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-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.

Circumferential membranous GPC3 immunoreactivity in HCC indicates poorer prognosis particularly in patients with HCV infection.

SummaryThe 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.

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.The Dickkopf (DKK) genes are Wnt antagonists that were originally identified as inducers of head formation in Xenopus.1 Wnt glycoproteins participate in tumor development and progression by binding to frizzled receptors and signaling through the canonical and noncanonical Wnt pathways. 2The DKK gene family consists of DKK-1, -2, -3, -4, and a unique DKK-3-related gene soggy.2 The expression of these genes is temporally and spatially regulated, and all DKK proteins show distinct and elevated expression patterns in tissues that mediate the epithelial-mesenchymal transformation. 3 This finding suggests that they may participate in the epithelial to mesenchymal transition that is important not only in embryogenesis, but also in cancer progression.4 DKK-1 is a secreted protein that has been clearly defined as a direct inhibitor of Wnt binding to LRP5/6 coreceptors of frizzled. 2Evidence for the potential involvement of DKK-1 inactivation in human cancers is accumulating. The DKK-1 gene is frequently hypermethylated in colorectal cancers, whereas overexpression of DKK-1 reduced the growth of colorectal carcinoma cells. 5,6 The expression of DKK-1 is also reportedly downregulated in melanoma cells, 7 lung cancer cells with neuroendocrine differentiation 8 and in a metastatic subline of hepatocellular carcinoma cell line. 9 In contrast, overexpression of DKK-1 has also been found in some tumors, such as Wilms' tumor, hepatoblastoma, hepatocellular carcinoma and ovarian e...

Recent advances in bio-medical research, such as the production of regenerative organs from stem cells, require three-dimensional analysis of cell/tissue architectures. High-resolution imaging by electron microscopy is the best way to elucidate complex cell/tissue architectures, but the conventional method requires a skillful and time-consuming preparation. The present study developed a three-dimensional survey method for assessing cell/tissue architectures in 30-µm-thick paraffin sections by taking advantage of backscattered electron imaging in a low-vacuum scanning electron microscope. As a result, in the kidney, the podocytes and their processes were clearly observed to cover the glomerulus. The 30 µm thickness facilitated an investigation on face-side (instead of sectioned) images of the epithelium and endothelium, which are rarely seen within conventional thin sections. In the testis, differentiated spermatozoa were three-dimensionally assembled in the middle of the seminiferous tubule. Further application to vascular-injury thrombus formation revealed the distinctive networks of fibrin fibres and platelets, capturing the erythrocytes into the thrombus. The four-segmented BSE detector provided topographic bird’s-eye images that allowed a three-dimensional understanding of the cell/tissue architectures at the electron-microscopic level. Here, we describe the precise procedures of this imaging method and provide representative electron micrographs of normal rat organs, experimental thrombus formation, and three-dimensionally cultured tumour cells.

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.

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.