrating viral vectors have low efficiency, making viral vectors unsuitable for clinical application. Therefore, we focused on developing a transposon system to establish a non-viral reprogramming method. Transposons are unique DNA elements that can be integrated into and removed from chromosomes. PiggyBac, a type of transposon, has high transduction efficiency and cargo capacity, and the integrated transgene can be precisely excised in the presence of transposase. This feature enables the piggyBac vector to achieve efficient transgene expression and a transgene-free state, thus making it a promising method for cell reprogramming. Here, we attempted to utilize the piggyBac transposon system to generate iHeps by integrating a transgene consisting of Hnf4a and Foxa3, and successfully obtained functional iHeps. We then demonstrated removal of the transgene to obtain transgene-free iHeps, which still maintained hepatocyte functions. This non-viral, transgene-free reprogramming method using the piggyBac vector may facilitate clinical applications of iHeps in upcoming cell therapy.