Takashi Kanematsu

Liver Repopulation: A New Concept of Hepatocyte Transplantation

Hepatocyte transplantation has been recognized as an alternative strategy for organ transplantation because the supply of donor livers is limited. However, in conventional hepatocyte transplantation, only 1%–10% of the liver replaced with transplanted hepatocytes. Recently a novel concept termed “liver repopulation” has been established, where the whole recipient liver can be replaced by a small number of donor hepatocytes. To induce liver repopulation, growth advantage of the donor hepatocytes against the host liver seems to be required according to the data of previous studies. Additionally, various cell sources, including bone marrow cells and other stem cells, could potentially be used as donor cells for liver repopulation. In this article, we discuss recent progress and future perspectives of this emerging technology.

A secured technique for bile duct division during living donor right hepatectomy

Accordingly, we should cutthe bile duct as close as possible to the commonhepatic duct, but biliary stricture in the remnant liverof the donor is a great concern. To overcome theseproblems, we describe our technical inventions forsafe and accurate bile duct division during living do-nor right hepatectomy.During hilar dissection, the right hepatic artery andright portal vein are fully exposed and isolated fromthe hilar plate. At the final step of subsequent paren-chymal transection, the right hilar plate is fully ex-posed and encircled with radiopaque marker fila-ment, which is obtained from surgical gauze (Fig. 1).Intraoperative cholangiography is then performed viaa catheter placed in the cystic duct (Fig. 2A). C-armfluoroscopy is adapted during this procedure to en-able us to check the optimal cutting point of the bileduct, which is made clear by pulling the filament andadjusting the accurate angle (Fig. 2B). The right hilarplate including the right hepatic duct is then sharplydivided with scissors, and the stump of the remnantbile duct is closed with continuous 6-0 absorbablemonofilament sutures ([Polydioxanone] Suture II,Ethicon, Somerville, NJ). Cholangiography with C-arm fluoroscopy is performed again to check the bil-iary leakage or stricture in the remnant bile duct (Fig.2C). The right liver graft is then removed after theright hepatic artery, portal vein, and hepatic veinhave been divided (Fig. 2D).Of 54 living donor hepatectomies from August 1997to December 2005, 38 underwent right hepatectomy,and the present procedure was adapted for use in thelast 10 cases. Compared to the first 28 cases withordinary cholangiography, the incidence of multipleducts in the graft was significantly reduced (3/10 vs.20/28, respectively;

Eradication of hepatocellular carcinoma xenografts by radiolabelled, lipiodol-inducible gene therapy

The promoter region of the early-growth response-1(Egr-1) gene has been shown to be activated by external radiation, thus making a selective tumoricidal effect possible. A previous experiment showed that the Egr-1 promoter can be activated by internal radiation using radioisotopes as well as external radiation. Internal radiation using I-131 lipiodol (I-131-Lip) has been established as one of the most useful therapeutic strategies against hepatoma. We herein linked the Egr-1 promoter to the herpes simplex virus-thymidine kinase (HSV-TK) gene, and investigated its efficacy in hepatoma gene therapy in combination with I-131-Lip. A luciferase assay showed the Egr-1-promoter activity to be markedly increased in hepatoma tissue specimens in an I-131-dose-dependent manner, whereas a less than two-fold increase in this activity was observed in other organs. In addition, the radioactivity derived from I-131 was selectively accumulated in the tumor tissue specimens. To examine the efficacy of EgrTK/ganciclovir (GCV) gene therapy in vivo, subcutaneous hepatoma xenografts in nude mice were transfected using a hemagglutinating virus of Japan (HVJ)-liposome vector. Complete tumor regression was observed in all the EgrTK-transfected tumors following combination treatment with I-131-Lip and GCV 42 days after treatment without any side effects (n=8). In contrast, the tumors continued to grow in all control mice (n=10). Furthermore, the serum α-fetoprotein levels decreased in the combination therapy group, while they increased in the controls. In conclusion, these data indicate that Egr-1 promoter-based gene therapy combined with internal radiation has a selective effect on hepatoma tumors while also showing an improved in vivo efficacy. This combination therapy might, therefore, be an effective human hepatoma gene therapy, even in advanced multiple cases.

The efficacy and safety of gene transfer into the porcine liver in vivo by HVJ (sendai virus) liposome

Background. Gene transfer systems using viral vectors are efficient; however, most viral vectors also tend to evoke immunologic reactions, thereby clinically causing serial side effects. HVJ-liposome vector is a hybrid vector consisting of liposome and an inactivated Sendai virus (Hemmagglutinating Virus of Japan [HVJ]), which has been reported to be less immunogenic and can also be repeatedly administered. We examined the usefulness of this vector for hepatic gene therapy in a pig model. Methods. Genes encoding &bgr;-galactosidase and luciferase were used as reporter genes. The pigs were injected with the reporter gene loaded-HVJ-liposome into the portal vein under total vascular exclusion of the liver. The transfection efficiencies were then assessed by &bgr;-galactosidase staining, a luciferase assay, and RT-PCR for LacZ mRNA. Biochemical and histologic analyses were performed to evaluate tissue toxicity after gene transfer. Results. The luciferase gene expression in the liver reached its highest level at 7 days after transfection. It continued to be detected up to 28 days after transfection, while all pigs remained healthy throughout the observation period. The transfection efficiency was 15% in the hepatocytes according to &bgr;-galactosidase staining. Extrahepatic transgene expression was slightly observed in the lung and kidney, but not in the spleen or ovary. Conclusions. These data suggest for the first time that the use of the HVJ-liposome vector is a safe and feasible modality for liver-directed gene transfer in pigs, and it might therefore be suitable for clinical gene therapy trials.