Long-Term Fate of Human Fetal Liver Progenitor Cells Transplanted in Injured Mouse Livers.

Liver progenitor cells have the potential to repair and regenerate a diseased liver. The success of any translational efforts, however, hinges on thorough understanding of the fate of these cells after transplant, especially in terms of long-term safety and efficacy. Here, we report transplantation of a liver progenitor population isolated from human fetal livers into immune-permissive mice with follow-up up to 36 weeks after transplant. We found that human progenitor cells engraft and differentiate into functional human hepatocytes in the mouse, producing albumin, alpha-1-antitrypsin, and glycogen. They create tight junctions with mouse hepatocytes, with no evidence of cell fusion. Interestingly, they also differentiate into functional endothelial cell and bile duct cells. Transplantation of progenitor cells abrogated carbon tetrachloride-induced fibrosis in recipient mice, with downregulation of procollagen and anti-smooth muscle actin. Paradoxically, the degree of engraftment of human hepatocytes correlated negatively with the anti-fibrotic effect. Progenitor cell expansion was most prominent in cirrhotic animals, and correlated with transcript levels of pro-fibrotic genes. Animals that had resolution of fibrosis had quiescent native progenitor cells in their livers. No evidence of neoplasia was observed, even up to 9 months after transplantation. Human fetal liver progenitor cells successfully attenuate liver fibrosis in mice. They are activated in the setting of liver injury, but become quiescent when injury resolves, mimicking the behavior of de novo progenitor cells. Our data suggest that liver progenitor cells transplanted into injured livers maintain a functional role in the repair and regeneration of the liver. Stem Cells 2018;36:103-113.

A cellular system for quantitation of vitamin K cycle activity: structure-activity effects on vitamin K antagonism by warfarin metabolites.

Warfarin and other 4-hydroxycoumarins inhibit vitamin K epoxide reductase (VKOR) by depleting reduced vitamin K that is required for posttranslational modification of vitamin K-dependent clotting factors. In vitro prediction of the in vivo potency of vitamin K antagonists is complicated by the complex multicomponent nature of the vitamin K cycle. Here we describe a sensitive assay that enables quantitative analysis of γ-glutamyl carboxylation and its antagonism in live cells. We engineered a human embryonic kidney (HEK) 293-derived cell line (HEK 293-C3) to express a chimeric protein (F9CH) comprising the Gla domain of factor IX fused to the transmembrane and cytoplasmic regions of proline-rich Gla protein 2. Maximal γ-glutamyl carboxylation of F9CH required vitamin K supplementation, and was dose-dependently inhibited by racemic warfarin at a physiologically relevant concentration. Cellular γ-glutamyl carboxylation also exhibited differential VKOR inhibition by warfarin enantiomers (S > R) consistent with their in vivo potencies. We further analyzed the structure-activity relationship for inhibition of γ-glutamyl carboxylation by warfarin metabolites, observing tolerance to phenolic substitution at the C-5 and especially C-6, but not C-7 or C-8, positions on the 4-hydroxycoumarin nucleus. After correction for in vivo concentration and protein binding, 10-hydroxywarfarin and warfarin alcohols were predicted to be the most potent inhibitory metabolites in vivo.

Sustained Glutathione Deficiency Interferes with the Liver Response to TNF-α and Liver Regeneration after Partial Hepatectomy in Mice.

Glutathione (GSH) is a critical intracellular antioxidant that is active in free radical scavenging and as a reducing equivalent in biological reactions. Recent studies have suggested that GSH can affect cellular function at the level of gene transcription as well, in particular by affecting NF-κB activation. Additionally, increased or decreased GSH levels in vitro have been tied to increased or decreased hepatocyte proliferation, respectively. Here, we investigated the effect of GSH on the liver's response to TNF-α injection and 2/3 partial hepatectomy (PH), using mice deficient for the modifier subunit of glutamate-cysteine ligase (GCLM), the rate-limiting enzyme in de novo GSH synthesis. We demonstrate that Gclm-/- mice have a delay in IκBα degradation after TNF-α injection, resulting in delayed NF-κB nuclear translocation. These mice display profound deficiencies in GSH levels both before and during regeneration, and after PH, Gclm-/- mice have an overall delay in cell cycle progression, with slower DNA synthesis, mitosis, and expression of cell cycle proteins. Moreover, there is a delay in expression of downstream targets of NF-κB in the regenerating liver in Gclm-/- mice. These data suggest that GSH may play a role in hepatic NF-κB activation in vivo, which is necessary for accurate timing of liver regeneration.

Toll-like receptor 4 and myeloid differentiation factor 88 provide mechanistic insights into the cause and effects of interleukin-6 activation in mouse liver regeneration.

UNLABELLED: Partial hepatectomy (PH) consistently results in an early increase of circulating interleukin-6 (IL-6), which is thought to play a major role in liver regeneration. Activation of this cytokine after PH requires the adaptor protein, MyD88, but the specific MyD88-related receptors involved remain unidentified. It is also unknown whether the magnitude of IL-6 elevation determines the extent of subsequent hepatocyte proliferation. Here, we uncovered artifacts in the assessment of circulating IL-6 levels when using cardiac puncture in mice after PH. By using retro-orbital bleed sampling, we show that the circulating levels of IL-6 after PH were not directly correlated with the extent of hepatocyte DNA synthesis in individual mice. The IL-6 increase after PH was attenuated in all lipopolysaccharide-hyporesponsive mouse strains studied (e.g., C3H/HeJ, Tlr4 null, Cd14 null, Tlr2,4,9 null, and Tlr2,4-Caspase1 null) and was severely abrogated in Myd88 null mice. Despite attenuated IL-6 levels, Tlr4 null mice showed normal signaling downstream of IL-6 and normal hepatocyte proliferation. In contrast, Myd88 null mice showed severe impairments in signal transducer and activator of transcription 3 phosphorylation and Socs3 induction, but had enhanced and prolonged extracellular signal-related kinase 1 and 2 phosphorylation in the first 6 hours after PH. Unexpectedly, these changes were associated with accelerated initiation of hepatocyte proliferation, as assessed by hepatocyte bromodeoxyuridine incorporation, phospho-histone H3 immunostaining, and cyclin E and A protein expression. CONCLUSION: TLR-4 signaling contributes to IL-6 activation after PH, but the Tlr4-independent component appears sufficient for ensuring intact signaling downstream of IL-6. The lack of correlation between IL-6 levels and hepatocyte proliferation after PH, and the accelerated start of hepatocyte proliferation in Myd88 null mice despite abrogated cytokine activation, may highlight relevant antiproliferative effects of IL-6 signaling, possibly via Socs3, in the regulation of liver regeneration.

Attenuated progression of diet-induced steatohepatitis in glutathione-deficient mice.

In nonalcoholic fatty liver disease (NAFLD), depletion of hepatic antioxidants may contribute to the progression of steatosis to nonalcoholic steatohepatitis (NASH) by increasing oxidative stress that produces lipid peroxidation, inflammation, and fibrosis. We investigated whether depletion of glutathione (GSH) increases NASH-associated hepatic pathology in mice fed a diet deficient in methionine and choline (MCD diet). Wild-type (wt) mice and genetically GSH-deficient mice lacking the modifier subunit of glutamate cysteine ligase (Gclm null mice), the rate-limiting enzyme for de novo synthesis of GSH, were fed the MCD diet, a methionine/choline-sufficient diet, or standard chow for 21 days. We assessed NASH-associated hepatic pathology, including steatosis, fibrosis, inflammation, and hepatocyte ballooning, and used the NAFLD Scoring System to evaluate the extent of changes. We measured triglyceride levels, determined the level of lipid peroxidation products, and measured by qPCR the expression of mRNAs for several proteins associated with lipid metabolism, oxidative stress, and fibrosis. MCD-fed GSH-deficient Gclm null mice were to a large extent protected from MCD diet-induced excessive fat accumulation, hepatocyte injury, inflammation, and fibrosis. Compared with wt animals, MCD-fed Gclm null mice had much lower levels of F2-isoprostanes, lower expression of acyl-CoA oxidase, carnitine palmitoyltransferase 1a, uncoupling protein-2, stearoyl-coenzyme A desaturase-1, transforming growth factor-ß, and plasminogen activator inhibitor-1 mRNAs, and higher activity of catalase, indicative of low oxidative stress, inhibition of triglyceride synthesis, and lower expression of profibrotic proteins. Global gene analysis of hepatic RNA showed that compared with wt mice, the livers of Gclm null mice have a high capacity to metabolize endogenous and exogenous compounds, have lower levels of lipogenic proteins, and increased antioxidant activity. Thus, metabolic adaptations resulting from severe GSH deficiency seem to protect against the development of steatohepatitis.

Defective DNA strand break repair causes chromosomal instability and accelerates liver carcinogenesis in mice.

UNLABELLED: Chromosomal instability is a characteristic feature of hepatocellular carcinoma (HCC) but its origin and role in liver carcinogenesis are undefined. We tested whether a defect in the nonhomologous end-joining (NHEJ) DNA repair gene Ku70 was associated with chromosomal abnormalities and enhanced liver carcinogenesis. Male Ku70 NHEJ-deficient (Ku70-/-), heterozygote (Ku70 +/-), and wild-type (WT) mice were injected with diethylnitrosamine (DEN), a liver carcinogen, at age 15 days. Animals were killed at 3, 6, and 9 months for assessment of tumorigenesis and hepatocellular proliferation. For karyotype analysis, primary liver tumor cell cultures were prepared from HCCs arising in Ku70 mice of all genotypes. Compared to WT littermates, Ku70-/- mice injected with DEN displayed accelerated HCC development. Ku70-/- HCCs harbored clonal increases in numerical and structural aberrations of chromosomes 4, 5, 7, 8, 10, 14, and 19, many of which recapitulated the spectrum of equivalent chromosomal abnormalities observed in human HCC. Ku70-/- HCCs showed high proliferative activity with increased cyclin D1 and proliferating cell nuclear antigen expression, Aurora A kinase activity, enhanced ataxia telangiectasia mutated kinase and ubiquitination, and loss of p53 via proteasomal degradation, features which closely resemble those of human HCC. CONCLUSION: These findings demonstrate that defects in the NHEJ DNA repair pathway may participate in the disruption of cell cycle checkpoints leading to chromosomal instability and accelerated development of HCC.

Pescadillo is essential for nucleolar assembly, ribosome biogenesis, and mammalian cell proliferation.

Mutation of the zebrafish pescadillo gene blocks expansion of a number of tissues in the developing embryo, suggesting roles for its gene product in controlling cell proliferation. We report that levels of the pescadillo protein increase in rodent hepatocytes as they enter the cell cycle. Pescadillo protein localizes to distinct substructures of the interphase nucleus including nucleoli, the site of ribosome biogenesis. During mitosis pescadillo closely associates with the periphery of metaphase chromosomes and by late anaphase is associated with nucleolus-derived foci and prenucleolar bodies. Blastomeres in mouse embryos lacking pescadillo arrest at morula stages of development, the nucleoli fail to differentiate and accumulation of ribosomes is inhibited. We propose that in mammalian cells pescadillo is essential for ribosome biogenesis and nucleologenesis and that disruption to its function results in cell cycle arrest.