Hiroaki Motoyama

In vitro reprogramming of adult hepatocytes into insulin-producing cells without viral vectors.

The pancreas and the liver share the same endodermal origin. We have been studying whether mature hepatocytes can be induced to differentiate into pancreatic beta-cells by in vitro delivery of transcriptional factors using a non-viral approach. Here we showed that nucleofection allowed suitable transfection of primary hepatocytes employing various non-viral methods. We introduced either pancreatic and duodenal homeobox 1 (Pdx1) or neurogenin 3 (Ngn3), or both, into the mature cells using nucleofection. Co-expression of pdx1 and ngn3 using a bicistronic vector activated the transcription of various islet-related genes, and the transfected hepatocytes acquired the ability to synthesize and secrete insulin. Our results suggest that simultaneous expression of Pdx1 and Ngn3 is an excellent inducer of liver-to-pancreas reprogramming, and that reprogramming will occur even in mature somatic cells without the need for viral vectors. These findings are of considerable significance for further therapeutic development for various intractable diseases including diabetes.

Impact of advanced age on the short- and long-term outcomes in patients undergoing hepatectomy for hepatocellular carcinoma: a single-center analysis over a 20-year period

BACKGROUND The purpose of this study was to analyze the influence of age on both the risk of hepatectomy and the prognosis in patients with hepatocellular carcinoma (HCC). METHODS Patients undergoing an initial hepatectomy for HCC were classified into 2 age groups: 75 years or over (n = 113) and less than 75 years (n = 499). RESULTS A zero 90-day mortality was achieved in the elderly. Although the recurrence rate and recurrence sites were almost similar between the 2 groups, the 5-year survival rate in the elderly patients was significantly lower than that in the younger patients (46.0% vs 57.6%; P = .018), possibly because of the higher incidence of deaths from other causes (26.8% vs 10.4%; P = .011) in the elderly. CONCLUSION Selected elderly HCC patients can undergo a hepatectomy safely and can benefit from long-term HCC control comparable with that of their younger counterparts.

Isolation and characterization of portal branch ligation-stimulated Hmga2-positive bipotent hepatic progenitor cells.

Hepatic stem/progenitor cells are one of several cell sources that show promise for restoration of liver mass and function. Although hepatic progenitor cells (HPCs), including oval cells, are induced by administration of certain hepatotoxins in experimental animals, such a strategy would be inappropriate in a clinical setting. Here, we investigated the possibility of isolating HPCs in a portal branch-ligated liver model without administration of any chemical agents. A non-parenchymal cell fraction was prepared from the portal branch-ligated or non-ligated lobe, and seeded onto plates coated with laminin. Most of the cells died, but a small number were able to proliferate. These proliferating cells were cloned as portal branch ligation-stimulated hepatic cells (PBLHCs) by the limiting dilution method. The PBLHCs expressed cytokeratin19, albumin, and Hmga2. The PBLHCs exhibited metabolic functions such as detoxification of ammonium ions and synthesis of urea on Matrigel-coated plates in the presence of oncostatin M. In Matrigel mixed with type I collagen, the PBLHCs became rearranged into cystic and tubular structures. Immunohistochemical staining demonstrated the presence of Hmga2-positive cells around the interlobular bile ducts in the portal branch-ligated liver lobes. In conclusion, successful isolation of bipotent hepatic progenitor cell clones, PBLHCs, from the portal branch-ligated liver lobes of mice provides the possibility of future clinical application of portal vein ligation to induce hepatic progenitor cells.

Hepatectomy preserving drainage veins of the posterior section for liver malignancy invading the right hepatic vein: an alternative to right hepatectomy.

BACKGROUND Although a right hepatectomy (RH) traditionally has been performed for liver tumors infiltrating the main trunk of the right hepatic vein (RHV), the presence of drainage veins of the posterior section (DVPS) beside the RHV provides a chance to preserve their draining area even if the main trunk of the RHV is removed. METHODS Since 2005, we systematically have performed DVPS-preserving hepatectomies whenever possible. In the present study, we describe our experience treating 12 consecutive patients who underwent this procedure. RESULTS We performed the following types of liver resections concomitant with the main trunk of the RHV without packed red cell transfusion, liver failure, or 90-day mortality: extended right anterior sectionectomy in 2 patients, extended segmentectomy 7 in 3, extended segmentectomy 8 in 2, and partial resection of segment 7 in 2 and segment 8 in 3. Postoperative morbidity was observed in 4 (33%) cases, all of which had pleural effusion requiring a tap. A free resection margin was obtained in all patients. CONCLUSIONS This procedure could be a useful alternative to RH, providing a chance for radical liver resection with minimal parenchymal sacrifice in selected patients with DVPS.

Impaired degradation followed by enhanced recycling of epidermal growth factor receptor caused by hypo-phosphorylation of tyrosine 1045 in RBE cells

BackgroundSince cholangiocarcinoma has a poor prognosis, several epidermal growth factor receptor (EGFR)-targeted therapies with antibody or small molecule inhibitor treatment have been proposed. However, their effect remains limited. The present study sought to understand the molecular genetic characteristics of cholangiocarcinoma related to EGFR, with emphasis on its degradation and recycling.MethodsWe evaluated EGFR expression and colocalization by immunoblotting and immunofluorescence, cell surface EGFR expression by fluorescence-activated cell sorting (FACS), and EGFR ubiquitination and protein binding by immunoprecipitation in the human cholangiocarcinoma RBE and immortalized cholangiocyte MMNK-1 cell lines. Monensin treatment and Rab11a depletion by siRNA were adopted for inhibition of EGFR recycling.ResultsUpon stimulation with EGF, ligand-induced EGFR degradation was impaired and the expression of phospho-tyrosine 1068 and phospho-p44/42 MAPK was sustained in RBE cells as compared with MMNK-1 cells. In RBE cells, the process of EGFR sorting for lysosomal degradation was blocked at the early endosome stage, and non-degradated EGFR was recycled to the cell surface. A disrupted association between EGFR and the E3 ubiquitin ligase c-Cbl, as well as hypo-phosphorylation of EGFR at tyrosine 1045 (Tyr1045), were also observed in RBE cells.ConclusionIn RBE cells, up-regulation of EGFR Tyr1045 phosphorylation is a potentially useful molecular alteration in EGFR-targeted therapy. The combination of molecular-targeted therapy determined by the characteristics of individual EGFR phosphorylation events and EGFR recycling inhibition show promise in future treatments of cholangiocarcinoma.

Biliary tract variations of the left liver with special reference to the left medial sectional bile duct in 500 patients

BACKGROUND Among the intrahepatic bile ducts, the biliary system of the left medial sectional bile duct (B4) is known to have relatively complex patterns. METHODS The records of 500 patients who had been diagnosed as having hepato-pancreatico-biliary disease were retrospectively studied for anatomical biliary variations of the left liver with special reference to the drainage system of B4 using magnetic resonance images. RESULTS The left hepatic duct was present in 494 patients (98.8%), whereas it was lacking in 6 patients (1.2%), and these patients exhibited the following B4 confluence patterns: B4 drained into the common hepatic duct in 2 patients (.4%), the right anterior sectional bile duct in 3 patients (.6%), and the right posterior sectional bile duct in 1 patient (.2%). The left hepatic duct was absent more frequently in patients with portal venous variations than in patients with a common branching pattern (8.2% vs .4%, P = .0011). CONCLUSION The presently reported data are useful for obtaining a better understanding of the surgical anatomy of the biliary system of the left liver.

Survival pathway of cholangiocarcinoma via AKT/mTOR signaling to escape RAF/MEK/ERKpathway inhibition by sorafenib

Cholangiocarcinoma (CCC) is a strongly aggressive malignancy for which surgical resection is the only potential curative therapy. Sorafenib, a multikinase inhibitor of the RAF/MEK/ERK pathway, is a molecular-targeted drug that is approved for hepatocellular carcinoma (HCC) but not for CCC. The differences in signaling pathway characteristics under sorafenib treatment between HCC (HLF, Huh7, PLC/PRF/5) and CCC (RBE, YSCCC, Huh28) cell lines were therefore investigated using cell proliferation, western blotting, and apoptosis analyses. Sorafenib inhibited cell growth significantly less in CCC cells than in HCC cells, with lower suppression of ERK phosphorylation. Significantly decreased AKT Ser473 phosphorylation in HCC cells, and conversely enhanced phosphorylation of AKT Ser473 and mTORC2 in CCC cells, were observed with sorafenib treatment. Disassembly of the mTORC2 complex in RBE cells with siRNA targeting Rictor resulted in the downregulation of AKT Ser473 phosphorylation and enhanced apoptosis presumably via increased FOXO1, which consequently suppressed RBE cell proliferation. Phosphorylation of mTORC1 and autophagy were not influenced by sorafenib in CCC cells. Simultaneous administration of everolimus to suppress activated mTORC1 in RBE cells revealed that combined everolimus and sorafenib treatment under mTORC2 disassembly could enhance growth inhibition through the suppression of both sorafenib- and everolimus-dependent AKT Ser473 phosphorylation in addition to the inhibition of mTORC1 phosphorylation. Prevention of escape by AKT/mTOR signaling from the RAF/MEK/ERK pathway in sorafenib treatment by suppressing mTORC2 activity may lead to promising new approaches in CCC therapy.

Features of acute liver congestion on gadoxetate disodium-enhanced MRI in a rat model: Role of organic anion-transporting polypeptide 1A1

To evaluate the features of hepatic congestion on gadoxetate disodium (Gd‐EOB‐DTPA)‐enhanced magnetic resonance imaging (MRI) and the mechanisms responsible for the radiological findings in a rat model of partial liver congestion.

Treatment with specific soluble factors promotes the functional maturation of transcription factor-mediated, pancreatic transdifferentiated cells

Pancreatic lineage-specific transcription factors (TFs) display instructive roles in converting adult cells to endocrine pancreatic cells through a process known as transdifferentiation. However, little is known about potential factors capable of accelerating transdifferentiation following transduction to achieve the functional maturation of transdifferentiated cells. In this study, we demonstrated, using adult liver-derived progenitor cells, that soluble factors utilized in pancreatic differentiation protocols of pluripotent stem cells promote functional maturation of TFs-mediated transdifferentiated cells. Treatment with an N2 supplement in combination with three soluble factors (glucagon-like peptide-1 [GLP-1] receptor agonist, notch inhibitor, and transforming growth factor-β [TGF-β] inhibitor) enhanced liver-to-pancreas transdifferentiation based on the following findings: i) the incidence of c-peptide-positive cells increased by approximately 1.2-fold after the aforementioned treatment; ii) the c-peptide expression level in the treated cells increased by approximately 12-fold as compared with the level in the untreated cells; iii) the treated cells secreted insulin in a glucose-dependent manner, whereas the untreated cells did not; and iv) transplantation of treated-transdifferentiated cells into streptozotocin-induced immunodeficient diabetic mice led to the amelioration of hyperglycemia. These results suggest that treatment with specific soluble factors promotes the functional maturation of transdifferentiated cells. Our findings could facilitate the development of new modalities for cell-replacement therapy for patients with diabetes.

Tu1061 Impact of Multiple Tumors or Portal Hypertension on the Results of Second Hepatectomy for Recurrent Hepatocellular Carcinoma: A Single Center Experience

Background & Aims: Roayaie et al. reported a 5-year overall survival (OS) rate of 67% after second hepatectomy for recurrent hepatocellular carcinoma (HCC) in highly selected patients with a single nodule, preserved liver function, and noportal hypertension (PHT). The aim of this study was whether second liver resection can offer survival benefit for patients with multiple HCCs and/or PHT. Methods: We retrospectively studied 101 patients who had undergone second liver resection for recurrent HCC and stratified them into 3 groups according to the number of tumors and the presence of PHT, defined as a platelet count , 100,000/μL and/or the presence of esophageal varices: group A, patients with solitary tumor and no-PHT (n = 45); group B, those with either multiple tumor or PHT (n = 48); and Group C, those with both multiple tumor and PHT (n = 8). Overall survival (OS) and recurrence rate (RR) curves were constructed by the Kaplan-Meier method, and multivariate regression analysis was performed using the Cox proportional hazard model. Results: There was no 30-day mortality. The morbidity rate was comparable among the groups. The 5year OS rates and the 2-year recurrent rates were 67% and 60% in group A, 62%and 55% in group B, and 38% and 88% in group C, respectively, showing no significant differences among the three groups. In a multivariate analysis, neither multiple tumors nor presence of PHT was a predictive factor for poor prognosis. Conclusions: We can extend the indication of second hepatectomy for recurrent HCC, at least, to the patients with either multiple HCCs or PHT.