L. Longato

Decellularized human liver as a natural 3D-scaffold for liver bioengineering and transplantation.

Liver synthetic and metabolic function can only be optimised by the growth of cells within a supportive liver matrix. This can be achieved by the utilisation of decellularised human liver tissue. Here we demonstrate complete decellularization of whole human liver and lobes to form an extracellular matrix scaffold with a preserved architecture. Decellularized human liver cubic scaffolds were repopulated for up to 21 days using human cell lines hepatic stellate cells (LX2), hepatocellular carcinoma (Sk-Hep-1) and hepatoblastoma (HepG2), with excellent viability, motility and proliferation and remodelling of the extracellular matrix. Biocompatibility was demonstrated by either omental or subcutaneous xenotransplantation of liver scaffold cubes (5 × 5 × 5 mm) into immune competent mice resulting in absent foreign body responses. We demonstrate decellularization of human liver and repopulation with derived human liver cells. This is a key advance in bioartificial liver development.

Rapid production of human liver scaffolds for functional tissue engineering by high shear stress oscillation-decellularization

The development of human liver scaffolds retaining their 3-dimensional structure and extra-cellular matrix (ECM) composition is essential for the advancement of liver tissue engineering. We report the design and validation of a new methodology for the rapid and accurate production of human acellular liver tissue cubes (ALTCs) using normal liver tissue unsuitable for transplantation. The application of high shear stress is a key methodological determinant accelerating the process of tissue decellularization while maintaining ECM protein composition, 3D-architecture and physico-chemical properties of the native tissue. ALTCs were engineered with human parenchymal and non-parenchymal liver cell lines (HepG2 and LX2 cells, respectively), human umbilical vein endothelial cells (HUVEC), as well as primary human hepatocytes and hepatic stellate cells. Both parenchymal and non-parenchymal liver cells grown in ALTCs exhibited markedly different gene expression when compared to standard 2D cell cultures. Remarkably, HUVEC cells naturally migrated in the ECM scaffold and spontaneously repopulated the lining of decellularized vessels. The metabolic function and protein synthesis of engineered liver scaffolds with human primary hepatocytes reseeded under dynamic conditions were maintained. These results provide a solid basis for the establishment of effective protocols aimed at recreating human liver tissue in vitro.

Fibroblast growth factor 2 (FGF2) regulates cytoglobin expression and activation of human hepatic stellate cells via JNK signaling

Cytoglobin (CYGB) belongs to the mammalian globin family and is exclusively expressed in hepatic stellate cells (HSCs) in the liver. In addition to its gas-binding ability, CYGB is relevant to hepatic inflammation, fibrosis, and cancer because of its anti-oxidative properties; however, the regulation of CYGB gene expression remains unknown. Here, we sought to identify factors that induce CYGB expression in HSCs and to clarify the molecular mechanism involved. We used the human HSC cell line HHSteC and primary human HSCs isolated from intact human liver tissues. In HHSteC cells, treatment with a culture supplement solution that included fibroblast growth factor 2 (FGF2) increased CYGB expression with concomitant and time-dependent α-smooth muscle actin (αSMA) down-regulation. We found that FGF2 is a key factor in inducing the alteration in both CYGB and αSMA expression in HHSteCs and primary HSCs and that FGF2 triggered the rapid phosphorylation of both c-Jun N-terminal kinase (JNK) and c-JUN. Both the JNK inhibitor PS600125 and transfection of c-JUN–targeting siRNA abrogated FGF2-mediated CYGB induction, and conversely, c-JUN overexpression induced CYGB and reduced αSMA expression. Chromatin immunoprecipitation analyses revealed that upon FGF2 stimulation, phospho-c-JUN bound to its consensus motif (5′-TGA(C/G)TCA), located −218 to −222 bases from the transcription initiation site in the CYGB promoter. Of note, in bile duct–ligated mice, FGF2 administration ameliorated liver fibrosis and significantly reduced HSC activation. In conclusion, FGF2 triggers CYGB gene expression and deactivation of myofibroblastic human HSCs, indicating that FGF2 has therapeutic potential for managing liver fibrosis.

Reactive gamma-ketoaldehydes as novel activators of hepatic stellate cells in vitro

Aims Products of lipid oxidation, such as 4‐hydroxynonenal (4‐HNE), are key activators of hepatic stellate cells (HSC) to a pro‐fibrogenic phenotype. Isolevuglandins (IsoLG) are a family of acyclic &ggr;‐ketoaldehydes formed through oxidation of arachidonic acid or as by‐products of the cyclooxygenase pathway. IsoLGs are highly reactive aldehydes which are efficient at forming protein adducts and cross‐links at concentrations 100‐fold lower than 4‐hydroxynonenal. Since the contribution of IsoLGs to liver injury has not been studied, we synthesized 15‐E2‐IsoLG and used it to investigate whether IsoLG could induce activation of HSC. Results Primary human HSC were exposed to 15‐E2‐IsoLG for up to 48 h. Exposure to 5 &mgr;M 15‐E2‐IsoLG in HSCs promoted cytotoxicity and apoptosis. At non‐cytotoxic doses (50 pM‐500 nM) 15‐E2‐IsoLG promoted HSC activation, indicated by increased expression of &agr;‐SMA, sustained activation of ERK and JNK signaling pathways, and increased mRNA and/or protein expression of cytokines and chemokines, which was blocked by inhibitors of JNK and NF‐kB. In addition, IsoLG promoted formation of reactive oxygen species, and induced an early activation of ER stress, followed by autophagy. Inhibition of autophagy partially reduced the pro‐inflammatory effects of IsoLG, suggesting that it might serve as a cytoprotective response. Innovation This study is the first to describe the biological effects of IsoLG in primary HSC, the main drivers of hepatic fibrosis. Conclusions IsoLGs represent a newly identified class of activators of HSC in vitro, which are biologically active at concentrations as low as 500 pM, and are particularly effective at promoting a pro‐inflammatory response and autophagy. HighlightsIsolevuglandins (IsoLG) are described as novel activators of hepatic stellate cells.IsoLG are biologically active at concentrations as low as 500 pM.IsoLG promote activation of stress signaling pathways and redox changes.IsoLG induce a pro‐inflammatory effect, in part mediated by autophagy activation.Autophagy may reduce toxicity from IsoLG adduction to cellular proteins.