In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.

Epithelial-mesenchymal transition (EMT) is an important mechanism to initiate cancer invasion and metastasis. Bone morphogenetic protein (BMP)-9 is a member of the transforming growth factor (TGF)-b superfamily. It has been suggested to play a role in cancer development in some non-hepatic tumors. In the present study, two hepatocellular carcinoma (HCC) lines, HLE and HepG2, were treated with BMP-9 in vitro, and phenotypic changes and cell motility were analyzed. In situ hybridization (ISH) and immunohistochemical analyses were performed with human HCC tissue samples in order to assess expression levels of BMP-9. In vivo, BMP-9 protein and mRNA were expressed in all the tested patients to diverse degrees. At the protein level, mildly positive (1 + ) BMP-9 staining could be observed in 25 ⁄ 41 (61%), and moderately to strongly positive (2 + ) in 16 ⁄ 41 (39%) of the patients. In 27 ⁄ 41 (65%) patients, the BMP-9 protein expression level was consistent with the mRNA expression level as measured by ISH. In those patients with 2 + protein level, nuclear pSmad1 expression in cancer cells was also significantly increased. Expression of BMP-9 was positively related to nuclear Snail expression and reversely correlated to cell surface E-cadherin expression, although this did not reach statistical significance. Expression levels of BMP-9 were significantly associated with the T stages of the investigated tumors and high levels of BMP-9 were detected by immunofluorescence especially at the tumor borders in samples from an HCC mouse model. In vitro, BMP-9 treatment caused a reduction of E-cadherin and ZO-1 and an induction of Vimentin and Snail expression. Furthermore, cell migration was enhanced by BMP-9 in both HCC cell lines. These results imply that EMT induced by BMP-9 is related to invasiveness of HCC. (Cancer Sci 2013; 104: 398-408) E ighty to ninety percent of primary liver cancer cases originate from hepatocellular carcinoma (HCC), which has been regarded as the fifth most common cancer and the third most cancer-related death in the world.(1) Current therapeutic options including surgical resection, liver transplantation and chemoembolization are only used at early stages of HCC with limited efficacy.(2) Cancer recurrence occurs in around 50% of patients.(3,4) Despite extensive scientific efforts, the prognosis of HCC is nowadays still poor since HCC is inclined to tumor invasiveness and formation of intra-and extra-hepatic metastases.Epithelial-mesenchymal transition (EMT) is one major mechanism participating in malignant progression of cancer cells. Epithelial-mesenchymal transition is characterized by loss of differentiated traits in epithelial cells, for example, cell-cell contacts and cell polarity, as well as acquisition of mesenchymal appearances such as higher motility, invasiveness and resistance to apoptosis.(5) Properties typical for EMT comprise downregulation of epithelial markers like E-cadherin, ZO-1, nuclear translocation of b-catenin and upregulation of mesenchymal markers like Vimentin, N-cadh...

0 AMP-activated protein kinase; CHK2, check point kinase 2; DM, Dorsomorphin; ESA, epithelial cell adhesion molecule; GSH, glutathione; HSP27, heat shock protein 2; LKB1, liver kinase B1;

Key Points Fibrosis in the liver is a common cause of liver disease, partially mediated by platelet TGF-β1 as shown in a mouse model of liver injury. Depleting platelet TGF-β1 results in decreased liver fibrosis suggesting that blocking platelet TGF-β1 may ameliorate or prevent fibrosis.

Zonation of metabolic activities within specific structures and cell types is a phenomenon of liver organization and ensures complementarity of variant liver functions like protein production, glucose homeostasis and detoxification. To analyze damage and regeneration of liver tissue in response to a toxic agent, expression of liver specific enzymes was analyzed by in situ hybridization in mouse over a 6 days time course following carbon tetrachloride (CCl4) injection. CCl4 mixed with mineral oil was administered to BALB/c mice by intraperitoneal injection, and mice were sacrificed at different time points post injection. Changes in the expression of albumin (Alb), arginase (Arg1), glutaminase 2 (Gls2), Glutamine synthetase (Gs), glucose-6-phosphatase (G6pc), glycogen synthase 2 (Gys2), Glycerinaldehyd-3-phosphat-Dehydrogenase (Gapdh), Cytochrom p450 2E1 (Cyp2e1) and glucagon receptor (Gcgr) genes in the liver were studied by in situ hybridization and qPCR. We observed significant changes in gene expression of enzymes involved in nitrogen and glucose metabolism and their local distribution following CCl4 injury. We also found that Cyp2e1, the primary metabolizing enzyme for CCl4, was strongly expressed in the pericentral zone during recovery. Furthermore, cells in the damaged area displayed distinct gene expression profiles during the analyzed time course and showed complete recovery with strong albumin production 6 days after CCl4 injection. Our results indicate that despite severe damage, liver cells in the damaged area do not simply die but instead display locally adjusted gene expression supporting damage response and recovery.