New Approaches in the Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells toward Hepatocytes

Orthotropic liver transplantation is the only established treatment for end-stage liver diseases. Utilization of hepatocyte transplantation and bio-artificial liver devices as alternative therapeutic approaches requires an unlimited source of hepatocytes. Stem cells, especially embryonic stem cells, possessing the ability to produce functional hepatocytes for clinical applications and drug development, may provide the answer to this problem. New discoveries in the mechanisms of liver development and the emergence of induced pluripotent stem cells in 2006 have provided novel insights into hepatocyte differentiation and the use of stem cells for therapeutic applications. This review is aimed towards providing scientists and physicians with the latest advancements in this rapidly progressing field

The differentiation of hepatocyte-like cells from monkey embryonic stem cells.

Embryonic stem cells (ESC) hold great potential for the treatment of liver diseases. Here, we report the differentiation of rhesus macaque ESC along a hepatocyte lineage. The undifferentiated monkey ESC line, ORMES-6, was cultured in an optimal culture condition in an effort to differentiate them into hepatocyte-like cells in vitro. The functional efficacy of the differentiated hepatic cells was evaluated using RT-PCR for the expression of hepatocyte specific genes, and Western blot analysis and immunocytochemistry for hepatic proteins such as alpha-fetoprotein (AFP), albumin and alpha1-antitrypsin (alpha1-AT). Functional assays were performed using the periodic acid schiff (PAS) reaction and ELISA. The final yield of ESC-derived hepatocyte-like cells was measured by flow cytometry for cells that were transduced with a liver-specific lentivirus vector containing the alpha1-AT promoter driving the expression of green fluorescence protein (GFP). The treatment of monkey ESC with an optimal culture condition yielded hepatocyte-like cells that expressed albumin, alpha1-AT, AFP, hepatocyte nuclear factor 3beta, glucose-6-phophatase, and cytochrome P450 genes and proteins as determined by RT-PCR and Western blot analysis. Immunofluorescent staining showed the cells positive for albumin, AFP, and alpha1-AT. PAS staining demonstrated that the differentiated cells showed hepatocyte functional activity. Albumin could be detected in the medium after 20 days of differentiation. Flow cytometry data showed that 6.5 +/- 1.0% of the total differentiated cells were positive for GFP. These results suggest that by using a specific, empirically determined, culture condition, we were able to direct monkey ESC toward a hepatocyte lineage

The differentiation of hepatocyte-like cells from monkey embryonic stem cells.

Embryonic stem cells (ESC) hold great potential for the treatment of liver diseases. Here, we report the differentiation of rhesus macaque ESC along a hepatocyte lineage. The undifferentiated monkey ESC line, ORMES-6, was cultured in an optimal culture condition in an effort to differentiate them into hepatocyte-like cells in vitro. The functional efficacy of the differentiated hepatic cells was evaluated using RT-PCR for the expression of hepatocyte specific genes, and Western blot analysis and immunocytochemistry for hepatic proteins such as alpha-fetoprotein (AFP), albumin and alpha1-antitrypsin (alpha1-AT). Functional assays were performed using the periodic acid schiff (PAS) reaction and ELISA. The final yield of ESC-derived hepatocyte-like cells was measured by flow cytometry for cells that were transduced with a liver-specific lentivirus vector containing the alpha1-AT promoter driving the expression of green fluorescence protein (GFP). The treatment of monkey ESC with an optimal culture condition yielded hepatocyte-like cells that expressed albumin, alpha1-AT, AFP, hepatocyte nuclear factor 3beta, glucose-6-phophatase, and cytochrome P450 genes and proteins as determined by RT-PCR and Western blot analysis. Immunofluorescent staining showed the cells positive for albumin, AFP, and alpha1-AT. PAS staining demonstrated that the differentiated cells showed hepatocyte functional activity. Albumin could be detected in the medium after 20 days of differentiation. Flow cytometry data showed that 6.5 +/- 1.0% of the total differentiated cells were positive for GFP. These results suggest that by using a specific, empirically determined, culture condition, we were able to direct monkey ESC toward a hepatocyte lineage

Role of MAPK phosphatase-1 in sustained activation of JNK during ethanol-induced apoptosis in hepatocyte-like VL-17A cells

Ethanol metabolism plays a central role in activating the mitogen-activated protein kinase (MAPK) cascade leading to inflammation and apoptosis. Sustained activation of c-Jun N-terminal kinase (JNK), one of the MAPKs, has been shown to induce apoptosis in hepatocytes. MAPK phosphatase-1 (MKP-1) has been shown to dephosphorylate MAPKs in several cells. The aim of the study is to evaluate the role of MKP-1 in sustained JNK activation as a mechanism to explain ethanol-induced hepatocyte apoptosis. VL-17A cells (HepG2 cells overexpressing alcohol dehydrogenase and cytochrome P450-2E1) were exposed to ethanol for different time periods. Western blots were performed for MKP-1, phospho-JNK, phosphotyrosine, and protein kinase Cdelta (PKCdelta). Electrophoretic mobility shift assays for AP-1 were performed. Apoptosis was measured by caspase-3 activity assay, TUNEL, and 4',6-diamidino-2-phenylindole staining. Reactive oxygen species were neutralized by overexpressing both superoxide dismutase-3 and catalase genes using lentiviral vectors in VL-17A cells. Ethanol incubation markedly decreased the MKP-1 protein levels to 15% of control levels and was associated with sustained phosphorylation of p46 JNK and p54 JNK, as well as increased apoptosis. VL-17A cells overexpressing superoxide dismutase-3 and catalase, treatment with a tyrosine kinase inhibitor, or incubation of the cells with PKCdelta small interference RNAs significantly inhibited the ethanol-induced MKP-1 degradation and apoptosis. Ethanol-induced oxidative stress enhanced the tyrosine phosphorylation of PKCdelta, which in turn caused the proteasomal degradation of MKP-1, leading to sustained JNK activation and increased apoptosis in VL-17A cells

Salvianolic Acid B Enhances Hepatic Differentiation of Human Embryonic Stem Cells Through Upregulation of WNT Pathway and Inhibition of Notch Pathway

Hepatocytes differentiated from human embryonic stem cells (ESCs) could provide a powerful tool for enabling cell-based therapies, studying the mechanisms underlying human liver development and disease, and testing the efficacy and safety of pharmaceuticals. However, currently most in vitro protocols yield hepatocytes with low levels of liver function. In this study, we investigated the potential of Salvianolic acid B (Sal B), an active pharmaceutical compound present in Salvia miltiorrhiza, which has been shown to have an antifibrotic effect in previous studies, to enhance hepatocyte differentiation from human ESCs. After treatment with Sal B, albumin expression and secretion were consistently increased, indicating that Sal B could promote hepatocyte differentiation process. Expression of a large number of important phase 1 and 2 metabolizing enzymes and phase 3 transporters was also increased in treated cells, indicating an enhanced biotransformation function. Our investigations further revealed the activation of Wnt pathway in treated cells, as determined by upregulation of Wnts, which increased amounts of nuclear β-catenin. This increased nuclear β-catenin led in turn to the enhanced expression of T cell factor (TCF) 3 and lymphoid enhancer-binding factor (LEF) 1 which upregulated their downstream targets, cyclin D1 and c-Myc. Notch receptors (Notch1, Notch3), Notch ligand (Jagged2), and Notch receptor targets [hairy and enhancer of split (Hes) 1, 5] were downregulated in treated cells, suggesting that Notch pathway was inhibited. Consistent with the inhibition of Notch pathway, expression of cholangiocyte marker, CK7, was significantly reduced by treatment with Sal B. Numb, a direct transcriptional target of Wnt pathway and a negative regulator of Notch pathway, was upregulated, consistent with activation of Wnt signaling and suppression of Notch signaling. In conclusion, our study demonstrated that Sal B enhanced hepatocyte differentiation from human ESCs through activation of Wnt pathway and inhibition of Notch pathway. Therefore, this study suggests that Sal B can be used as a potential agent to generate more mature hepatocytes for cell-based therapeutics and pharmaceutical studies

Lentivirus-mediated superoxide dismutase1 gene delivery protects against oxidative stress-induced liver injury in mice

BACKGROUND: The exposure of liver to hepatotoxins, and their subsequent metabolism, results in increased reactive oxygen species (ROS), one of the major culprits in causing both acute liver cell injury and chronic liver diseases. The aim of this present study is to investigate the protective effects of lentiviral vector-mediated copper-zinc superoxide dismutase (LV-SOD1) gene transfer against ROS-induced cytotoxicity in Hep G2 cells and liver injury in mice. METHODS: In vitro SOD1 efficacy was tested against two ROS-generating systems: hypoxanthine/xanthine oxidase (HX/XO) and hydroxyethyl radicals (HER), whereas in vivo SOD1 efficacy was evaluated in carbon tetrachloride (CCl4)-induced liver injury in C57BL/6 mice. RESULTS: LV-SOD1 transduction in Hep G2 cells resulted in a significant increase in SOD activity in cell lysates, and it significantly decreased the toxicity induced by HX/XO and HER. High SOD1 expression in the liver was achieved via portal vein injection of LV-SOD1 in mice and these high levels were observed for 30 days, the length of the experiment to date. SOD1 overexpression significantly decreased the toxicity and restored liver function in the CCl4-treated mice. CONCLUSIONS: These findings demonstrate for the first time that LV transduction led to the long-term expression of fully functional transgene expression in both in vitro and in vivo systems

Label-free imaging and analysis of the effects of lipolysis products on primary hepatocytes.

The increased accumulation of intracellular lipid droplets within hepatocytes is a pathologic hallmark of liver injury of various etiologies, especially non-alcoholic steatohepatitis (NASH). The dynamics, subcellular origin, and chemical composition of lipid droplets under various pathophysiologic conditions, however, remain poorly understood. We used coherent Raman microscopy and spontaneous Raman spectroscopy to monitor and analyze the formation of lipid droplets in living primary rat hepatocytes exposed to triglyceride-rich lipoprotein (TGRL) lipolysis products. After exposure to the complex fatty acid mixture released during the lipolysis process for 30 minutes, new lipid droplets rapidly appeared within hepatocytes and increased in size and number over the total observation period of 205 minutes. Raman spectroscopic analysis of individual intracellular lipid droplets before and after exposure to lipolysis products reveals that the major components of these droplets are esterified unsaturated fatty acids. We find that the fatty acid unsaturation ratio increases with droplet size. Control experiments with defined fatty acid mixtures reveal the complexity of the cellular response to assault by combinations of lipids

Label-free imaging and analysis of the effects of lipolysis products on primary hepatocytes.

The increased accumulation of intracellular lipid droplets within hepatocytes is a pathologic hallmark of liver injury of various etiologies, especially non-alcoholic steatohepatitis (NASH). The dynamics, subcellular origin, and chemical composition of lipid droplets under various pathophysiologic conditions, however, remain poorly understood. We used coherent Raman microscopy and spontaneous Raman spectroscopy to monitor and analyze the formation of lipid droplets in living primary rat hepatocytes exposed to triglyceride-rich lipoprotein (TGRL) lipolysis products. After exposure to the complex fatty acid mixture released during the lipolysis process for 30 minutes, new lipid droplets rapidly appeared within hepatocytes and increased in size and number over the total observation period of 205 minutes. Raman spectroscopic analysis of individual intracellular lipid droplets before and after exposure to lipolysis products reveals that the major components of these droplets are esterified unsaturated fatty acids. We find that the fatty acid unsaturation ratio increases with droplet size. Control experiments with defined fatty acid mixtures reveal the complexity of the cellular response to assault by combinations of lipids