Masaya Iwamuro

Review of the diagnosis and management of gastrointestinal bezoars

The formation of a bezoar is a relatively infrequent disorder that affects the gastrointestinal system. Bezoars are mainly classified into four types depending on the material constituting the indigestible mass of the bezoar: phytobezoars, trichobezoars, pharmacobezoars, and lactobezoars. Gastric bezoars often cause ulcerative lesions in the stomach and subsequent bleeding, whereas small intestinal bezoars present with small bowel obstruction and ileus. A number of articles have emphasized the usefulness of Coca-Cola(®) administration for the dissolution of phytobezoars. However, persimmon phytobezoars may be resistant to such dissolution treatment because of their harder consistency compared to other types of phytobezoars. Better understanding of the etiology and epidemiology of each type of bezoar will facilitate prompt diagnosis and management. Here we provide an overview of the prevalence, classification, predisposing factors, and manifestations of bezoars. Diagnosis and management strategies are also discussed, reviewing mainly our own case series. Recent progress in basic research regarding persimmon phytobezoars is also briefly reviewed.

COMBINED DUODENAL STENT PLACEMENT AND ENDOSCOPIC ULTRASONOGRAPHY-GUIDED BILIARY DRAINAGE FOR MALIGNANT DUODENAL OBSTRUCTION WITH BILIARY STRICTURE

Patients with malignant pancreatobiliary neoplasm sometimes manifest duodenal obstruction and biliary stricture synchronously or metachronously. In this paper, we reviewed our experience with and technique for combined endoscopic duodenal stent placement and endoscopic ultrasonography (EUS)‐guided biliary drainage. Between May 2007 and September 2009, this combined technique was performed on seven patients with distal biliary strictures and duodenal obstructions. The clinical success rate of the procedure, complications, patency periods of duodenal stents and patency periods of biliary stents were retrospectively evaluated. Clinical success was achieved in all seven cases for both procedures. Complications related to EUS‐biliary drainage, namely localized peritonitis due to bile leakage, occurred in two cases. Both patients recovered without additional interventions. Occlusion of a duodenal stent was observed in one patient, but additional intervention could not be performed due to sepsis. Occlusion of both a duodenal stent and a biliary stent was also observed in one patient, and this was resolved with the insertion of an additional duodenal stent and a biliary stent exchange. In conclusion, combined duodenal stent placement and EUS‐guided biliary drainage is a therapeutic option in case of failed endoscopic retrograde cannulation of malignant strictures with a malignant duodenal obstruction.

Intramuscular transplantation of engineered hepatic tissue constructs corrects acute and chronic liver failure in mice.

BACKGROUND & AIMS Transplantation of isolated hepatocytes holds great promise as an alternative to whole organ liver transplantation. For treatment of liver failure, access to the portal circulation has significant risks and intrahepatic hepatocyte engraftment is poor. In advanced cirrhosis, transplantation into an extrahepatic site is necessary and intrasplenic engraftment is short-lived. Strategies that allow repeated extrahepatic infusion of hepatocytes could improve the efficacy and safety of hepatocyte transplantation for the treatment of liver failure. METHODS A non-immunogenic self-assembling peptide nanofiber (SAPNF) was developed as a three-dimensional scaffold and combined with growth factors derived from a conditionally immortalized human hepatocyte cell line to engineer a hepatic tissue graft that would allow hepatocyte engraftment outside the liver. RESULTS The hepatic tissue constructs maintained hepatocyte-specific gene expression and functionality in vitro. When transplanted into skeletal muscle as an extrahepatic site for engraftment, the engineered hepatic grafts provided life-saving support in models of acute, fulminant, and chronic liver failure that recapitulates these clinical diseases. CONCLUSIONS SAPNF-engineered hepatic constructs engrafted and functioned as hepatic tissues within the muscle to provide life-sustaining liver support. These engineered tissue constructs contained no animal products that would limit their development as a therapeutic approach.

Hepatic differentiation of mouse iPS cells in vitro.

Induced pluripotent stem (iPS) cells are pluripotent and are able to unlimitedly proliferate in vitro. This technical breakthrough in creating iPS cells from somatic cells has noteworthy implications for overcoming the immunological rejection and the ethical issues associated with the derivation of embryonic stem cells from embryos. In the current work, we present an efficient hepatic differentiation of mouse iPS cells in vitro. iPS cells were cultured free floating to induce the formation of embryoid bodies (EB) for 5 days. EB were transferred to a gelatin-coated plate and treated with 100 ng/ml activin A and 100 ng/ml basic fibroblast growth factor (bFGF) for 3 days to induce definitive endoderm. Cells were further cultured for 8 days with 100 ng/ml hepatocyte growth factor (HGF) to generate hepatocytes. Characterization was performed by RT-PCR assay. Functional analysis for albumin secretion and ammonia removal was also carried out. iPS cell-derived hepatocyte-like cells (iPS-Heps) were obtained at the end of the differentiation program. Expression levels of a gestational hepatocyte gene and lineage-specific hepatic genes intensified in iPS-Heps. The production of albumin increased in a time-dependent manner. iPS-Heps were capable of metabolizing ammonia. We present here instant hepatic differentiation of mouse iPS cells using combined 3-day treatments of activin A and bFGF with subsequent 8-day HGF. Our study will be an important step to generate hepatocytes from human iPS cells as a new source for liver-targeted cell therapies.

Characteristics of CD133(+) human colon cancer SW620 cells.

Worldwide, colorectal cancer is the third most common type of cancer affecting both sexes. It has been proposed that a small subset of cancer cells (cancer stem cells) within each tumor is able to initiate tumor growth. In 2007, two research groups simultaneously identified a colon cancer stem cell population in human tumors by the use of CD133 expression. In the present study, we used a human colon cancer cell line, SW620, to analyze the cancer stem cell-like characteristics of CD133+ cells in vitro and in vivo. In vitro, CD133+ SW620 cells had a higher proliferative capacity, were more irradiation- and chemotherapy-resistant, and had a higher expression of β-catenin compared with CD133- cells. Injections of either CD133+ or CD133- cells into the skin or rectal mucosa of NOD/SCID mice led to tumors; however, injection of CD133+ cells resulted in the formation of larger tumors. Tumors derived from injections of CD133- cells did not contain any CD133+ cells, whereas tumors derived from injections of CD133+ cells did contain CD133+ cells, suggesting self-renewing capability. However, the proportion of CD133+ cells in the newly formed tumors in vivo was lower than the proportion of CD133+ cells in vitro. In conclusion, the human colon cancer cell line, SW620, contains both CD133+ and CD133- phenotypes, and the CD133+ phenotype has characteristics consistent with those of cancer stem cells.

Duodenal follicular lymphoma: comprehensive gene expression analysis with insights into pathogenesis.

Follicular lymphoma (FL) of the gastrointestinal tract, particularly duodenal follicular lymphoma (DFL), is a rare variant of FL with indolent clinical behavior, and this disease is included in the 2008 World Health Organization classification system. In contrast to nodal follicular lymphoma (NFL), DFL occurs most frequently in the second part of the duodenum, lacks follicular dendritic cell meshworks and has memory B‐cell characteristics. However, its molecular pathogenesis is still unclear. In the present study, we examined 10 DFL, 18 NFL and 10 gastric MALT lymphoma samples using gene expression analysis. Quantitative RT‐PCR experiments and immunohistochemical analysis for 72 formalin‐fixed, paraffin‐embedded tissues from an independent series, including 32 DFL, 19 gastric MALT lymphoma and 27 NFL samples, were performed for validation of microarray data. Gene expression profiles of the three lymphoma types were compared using 2918 differentially expressed genes (DEG) and results suggested that DFL shares characteristics of MALT lymphoma. Among these DEG, CCL20 and MAdCAM‐1 were upregulated in DFL and MALT but downregulated in NFL. In contrast, protocadherin gamma subfamily genes were upregulated in DFL and NFL. Quantitative RT‐PCR and immunohistochemical studies demonstrated concordant results. Double immunofluorescence studies revealed that CCL20 and CCR6 were co‐expressed in both DFL and MALT. We hypothesize that increased expression of CCL20 and MAdCAM‐1 and co‐expression of CCL20 and CCR6 may play an important role in tumorigenesis.

Des-γ-carboxyl prothrombin is associated with tumor angiogenesis in hepatocellular carcinoma

Background and Aim:  Hepatocellular carcinoma (HCC) is a hypervascular tumor, and angiogenesis plays an important role in its development. Previously, we demonstrated that des‐γ‐carboxyl prothrombin (DCP) promotes both cell proliferation and migration of human umbilical vein endothelial cells (HUVECs) by inducing the autophosphorylation of kinase insert domain receptor (KDR). In the present study, DCP‐associated tumor angiogenesis was assessed by comparing hypovascular and common hypervascular HCC.

A preliminary study for constructing a bioartificial liver device with induced pluripotent stem cell-derived hepatocytes

BackgroundBioartificial liver systems, designed to support patients with liver failure, are composed of bioreactors and functional hepatocytes. Immunological rejection of the embedded hepatocytes by the host immune system is a serious concern that crucially degrades the performance of the device. Induced pluripotent stem (iPS) cells are considered a desirable source for bioartificial liver systems, because patient-derived iPS cells are free from immunological rejection. The purpose of this paper was to test the feasibility of a bioartificial liver system with iPS cell-derived hepatocyte-like cells.MethodsMouse iPS cells were differentiated into hepatocyte-like cells by a multi-step differentiation protocol via embryoid bodies and definitive endoderm. Differentiation of iPS cells was evaluated by morphology, PCR assay, and functional assays. iPS cell-derived hepatocyte-like cells were cultured in a bioreactor module with a pore size of 0.2 μm for 7 days. The amount of albumin secreted into the circulating medium was analyzed by ELISA. Additionally, after a 7-day culture in a bioreactor module, cells were observed by a scanning electron microscope.ResultsAt the final stage of the differentiation program, iPS cells changed their morphology to a polygonal shape with two nucleoli and enriched cytoplasmic granules. Transmission electron microscope analysis revealed their polygonal shape, glycogen deposition in the cytoplasm, microvilli on their surfaces, and a duct-like arrangement. PCR analysis showed increased expression of albumin mRNA over the course of the differentiation program. Albumin and urea production was also observed. iPS-Heps culture in bioreactor modules showed the accumulation of albumin in the medium for up to 7 days. Scanning electron microscopy revealed the attachment of cell clusters to the hollow fibers of the module. These results indicated that iPS cells were differentiated into hepatocyte-like cells after culture for 7 days in a bioreactor module with a pore size of 0.2 μm.ConclusionWe consider the combination of a bioreactor module with a 0.2-μm pore membrane and embedded hepatocytes differentiated from iPS cells to be a promising option for bioartificial liver systems. This paper provides the basic concept and preliminary data for an iPS cell-oriented bioartificial liver system.PACS code: 87. Biological and medical physics, 87.85.-d Biomedical engineering, 87.85.Lf Tissue engineering, 87.85.Tu Modeling biomedical systems.

Treatment of Acute Liver Failure in Mice by Hepatocyte Xenotransplantation

Liver diseases still have a high mortality even though liver transplantation has become a standard treatment. Currently, hepatocyte transplantation has been proposed as another promising strategy. One limitation is the availability of human livers as a source of hepatocytes. Because of an unlimited supply, the use of porcine hepatocytes might address this problem. Regardless of the source, once isolated hepatocytes lose specific functionality due to the loss of the natural microenvironment. For this reason, we tested the ability of a self-assembling peptide nanofiber (SAPNF) to provide a provisional three-dimensional (3D) support to interact with cells to control their function in vivo. Isolated porcine hepatocytes were embedded in SAPNF, or collagen type I and transplanted by direct injection into the splenic pulp of SCID mice suffering from acute liver failure (ALF) by 90% hepatectomy. SAPNF porcine hepatocyte transplantation produced engraftment that was far superior to that obtained using collagen and prolonged the survival of mice with ALF, in contrast with controls. An ultrastructural evaluation using transmission electron microscopy indicated extensive cell–cell communication and preservation of hepatocyte architecture. The transplanted SAPNF hepatocytes showed higher expression of albumin and PAS and lower apoptotic events assessed by TUNEL staining. Hepatocytes culture in a truly 3D network allows in vivo maintaining of differentiated functions, and once transplanted between widely divergent species can function to correct acute liver failure in mice and prolong their survival.

Comparative analysis of endoderm formation efficiency between mouse ES cells and iPS cells.

Definitive endoderm (DE) derived from stem cells holds potential to differentiate into hepatocytes. Stem cell therapy using those cells has potential for a treatment of liver disease. To date, various ways of inducing hepatocytes from embryonic stem (ES) cells have been reported by researchers. However, it has not been proved enough that induced pluripotent stem (iPS) cells behave in the same manner as ES cells in endoderm differentiation. The purpose of this study was to establish an efficient method to induce DE from iPS cells, through comparatively analyzing the efficacy of endoderm formation from mouse ES cells. Furthermore, the efficiency of a serum-free medium in the differentiation into DE was investigated. Mouse ES cells and iPS cells were floated in culture medium for 2 or 5 days and embryoid bodies (EB) were formed. Subsequently, DE was induced with 100 ng/ml activin A and 100 ng/ml basic fibroblast growth factor (bFGF). RT-PCR and real-time PCR analyses were carried out at each step to determine the gene expression of EB markers. The difference in cellular proliferation between serum-containing and serum-free media was examined by an MTS assay in EB and DE induction. iPS cells showed the paralleled mRNA expression to ES cells in each step of differentiation into EB, but the levels of expression of Sox17 and Foxa2 were relatively higher in ES cell-derived DE, whereas Cxcr4 expression was higher in iPS cell-derived DE. The utilization of serum-free medium for iPS cells showed significantly favorable cellular proliferation during EB formation and subsequent DE induction. Forming EB for 5 days and subsequently DE induction with activin A and bFGF with serum-free medium was an appropriate protocol in iPS cells. This may represent an important step for generating hepatocytes from iPS cells for the development of cell therapy.