SOX9 is a novel cancer stem cell marker surrogated by osteopontin in human hepatocellular carcinoma

The current lack of cancer stem cell (CSC) markers that are easily evaluated by blood samples prevents the establishment of new therapeutic strategies in hepatocellular carcinoma (HCC). Herein, we examined whether sex determining region Y-box 9 (SOX9) represents a new CSC marker, and whether osteopontin (OPN) can be used as a surrogate marker of SOX9 in HCC. In HCC cell lines transfected with a SOX9 promoter-driven enhanced green fluorescence protein gene, FACS-isolated SOX9+ cells were capable of self-renewal and differentiation into SOX9-cells, and displayed high proliferation capacity in vitro. Xenotransplantation experiments revealed that SOX9+ cells reproduced, differentiated into SOX9-cells, and generated tumors at a high frequency in vivo. Moreover, SOX9+ cells were found to be involved in epithelial-mesenchymal transition (EMT) and activation of TGFb/Smad signaling. Gain/loss of function experiments showed that SOX9 regulates Wnt/beta-catenin signaling, including cyclin D1 and OPN. Immunohistochemistry of 166 HCC surgical specimens and serum OPN measurements showed that compared to SOX9-patients, SOX9+ patients had significantly poorer recurrence-free survival, stronger venous invasion, and higher serum OPN levels. In conclusion, SOX9 is a novel HCCCSC marker regulating the Wnt/beta-catenin pathway and its downstream target, OPN. OPN is a useful surrogate marker of SOX9 in HCC

ヒト肝内胆管癌におけるCD90発現はリンパ節転移と予後不良に関与する

京都大学0048新制・課程博士博士(医学)甲第21642号医博第4448号新制||医||1034(附属図書館)京都大学大学院医学研究科医学専攻(主査)教授 妹尾 浩, 教授 小川 誠司, 教授 坂井 義治学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

Secular and coseismic changes in S-wave velocity detected using ACROSS in the Tokai region

Abstract We discovered a secular change in the travel time of direct S-waves over a 10-year observation period by means of continuous operation of an artificial and stable seismic source, called Accurately Controlled Routinely Operated Signal System (ACROSS), which is deployed in the central part of Japan along the Nankai Trough. We used 13 High Sensitivity Seismograph Network Japan (Hi-net) stations around the ACROSS source to monitor the temporal variation in travel time. Green’s functions were calculated for each station daily from March 29, 2007, through October 30, 2017. Secular advance in the temporal variation in travel time was seen for the whole operation period, in addition to a steplike delay associated with the 2011 Tohoku earthquake. We estimated the rate of secular change and the amount of coseismic step by modeling the transfer function of S-waves with a linear trend and the coseismic step of the 2011 Tohoku earthquake. Distance dependences of the travel time changes can be explained as a combination of common bias and dispersion for each station, for both the secular and coseismic changes. This can be interpreted as a randomly distributed change in seismic velocity over the range of the observation region. An azimuthal dependence exists for both changes and shows larger changes in the NE–SW direction than in the NW–SE direction from the ACROSS source

Variations of fluid pressure within the subducting oceanic crust and slow earthquakes

We show fine‐scale variations of seismic velocities and converted teleseismic waves that reveal the presence of zones of high‐pressure fluids released by progressive metamorphic dehydration reactions in the subducting Philippine Sea plate in Tokai district, Japan. These zones have a strong correlation with the distribution of slow earthquakes, including long-term slow slip (LTSS) and low-frequency earthquakes (LFEs). Overpressured fluids in the LTSS region appear to be trapped within the oceanic crust by an impermeable cap rock in the fore-arc, and impede intraslab earthquakes therein. In contrast, fluid pressures are reduced in the LFE zone, which is deeper than the centroid of the LTSS, because there fluids are able to infiltrate into the narrow corner of the mantle wedge, leading to mantle serpentinization. The combination of fluids released from the subducting oceanic crust with heterogeneous fluid transport properties in the hanging wall generates variations of fluid pressures along the downgoing plate boundary, which in turn control the occurrence of slow earthquakes

Evaluation of Temperature Characteristics of a Data Logger LS-7000 and Fabrication of Preamplifier Exclusive to the Data Logger

＜短報

Efficient recellularisation of decellularised whole-liver grafts using biliary tree and foetal hepatocytes

A whole-organ regeneration approach, using a decellularised xenogeneic liver as a scaffold for the construction of a transplantable liver was recently reported. Deriving suitable scaffolds was the first step towards clinical application; however, effective recellularisation remains to be achieved. This report presents a strategy for the improvement of the recellularisation process, using novel cell-seeding technique and cell source. We evaluated recellularised liver grafts repopulated through the portal vein or the biliary duct with mice adult hepatocytes or E14.5 foetal hepatocytes. More than 80% of the cells seeded through the biliary tree entered the parenchyma beyond the ductule-lining matrix barrier and distributed throughout the liver lobule. In contrast, about 20% of the cells seeded through the portal tree entered the parenchyma. The gene expression levels of foetal hepatocyte albumin, glucose 6-phosphatase, transferrin, cytokeratin 19, and gamma-glutamyl transpeptidase were increased in three-dimensional cultures in the native liver-derived scaffolds, and the activation of liver detoxification enzymes and formation of biliary duct-like structures were supported. The metabolic functions of liver grafts recellularised with different cell types were similar. These results suggest that biliary tree cell-seeding approach is promising, and that liver progenitor cells represent a good cell source candidate

Novel hybrid three-dimensional artificial liver using human induced pluripotent stem cells and a rat decellularized liver scaffold

Introduction: Liver transplantation is currently the only curative therapy for end-stage liver failure; however, establishment of alternative treatments is required owing to the serious donor organ shortage. Here, we propose a novel model of hybrid three-dimensional artificial livers using both human induced pluripotent stem cells (hiPSCs) and a rat decellularized liver serving as a scaffold. Methods: Rat liver harvesting and decellularization were performed as reported in our previous studies. The decellularized liver scaffold was recellularized with hiPSC-derived hepatocyte-like cells (hiPSC-HLCs) through the biliary duct. The recellularized liver graft was continuously perfused with the culture medium using a pump at a flow rate of 0.5 mL/min in a standard CO2 (5%) cell incubator at 37 °C. Results: After 48 h of continuous perfusion culture, the hiPSC-HLCs of the recellularized liver distributed into the parenchymal space. Furthermore, the recellularized liver expressed the albumin (ALB) and CYP3A4 genes, and secreted human ALB into the culture medium. Conclusion: Novel hybrid artificial livers using hiPSCs and rat decellularized liver scaffolds were successfully generated, which possessed human hepatic functions. Keywords: Decellularized liver, Recellularization, Human iPSC, Hepatocyte, Artificial live

Generation of non-viral, transgene-free hepatocyte like cells with piggyBac transposon

Somatic cells can be reprogrammed to induced hepatocyte-like cells (iHeps) by overexpressing certain defined factors in direct reprogramming techniques. Of the various methods to deliver genes into cells, typically used genome-integrating viral vectors are associated with integration-related adverse events such as mutagenesis, whereas non-integrating viral vectors have low efficiency, making viral vectors unsuitable for clinical application. Therefore, we focused on developing a transposon system to establish a non-viral reprogramming method. Transposons are unique DNA elements that can be integrated into and removed from chromosomes. PiggyBac, a type of transposon, has high transduction efficiency and cargo capacity, and the integrated transgene can be precisely excised in the presence of transposase. This feature enables the piggyBac vector to achieve efficient transgene expression and a transgene-free state, thus making it a promising method for cell reprogramming. Here, we attempted to utilize the piggyBac transposon system to generate iHeps by integrating a transgene consisting of Hnf4a and Foxa3, and successfully obtained functional iHeps. We then demonstrated removal of the transgene to obtain transgene-free iHeps, which still maintained hepatocyte functions. This non-viral, transgene-free reprogramming method using the piggyBac vector may facilitate clinical applications of iHeps in upcoming cell therapy

A novel three-dimensional culture system maintaining the physiological extracellular matrix of fibrotic model livers accelerates progression of hepatocellular carcinoma cells

Abstract Liver fibrosis is characterized by the progressive accumulation of extracellular matrix (ECM) and is a strong predictor of hepatocellular carcinoma (HCC) development and progression. However, the effect of ECM in fibrotic livers on HCC cells is poorly understood. The aims of this study were to create a new culture system that retained the natural ECM of fibrotic model livers and to establish whether natural ECM regulated the characteristics of HCC cells. Using an organ decellularization technique, we created a new culture system that preserved the tissue-specific ECM of fibrotic model livers from CCl4-treated rats. The content of ECM in fibrotic model liver scaffolds was increased and the ECM microstructure was distorted. Quantitative polymerase chain reaction and immunofluorescence assays of HCC cells cultured in fibrotic model liver scaffolds for 7 days showed an epithelial-mesenchymal transition phenotype. Moreover, the ECM of fibrotic model livers promoted proliferation and chemoresistance of HCC cells. These results showed a novel effect of natural ECM in fibrotic model livers on the malignant behaviour of HCC cells. This new culture system will be useful for both understanding the cell biology of fibrotic livers and developing novel anti-cancer drugs