Nobuyasu Takahashi

Expandable Megakaryocyte Cell Lines Enable Clinically Applicable Generation of Platelets from Human Induced Pluripotent Stem Cells

The donor-dependent supply of platelets is frequently insufficient to meet transfusion needs. To address this issue, we developed a clinically applicable strategy for the derivation of functional platelets from human pluripotent stem cells (PSCs). This approach involves the establishment of stable immortalized megakaryocyte progenitor cell lines (imMKCLs) from PSC-derived hematopoietic progenitors through the overexpression of BMI1 and BCL-XL to respectively suppress senescence and apoptosis and the constrained overexpression of c-MYC to promote proliferation. The resulting imMKCLs can be expanded in culture over extended periods (4-5 months), even after cryopreservation. Halting the overexpression of c-MYC, BMI1, and BCL-XL in growing imMKCLs led to the production of CD42b(+) platelets with functionality comparable to that of native platelets on the basis of a range of assays in vitro and in vivo. The combination of robust expansion capacity and efficient platelet production means that appropriately selected imMKCL clones represent a potentially inexhaustible source of hPSC-derived platelets for clinical application.

Hepatocyte Growth Factor Activator Inhibitor Type 1 Regulates Epithelial to Mesenchymal Transition through Membrane-Bound Serine Proteinases

Hepatocyte growth factor activator inhibitor-1 (HAI-1), encoded by the serine protease inhibitor Kunitz type 1 (SPINT1) gene, is a membrane-associated proteinase inhibitor that potently inhibits a variety of serine proteinases, including those that are membrane bound. Although HAI-1/SPINT1 is widely expressed by epithelial cells and cancer cells, its functional role is still unclear, particularly in cancer. Here, we show that stable knockdown of HAI-1/SPINT1 in the human pancreatic cancer cell line SUIT-2 induces an elongated spindle-like morphology associated with accelerated invasion, thereby mimicking an epithelial to mesenchymal transition (EMT). We found that HAI-1/SPINT1 knockdown significantly reduced the expression of E-cadherin and was accompanied by up-regulation of Smad-interacting protein 1 (SIP1), an E-cadherin transcriptional repressor. In addition, matrix metalloproteinase-9 (MMP-9) was up-regulated. Similar results were obtained in the HLC-1 lung carcinoma cell line. Moreover, a metastatic variant of SUIT-2 (S2-CP8) that showed loss of E-cadherin expression also showed a significantly reduced level of HAI-1/SPINT1. Engineered overexpression of HAI-1/SPINT1 in S2-CP8 resulted in reversion of E-cadherin expression and SIP1 down-regulation, which accompanied reestablishment of epithelial morphology in culture. The EMT caused by HAI-1/SPINT1 knockdown seemed to be mediated, at least partly, by membrane-bound serine proteinases, matriptase/ST14 and TMPRSS4, as knockdown of matriptase/ST14 or TMPRSS4 in HAI-1/SPINT1 knockdown SUIT-2 cells and HLC-1 cells resulted in reversion of SIP1 and/or MMP-9 expression levels. We suggest that interactions between HAI-1/SPINT1 and membrane-bound serine proteinases contribute to transcriptional and functional changes involved in EMT in certain carcinoma cells.

Prognostic significance of circumferential cell surface immunoreactivity of glypican‐3 in hepatocellular carcinoma

Background: GC33 is a recently developed monoclonal antibody against human glypican‐3 (GPC3), which is significantly upregulated in hepatocellular carcinoma (HCC). GC33 recognizes a GPC3 ectodomain and shows significant antitumour activity in vivo. Thus, humanized GC33 antibody may be a promising tool for treating HCC having cell surface GPC3 expression.

Immortalization of Erythroblasts by c-MYC and BCL-XL Enables Large-Scale Erythrocyte Production from Human Pluripotent Stem Cells

Summary The lack of knowledge about the mechanism of erythrocyte biogenesis through self-replication makes the in vitro generation of large quantities of cells difficult. We show that transduction of c-MYC and BCL-XL into multipotent hematopoietic progenitor cells derived from pluripotent stem cells and gene overexpression enable sustained exponential self-replication of glycophorin A+ erythroblasts, which we term immortalized erythrocyte progenitor cells (imERYPCs). In an inducible expression system, turning off the overexpression of c-MYC and BCL-XL enabled imERYPCs to mature with chromatin condensation and reduced cell size, hemoglobin synthesis, downregulation of GCN5, upregulation of GATA1, and endogenous BCL-XL and RAF1, all of which appeared to recapitulate normal erythropoiesis. imERYPCs mostly displayed fetal-type hemoglobin and normal oxygen dissociation in vitro and circulation in immunodeficient mice following transfusion. Using critical factors to induce imERYPCs provides a model of erythrocyte biogenesis that could potentially contribute to a stable supply of erythrocytes for donor-independent transfusion.

Dickkopf-1 is overexpressed in human pancreatic ductal adenocarcinoma cells and is involved in invasive growth

The protein products of the Dickkopf (DKK) genes are antagonists of Wnt glycoproteins, which participate in tumor development and progression by binding to frizzled receptors. In this study, the expression of DKK‐1 was analyzed in a panel of 43 human cultured carcinoma cell lines. DKK‐1 expression was consistently and significantly upregulated in pancreatic carcinoma cell lines. Low level of DKK‐3 expression was also seen. In contrast, the expression of DKK‐2 and ‐4 was not detectable in most pancreatic carcinoma cell lines. The overexpression of DKK‐1 was confirmed in surgically resected human pancreatic cancer tissues, in which the mRNA level was evaluated in paired samples from cancerous and noncancerous pancreatic tissues. In ductal adenocarcinomas (23 cases), DKK‐1 mRNA levels were significantly upregulated compared to corresponding noncancerous tissues in a statistically significant level. To test the biological role of DKK‐1 in pancreatic carcinoma cells, we performed a knockdown of DKK‐1 in SUIT‐2 human pancreatic adenocarcinoma cell line and S2‐CP8, its metastatic subline, using a retroviral short hairpin RNA expression vector. DKK‐1 knockdown resulted in reduced migratory activity of SUIT‐2 in vitro. The in vitro growth rate and Matrigel invasion were also suppressed by DKK‐1 knockdown in S2‐CP8 cells. Collectively, the evidence suggests that, despite of its presumed antagonistic role in Wnt signaling, DKK‐1 may have a role in the aggressiveness of pancreatic carcinoma cells and could, therefore, serve as a novel biomarker of pancreatic cancer.

Synchronous adenocarcinoma and gastrointestinal stromal tumors of the stomach treated laparoscopically

Gastric adenocarcinomas account for approximately 95% of primary gastric tumors, and gastrointestinal stromal tumor (GIST) is the most common gastrointestinal mesenchymal tumor, accounting for 1%–3% of primary gastric tumors. However, the synchronous occurrence of GIST and gastric epithelial tumor is rare. We herein report a case of synchronous occurrence of gastric adenocarcinoma and two GISTs of the stomach. All lesions were resected laparoscopically. We discuss this case and review the literature.

Activation of MET receptor tyrosine kinase in ulcer surface epithelial cells undergoing restitution.

To the Editor: Hepatocyte growth factor (HGF) is a multifunctional cytokine that was initially identified as a potent inducer of hepatocyte growth. HGF was also independently identified as a scatter factor (SF) due to its capacity to induce dissociation and migration of epithelial cells. It is now well documented that HGF/SF is essential for the development and regeneration of various tissues and organs. In general, HGF/SF is produced by stromal cells as an inactive proform (pro-HGF/SF) and requires proteolytic activation to exhibit its biological activity. Activation occurs in response to tissue injury by serum proteases such as HGF activator (HGFA) or cellular proteases such as matriptase and hepsin. Activated HGF/SF subsequently stimulates epithelial and endothelial cells that express the MET receptor tyrosine kinase, a protein product of c-met proto-oncogene, to facilitate the repair and regeneration of injured tissues. It has been postulated that growth factors, such as epidermal growth factor, platelet-derived growth factor, basic fibroblast growth factor and HGF/SF, stimulate healing of gastrointestinal ulcers. Restitution is the initial phase of mucosal regeneration during which epithelial cells migrate and cover the ulcer base. Our previous study of HGFA knockout mice showed that restitution of severely damaged mucosa is significantly delayed in HGFA mice. Because HGFA is a potent activator of pro-HGF/SF, impaired restitution may be mediated by inefficient activation of pro-HGF/SF in the injured mucosa of HGFA mice. In fact, increased HGF/SF and MET levels at the ulcer edge suggest that the HGF/SF-MET system plays an important role in the in vivo regeneration of human gastrointestinal mucosa tissues. A block of HGF/SF signaling in experimental mouse models leads to mucosal injury, which is healed upon subsequent administration of HGF/SF. But it is not known if MET signaling is in fact activated in vivo in epithelial cells involved in the process of restitution. Enhanced MET activation has been successfully visualized in cancer cells using an antibody that specifically recognizes a phosphorylated tyrosine residue, Y1235, the major phosphorylation site of MET. Therefore, we used this antibody for immunohistochemical examination of MET receptor activation in epithelial cells showing restitution on the gastric ulcer base. The representative results are shown in Fig. 1. In this study we initially stained the serial sections of a regenerating ulcer after endoscopic mucosal resection (EMR) or submucosal dissection (ESD) of early gastric cancer (Table 1) with HE, antihuman MET (total MET) polyclonal antibody (C-28, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and antiphosphorylated MET (anti-p-MET) polyclonal antibody as described previously. With HE stain, formation of a healing zone along the ulcer margin and migration of epithelial cells from the ulcer margin onto granulation tissue to re-epithelialize the ulcer base (i.e. restitution) are observed (Fig. 1a). The immunoreactivity for total MET was observed in most epithelial cells (Fig. 1a). In contrast, p-MET immunoreactivity was primarily observed in migrating epithelial cells (Fig. 1a). Similar to previous observations, the p-MET staining did not localize at the cell surface but rather localized at the inner part of the cells (Fig. 1b). It appears likely that p-MET may be internalized to endosomes, consistent with our previous result obtained with cultured HepG2 cells. The epithelial cells showing restitution are known to be actively migrating but relatively low proliferating subpopulations. Figure 1(c) shows comparative immunohistochemistry for p-MET and Ki-67 (clone MIB-1; Dako, Glostrup, Denmark) in another EMR patient. The activation of MET was observed not only in Ki-67-positive, proliferating epithelial cells of the ulcer margin, but also in actively migrating Ki-67negative epithelial cells at the restitution front. Moreover, there was a tendency for the cells at the restitution front to show more intense p-MET immunoreactivity, and in this case membranous localization was also seen in part. We also immunostained intestinal ulcers of ischemic colitis, ulcerative colitis, Crohn’s disease and post-EMR adenocarcinoma (Table 1). As shown in Fig. 1(d), similar results were obtained in the intestinal specimens, showing enhanced MET phosphorylation in cells at the restitution front (Fig. 1d). But after re-epithelialization, MET phosphorylation in the surface epithelium was decreased along with the progress of mucosal regeneration, and most p-MET-positive epithelial cells were Ki-67-positive proliferating subpopulations (Fig. 1e). The immunohistochemistry data are summarized in Table 1. These findings are consistent with previous observations that activation of the HGF/SF-MET axis occurs in the injured tissue, and that mice lacking HGFA show significantly delayed epithelial restitution in an experimental colitis model. In contrast, endothelial cells had only focal and faint immunoreactivity for p-MET in the present study (data not shown). Growth factors activate epithelial cell migration and proliferation, and accelerate in vivo ulcer healing via receptor-specific binding on the cell surface. This observation confirms, for the first time, that MET is specifically activated in Pathology International 2008; 58: 462–464 doi:10.1111/j.1440-1827.2008.02255.x

Establishment and biological characterization of a novel cell line derived from hepatoid adenocarcinoma originated at the ampulla of Vater

Hepatoid adenocarcinoma is a rare gastrointestinal tumor and mostly reported in the stomach. Effective chemotherapy has yet to be developed to improve poor prognosis. The present study was undertaken to establish a useful cell line derived from a hepatoid adenocarcinoma, possibly leading to a new therapeutic strategy. The new human cell line VAT-39 was established from a metastatic lymph node of a 69-year-old Japanese male patient with hepatoid adenocarcinoma of the ampulla of Vater. The primary tumor and metastatic lymph node were composed of hepatoid adenocarcinoma cells exhibiting immunohistochemical reactivity for alpha-fetoprotein (AFP) and glypican-3 (GPC3). In the metastatic lymph node, Periodic acid-Schiff (PAS) staining clarified diffuse deposition of glycogen in the cytoplasm, indicating analogous characteristics to the primary hepatoid adenocarcinoma. Moreover, VAT-39 cells produced high levels of AFP in the cultured medium, and reverse-transcriptase polymerase chain reaction (RT-PCR) verified increased expression of GPC3 mRNA in this cell line. Further, we evaluated the sensitivity to major chemotherapeutic drugs against the bile duct cancer. Neither 5-fluorouracil nor gemcitabine showed particular sensitivity to this cell line. The tumorigenicity of the cultured cells was confirmed in athymic nude mice and the histological features of the explanted tumor were similar to the VAT-39 cell line. The present VAT-39 is the first hepatoid adenocarcinoma cell line that originates from the ampulla of Vater and it will be applicable for basic biological studies searching for new strategies of molecular targeted chemotherapy to this disease.

Primary pulmonary angiosarcoma: A case report.

Primary sarcoma is uncommon in the lung, and primary angiosarcoma is exceedingly rare. We report a case of primary pulmonary angiosarcoma of the left lung with emphasis on its growth pattern in the lung. A 48‐year‐old Japanese man was admitted to our hospital because of dyspnea on exertion. He was subsequently found to have left pleural effusion. Computed tomography shows a nodular lesion measuring 7 × 4 cm in his left lung. Obstruction of the left inferior lobar bronchus was observed, and endobronchial biopsy suggested angiosarcoma. Left pneumonectomy was performed. On macroscopic examination of the cut surface, multiple nodular lesions were observed particularly in portions around branches of pulmonary artery along bronchioles. Histological examination revealed vascular channel‐like structure with vague lumen formations by atypical polygonal or spindle‐shaped neoplastic cells. Immunohistochemically, the neoplastic cells are positive for FLI‐1, ERG, CD31 and von Willebrand factor/factor VIII‐related antigen, but not CD34. Angiosarcoma is a particularly rare form of primary pulmonary tumors, and this case report describes its unique macroscopic growth pattern in the lung.

Synchronous solid pseudopapillary neoplasm and intraductal papillary mucinous neoplasm of the pancreas: report of a case.

Solid pseudopapillary neoplasm (SPN) of the pancreas, most commonly found in young female subjects, is a rare neoplasm with low potential for malignancy. We report an unusual case of a 66-year-old male patient who had a simultaneous malignant SPN and an intraductal papillary mucinous adenoma (IPMA) of the pancreas. The patient was admitted to our department for the evaluation of the main solid tumor with calcification and small multilocular cystic lesions apart from the main tumor in the pancreatic head. We performed pylorus-preserving pancreaticoduodenectomy to treat the calcified tumor and multilocular cystic lesions. The diagnosis of malignant SPN was confirmed on the basis of histological invasion to the adjacent structures. The separate cystic lesions were diagnosed as a branch-type IPMA. The synchronous occurrence of IPMA and SPN in the present case did not demonstrate that there were tumors maintained through the common abnormal Wnt signaling pathway by immunohistochemical study. To our knowledge, this is the first known case of synchronous SPN and IPMA of the pancreas.