Kenichi Teramoto

Human Umbilical Cord Blood as a Source of Transplantable Hepatic Progenitor Cells

Human umbilical cord blood (UCB) cells have many advantages as grafts for cell transplantation because of the immaturity of newborn cells compared with adult cells. In contrast to their hematopoietic and mesenchymal potential, it remains unclear whether UCB cells have endodermal competence. Here, with a view to utilize UCB cells for cell transplantation into injured liver, we investigated the hepatic potential of UCB cells both in vitro and in vivo. We determined the most efficient conditions leading UCB cells to produce albumin (ALB). In a novel primary culture system supplemented with a combination of growth/differentiation factors, about 50% of UCB cells in 21‐day cultures expressed ALB, and the ALB+ cells coexpressed hepatocyte lineage markers. The ALB‐expressing cells were able to proliferate in the culture system. Moreover, in the cell‐transplantation model into liver‐injured severe combined immunodeficient mice, inoculated UCB cells developed into functional hepatocytes in the liver, which released human ALB into the sera of the recipient mice. In conclusion, this study demonstrates that human UCB is a source of transplantable hepatic progenitor cells. Our findings may have relevance to clinical application of UCB‐derived cell transplantation as a novel therapeutic option for liver failure.

Hepatic microcirculatory changes after reperfusion in fatty and normal liver transplantation in the rat

The hepatic microcirculation in fatty and normal liver grafts in ACI rats was investigated using in vivo microscopy. Six groups were studied. They were: normal and fatty control livers (sham operated), 6-hr cold University of Wisconsin solution (UW)-preserved fatty and normal liver grafts (survival conditions, fatty and normal liver grafts), 18-hr cold UW-preserved fatty livers (nonsurvival conditions, fatty liver graft), and 24-hr cold UW-preserved normal livers (nonsurvival conditions, normal liver grafts). Fatty livers in all groups were found to have narrow and irregular sinusoids with blood cell adhesions to endothelial cells. The number of adhesions increased as the preservation time increased. Sinusoidal blood flow area decreased as the preservation time increased and was correlated with survival in both normal and fatty liver grafts. The phagocytic activity of Kupffer cells (corrected for flow) increased as the preservation time increased. The phagocytic Kupffer cell activity of the 18-hr preserved fatty liver group was greater than the activity of any other group. These features may cause liver cell death and contribute to primary graft nonfunction after transplantation of a fatty liver.

Expression of the liver-specific gene Cyp7a1 reveals hepatic differentiation in embryoid bodies derived from mouse embryonic stem cells

Hepatic differentiation from mouse embryonic stem (ES) cells via the formation of embryoid bodies (EBs) has been revealed by the expression of hepatocyte‐related genes such as α‐fetoprotein and albumin. It is known, however, that the visceral endoderm differentiates in early EBs and expresses these hepatocyte‐related genes. Thus, it remains unclear whether ES cells are capable of differentiating into hepatocytes derived from definitive endoderm in vitro. In the present study, yolk sac tissues isolated from the foetal mouse were found to express many hepatocyte‐related genes. Among the hepatocyte‐related genes examined, cytochrome P450 7A1 (Cyp7a1) was identified as a liver‐specific gene that was not expressed in the yolk sac. Cyp7a1 was induced in developing EBs, and hepatic differentiation was preferentially observed in the developing EBs in attached culture as compared to those in suspension culture. Leukaemia inhibitory factor permitted the differentiation of visceral endoderm, but inhibited the expression of gastrulation‐related genes and the hepatic differentiation in cultured EBs. ES cells expressing green fluorescent protein (GFP) under the control of the Cyp7a1 enhancer/promoter showed that cultured EBs contained GFP‐positive epithelial‐like cells. These results demonstrate that ES cells can differentiate in vitro into hepatocytes derived from definitive endoderm.

A rat fatty liver transplant model

A rat model of fatty liver transplantation has been developed to study primary nonfunction in fatty liver grafts. ACI rats were fed with a diet deficient in choline and methionine for 7, 14, 28, and 42 days. Fat content in the pretransplant livers was examined by gas chromatography and histology. The main constituent of the fatty droplets was determined to be triglyceride. The triglyceride concentration reached a maximum by day 14 and remained constant for an additional 28 days. Histology revealed an absence of necrosis in 14- and 28-day fatty livers but scattered hepatocytic necrosis and inflammation in 42-day fatty livers. After being given cold (UW stored, 4 degrees C) or warm (37 degrees C) ischemia, the fatty liver was orthotopically transplanted into normal ACI rats. The one-week survival of fatty liver grafts after 6, 12, 18, and 24 hr cold preservation was 5/5, 5/6, 3/8, 0/6 for 14-day fatty liver and 5/5, 4/6, 0/8, 0/6 for 42-day fatty livers. The survival of normal liver grafts was 5/5, 6/6, 5/9, 2/8, respectively. Increased survival rate was correlated with the absence of hepatocytic necrosis. The survival after 15 and 30 min warm ischemia prior to transplant was 5/5, 2/6 for normal liver grafts and 4/7, 0/6 for 28-day fatty liver graft, respectively. Fatty livers were less resistant to damage induced by cold or warm ischemia.

Laparoscopic and Thoracoscopic Partial Hepatectomy for Hepatocellular Carcinoma

Several trials have been reported examining laparoscopic liver resections for the treatment of various kinds of liver tumors. However, there are no detailed reports on the use of laparoscopic (LH) and thoracoscopic (TH) hepatectomy for the treatment of hepatocellular carcinoma (HCC). Eleven laparoscopic and thoracoscopic partial liver resections were attempted for treating HCC. The indications for performing a laparoscopic or thoracoscopic partial hepatectomy were as follows: (1) the tumor was located on the surface of the liver; (2) the tumor was less than 3 cm in diameter; and (3) the tumor was not located adjacent to any large vessels. A TH was performed if the tumor was located in segment 8; an LH was performed if the tumor was located in segment 3, 4, or 5. Hand-assisted operations were performed in two patients. The mean operating time was 186.1 ± 44.0 minutes (range 130–310 minutes). The operative blood loss was 218.3 ± 197.6 ml (range 20–650 ml). The mean postoperative hospital stay was 11.3 ± 5.7 days (range 7–26 days). Two patients experienced postoperative complications (wound infection and ascites). No local recurrences have occurred to date. The overall 5-year survival rate and disease-free 5-year survival rate were 75.0% and 38.2%, respectively. Laparoscopic and thoracoscopic hepatic resections are less invasive than conventional surgical techniques and are useful for treating HCC in select patients.

In Vivo and in Vitro Analysis of the Effectiveness of Doxorubicin Combined with Temporary Arterial Occlusion in Liver Tumors

PURPOSE The authors evaluated the effects of daunomycin (daunorubicin)--an analogue of doxorubicin--ethiodized oil, and arterial occlusion on an in vitro hepatoma analogue and on in vivo rat liver tumors. MATERIALS AND METHODS A human Sk hepatoma cell monolayer sandwich system was used to determine uptake of 3H-daunomycin under normoxic/hypoxic conditions with use of autoradiography. Fluorescence microscopy was used to evaluate the biodistribution of doxorubicin in cell cultures (human Sk hepatoma and colon carcinoma). Microvascular flow adjacent to and within liver tumors and the intrahepatic effects of doxorubicin and ethiodized oil were studied with in vivo video microscopy on exteriorized rat livers containing peripheral hepatomas. RESULTS Increased uptake of 3H-daunomycin by hepatoma cells occurred under hypoxic conditions. Intrahepatic arterial administration of ethiodized oil caused temporary occlusion of peripheral sinusoids following passage through arterioportal anastomoses. Tumors received portal venous and neovascular blood supply and ethiodized oil occluded but did not enter the narrow neovasculature perfusing the tumors. CONCLUSION Hypoxia increases uptake of 3H-daunomycin by human Sk hepatoma and colon carcinoma cell cultures. Selective hepatic arterial occlusion (and perhaps the resultant hypoxia) may facilitate increased uptake of doxorubicin analogues into liver tumors. Hepatomas receive both arterial and portal venous blood supply, and ethiodized oil reaches the tumor via arterioportal anastomoses that perfuse the tumor periphery.

Resection of a cancer developing in the remnant pancreas after a pancreaticoduodenectomy for pancreas head cancer

We report a rare case of a curative resection performed on a carcinoma developing in the remnant pancreas at 3 years 7 months after a pancreaticoduodenectomy for pancreatic cancer. A 63-year-old man underwent a pancreaticoduodenectomy for pancreatic cancer on November 1999. Because the celiac trunk was occluded by atherosclerosis, an aortohepatic bypass with a saphenous vein graft was performed simultaneously. In May 2003, tumor marker levels increased, and a tumor was detected in the remnant pancreas on computed tomography. There were no findings such as invasion into the surrounding tissue or distant metastasis, and therefore we removed the remnant pancreas in July 2003. Histopathologically, the tumor consisted of a well-differentiated tubular adenocarcinoma and was limited to the pancreas. Moreover, the anastomotic site of the pancreaticojejunostomy was negative for cancer, and some foci of papillary hyperplasia and goblet cell metaplasia of the pancreatic ductal epithelium, which was thought to be the precursor of the pancreatic cancer, were seen. These findings suggested that the tumor was a second primary cancer developing in the remnant pancreas. This case provided suggestive evidence for the development of pancreatic cancer, and the surgical procedure for a pancreaticoduodenectomy with occlusion of the celiac trunk is discussed.

Embryonic Stem Cells: Hepatic Differentiation and Regenerative Medicine for the Treatment of Liver Disease

Hepatocyte transplantation is considered a potential treatment for liver diseases and a bridge for patients awaiting liver transplantation, but its application has been hampered by a limited supply of hepatocytes. Embryonic stem (ES) cells established from early mouse and human embryos are pluripotent, and proliferate indefinitely in an undifferentiated state in vitro. Since differentiation from ES cells seems to recapitulate early embryonic development, if hepatocytes could be efficiently generated in vitro, ES cells might become a source of transplantable hepatocytes for cell replacement therapy. Hepatocytes have been generated from ES cells in vitro, and the hepatocytes differentiated from ES cells have been found to express many hepatocyte-related genes and perform hepatic functions. However, it remains unclear whether the hepatocytes differentiated from ES cells are derived from definitive endoderm or primitive endoderm. Because visceral endoderm, which expresses many hepatocyte-related genes, is derived from primitive endoderm and is fated to form extraembryonic yolk sac tissues, not to form hepatocytes, ES cells must be directed to a definitive endoderm lineage in vitro. This article discusses the differentiation of ES cells into hepatocytes in vitro in comparison with early embryogenesis, and describes the efficacy of ES cell-derived hepatocyte transplantation.

Generation of hybrid hepatocytes by cell fusion from monkey embryoid body cells in the injured mouse liver

Monkey embryonic stem (ES) cells have characteristics that are similar to human ES cells, and might be useful as a substitute model for preclinical research. When embryoid bodies (EBs) formed from monkey ES cells were cultured, expression of many hepatocyte-related genes including cytochrome P450 (Cyp) 3a and Cyp7a1 was observed. Hepatocytes were immunocytochemically observed using antibodies against albumin (ALB), cytokeratin-8/18, and α1-antitrypsin in the developing EBs. The in vitro differentiation potential of monkey ES cells into the hepatic lineage prompted us to examine the transplantability of monkey EB cells. As an initial approach to assess the repopulation potential, we transplanted EB cells into immunodeficient urokinase-type plasminogen activator transgenic mice that undergo liver failure. After transplantation, the hepatocyte colonies expressing monkey ALB were observed in the mouse liver. Fluorescence in-situ hybridization revealed that the repopulating hepatocytes arise from cell fusion between transplanted monkey EB cells and recipient mouse hepatocytes. In contrast, neither cell fusion nor repopulation of hepatocytes was observed in the recipient liver after undifferentiated ES cell transplantation. These results indicate that the differentiated cells in developing monkey EBs, but not contaminating ES cells, generate functional hepatocytes by cell fusion with recipient mouse hepatocytes, and repopulate injured mouse liver.

Liver metastasis from rectal cancer with prominent intrabile duct growth.

Intrabiliary growth of liver metastases from colorectal cancer has rarely been studied. A surgically resected case of a metastatic liver tumor with prominent intrabiliary growth derived  from  rectal  cancer  is  reported.  The  patient  was  a 62‐year‐old man who had received a low anterior resection for rectal cancer in March 2000. He was re‐admitted due to obstructive jaundice in January 2003, and was diagnosed with hepatic malignancy in segment II of the liver with an intrabiliary tumor extending from the intrahepatic bile duct of segment II to the common hepatic duct. He underwent a left hepatectomy, a partial resection of segment VI, and an extrahepatic bile duct resection with reconstruction of the biliary tract. In the resected specimen, there were whitish tumors of 3 cm and 1.5 cm in diameter in segments II and VI, respectively, and an intrabiliary tumor originating from the main tumor in segment II extended to the common hepatic duct. Both the liver tumors and the intrabiliary tumor consisted of a well‐ to moderately differentiated adenocarcinoma, which showed the same histological features as the rectal cancer. The immunohistochemical findings strongly supported that these tumors, including the intrabiliary growth, were liver metastasis from the rectal cancer. The intrabiliary invasion and growth of metastatic liver tumors has generally been overlooked, notwithstanding their frequently observed biological behavior. The present case is informative, and further investigation into this type of metastatic liver tumor may be warranted.