Human embryonic stem cells (hESCs) are a valuable source of pluripotential primary cells. To date, however, their homogeneous cellular differentiation to specific cell types in vitro has proven difficult. Wnt signaling has been shown to play important roles in coordinating development, and we demonstrate that Wnt3a is differentially expressed at critical stages of human liver development in vivo. The essential role of Wnt3a in hepatocyte differentiation from hESCs is paralleled by our in vitro model, demonstrating the importance of a physiologic approach to cellular differentiation. Our studies provide compelling evidence that Wnt3a signaling is important for coordinated hepatocellular function in vitro and in vivo. In addition, we demonstrate that Wnt3a facilitates clonal plating of hESCs exhibiting functional hepatic differentiation. These studies represent an important step toward the use of hESC-derived hepatocytes in high-throughput metabolic analysis of human liver function.definitive endoderm ͉ function ͉ hepatocyte ͉ drug metabolism ͉ high throughput H uman embryonic stem cells (hESCs) are derived from the inner cell mass of preimplantation embryos and demonstrate pluripotency in vitro and in vivo (1, 2). Such attributes allow hESCs to be differentiated down all germ lineages in large numbers and offer significant advantages over their adult stem cell counterparts, which are generally limited in their capacity to differentiate and proliferate (3). Although these hESCs provide a valuable source of adult differentiated cells, homogeneous cellular differentiation to specific germ layers has proven difficult to achieve. One potential explanation for this failure is that cells do not receive sequential developmental cues that they do in vivo.Wnt signaling has been shown to play an important role in hESC self-renewal and differentiation and stimulates numerous intracellular signal transduction cascades, including the canonical pathway regulating gene expression in the nucleus and what seems to be a network of noncanonical pathways regulating many other aspects of cell biology [reviewed by Cadigan and Liu (4)]. In the absence of Wnt signaling, ␤-catenin is targeted for degradation; however, active Wnt signaling inhibits ␤-catenin destruction, resulting in its nuclear translocation (5, 6). After nuclear localization, ␤-catenin dimerizes with the nuclear proteins from the T cell factor/lymphoid enhancer factor (TCF/ LEF) family and transactivates gene expression. TCF/LEFs are not only present in transcriptional activator complexes; they also play a role in corepressor complex assembly (6).The important role played by Wnt signaling during gastrulation in vivo is evidenced by gene knockouts or dominant negatives (7, 8). Wnt3-mediated Brachyury expression is also important for migration of precursor cells through the anterior region of the primitive streak (PS). The subsequent specification of the anterior region of the PS to mesoderm or endoderm is likely to depend on the duration and magnitude of Nodal signaling (...

Liver transplantation is the accepted treatment for patients with acute liver failure and liver-based metabolic disorders. However, donor organ shortage and lifelong need for immunosuppression are the main limitations to liver transplantation. In addition, loss of the native liver as a target organ for future gene therapy for metabolic disorders limits the futuristic treatment options, resulting in the need for alternative therapeutic strategies. A potential alternative to liver transplantation is allogeneic hepatocyte transplantation. Over the last two decades, hepatocyte transplantation has made the transition from bench to bedside. Standardized techniques have been established for isolation, culture, and cryopreservation of human hepatocytes. Clinical hepatocyte transplantation safety and short-term efficacy have been proven; however, some major hurdles-mainly concerning shortage of donor organs, low cell engraftment, and lack of a long-lasting effect-need to be overcome to widen its clinical applications. Current research is aimed at addressing these problems, with the ultimate goal of increasing hepatocyte transplantation efficacy in clinical applications.

Background and AimIntraperitoneal transplantation of alginate-microencapsulated human hepatocytes is an attractive option for the management of acute liver failure (ALF) providing short-term support to allow native liver regeneration. The main aim of this study was to establish an optimised protocol for production of alginate-encapsulated human hepatocytes and evaluate their suitability for clinical use.MethodsHuman hepatocyte microbeads (HMBs) were prepared using sterile GMP grade materials. We determined physical stability, cell viability, and hepatocyte metabolic function of HMBs using different polymerisation times and cell densities. The immune activation of peripheral blood mononuclear cells (PBMCs) after co-culture with HMBs was studied. Rats with ALF induced by galactosamine were transplanted intraperitoneally with rat hepatocyte microbeads (RMBs) produced using a similar optimised protocol. Survival rate and biochemical profiles were determined. Retrieved microbeads were evaluated for morphology and functionality.ResultsThe optimised HMBs were of uniform size (583.5±3.3 µm) and mechanically stable using 15 min polymerisation time compared to 10 min and 20 min (p<0.001). 3D confocal microscopy images demonstrated that hepatocytes with similar cell viability were evenly distributed within HMBs. Cell density of 3.5×106 cells/ml provided the highest viability. HMBs incubated in human ascitic fluid showed better cell viability and function than controls. There was no significant activation of PBMCs co-cultured with empty or hepatocyte microbeads, compared to PBMCs alone. Intraperitoneal transplantation of RMBs was safe and significantly improved the severity of liver damage compared to control groups (empty microbeads and medium alone; p<0.01). Retrieved RMBs were intact and free of immune cell adherence and contained viable hepatocytes with preserved function.ConclusionAn optimised protocol to produce GMP grade alginate-encapsulated human hepatocytes has been established. Transplantation of microbeads provided effective metabolic function in ALF. These high quality HMBs should be suitable for use in clinical transplantation.

Biological processes require close cooperation of multiple transcription factors that integrate different signals. Thyroid hormone receptors (TRs) induce Kr€ uppel-like factor 9 (KLF9) to regulate neurogenesis. Here, we show that triiodothyronine (T3) also works through TR to induce KLF9 in HepG2 liver cells, mouse liver, and mouse and human primary hepatocytes and sought to understand TR/KLF9 network function in the hepatocyte lineage and stem cells. Knockdown experiments reveal that KLF9 regulates hundreds of HepG2 target genes and modulates T3 response. Together, T3 and KLF9 target genes influence pathways implicated in stem cell selfrenewal and differentiation, including Notch signaling, and we verify that T3 and KLF9 cooperate to regulate key Notch pathway genes and work independently to regulate others. T3 also induces KLF9 in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC) and this effect persists during differentiation to definitive endoderm and hiPSC-derived hepatocytes. Microarray analysis reveals that T3 regulates hundreds of hESC and hiPSC target genes that cluster into many of the same pathways implicated in TR and KLF9 regulation in HepG2 cells. KLF9 knockdown confirms that TR and KLF9 cooperate to regulate Notch pathway genes in hESC and hiPSC, albeit in a partly cell-specific manner. Broader analysis of T3 responsive hESC/hiPSC genes suggests that TRs regulate multiple early steps in ESC differentiation. We propose that TRs cooperate with KLF9 to regulate hepatocyte proliferation and differentiation and early stages of organogenesis and that TRs exert widespread and important influences on ESC biology. STEM CELLS 2015;33:416-428

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BackgroundThe development of effective therapies for acute liver failure (ALF) is limited by our knowledge of the pathophysiology of this condition, and the lack of suitable large animal models of acetaminophen toxicity. Our aim was to develop a reproducible invasively-monitored porcine model of acetaminophen-induced ALF.Method35kg pigs were maintained under general anaesthesia and invasively monitored. Control pigs received a saline infusion, whereas ALF pigs received acetaminophen intravenously for 12 hours to maintain blood concentrations between 200-300 mg/l. Animals surviving 28 hours were euthanased.ResultsCytochrome p450 levels in phenobarbital pre-treated animals were significantly higher than non pre-treated animals (300 vs 100 pmol/mg protein). Control pigs (n = 4) survived 28-hour anaesthesia without incident. Of nine pigs that received acetaminophen, four survived 20 hours and two survived 28 hours. Injured animals developed hypotension (mean arterial pressure; 40.8 +/- 5.9 vs 59 +/- 2.0 mmHg), increased cardiac output (7.26 +/- 1.86 vs 3.30 +/- 0.40 l/min) and decreased systemic vascular resistance (8.48 +/- 2.75 vs 16.2 +/- 1.76 mPa/s/m3). Dyspnoea developed as liver injury progressed and the increased pulmonary vascular resistance (636 +/- 95 vs 301 +/- 26.9 mPa/s/m3) observed may reflect the development of respiratory distress syndrome.Liver damage was confirmed by deterioration in pH (7.23 +/- 0.05 vs 7.45 +/- 0.02) and prothrombin time (36 +/- 2 vs 8.9 +/- 0.3 seconds) compared with controls. Factor V and VII levels were reduced to 9.3 and 15.5% of starting values in injured animals. A marked increase in serum AST (471.5 +/- 210 vs 42 +/- 8.14) coincided with a marked reduction in serum albumin (11.5 +/- 1.71 vs 25 +/- 1 g/dL) in injured animals. Animals displayed evidence of renal impairment; mean creatinine levels 280.2 +/- 36.5 vs 131.6 +/- 9.33 μmol/l. Liver histology revealed evidence of severe centrilobular necrosis with coagulative necrosis. Marked renal tubular necrosis was also seen. Methaemoglobin levels did not rise >5%. Intracranial hypertension was not seen (ICP monitoring), but there was biochemical evidence of encephalopathy by the reduction of Fischer's ratio from 5.6 +/- 1.1 to 0.45 +/- 0.06.ConclusionWe have developed a reproducible large animal model of acetaminophen-induced liver failure, which allows in-depth investigation of the pathophysiological basis of this condition. Furthermore, this represents an important large animal model for testing artificial liver support systems.

Our data indicate that ROS formation, rather than cellular steatosis per se, impairs mitochondrial function. Thus, reduction in cellular steatosis may not always be the desired outcome without concomitant improvement in mitochondrial function and/or reducing of ROS formation.

At sub-lethal concentrations, ZnO nanoparticles dramatically increased both gluconeogenesis and glycogenolysis, which warrants further in vivo studies.