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Hepatic inflammation is culpable for the evolution of asymptomatic steatosis to nonalcoholic steatohepatitis (NASH). Hepatic inflammation results from abnormal macrophage activation. We found that FoxO1 links overnutrition to hepatic inflammation by regulating macrophage polarization and activation. FoxO1 was upregulated in hepatic macrophages, correlating with hepatic inflammation, steatosis and fibrosis in mice and patients with NASH. Myeloid cell-conditional FoxO1 knockout skewed macrophage polarization from pro-inflammatory M1 to anti-inflammatory M2 phenotypes, accompanied by the reduction of macrophage infiltration in liver. These effects mitigated overnutrition-induced hepatic inflammation and insulin resistance, contributing to improved hepatic metabolism and increased energy expenditure in myeloid cell FoxO1 knockout mice on HFD. When fed a NASH-inducing diet, myeloid cell FoxO1 knockout mice were protected from developing NASH, culminating in the reduction of hepatic inflammation, steatosis and fibrosis. Mechanistically, FoxO1 counteracts Stat6 to skew macrophage polarization from M2 toward M1 signatures to perpetuate hepatic inflammation in NASH. FoxO1 appears as a pivotal mediator of macrophage activation in response to overnutrition and a therapeutic target for ameliorating hepatic inflammation to stem the disease progression from benign steatosis to NASH.

The protein diversity of mammalian cells is determined by arrays of isoforms from genes. Genetic mutation is essential in species evolution and cancer development. Accurate long-read transcriptome sequencing at single-cell level is required to decipher the spectrum of protein expressions in mammalian organisms. In this report, we developed a synthetic long-read single-cell sequencing technology based on LOOPSeq technique. We applied this technology to analyze 447 transcriptomes of hepatocellular carcinoma (HCC) and benign liver from an individual. Through Uniform Manifold Approximation and Projection analysis, we identified a panel of mutation mRNA isoforms highly specific to HCC cells. The evolution pathways that led to the hyper-mutation clusters in single human leukocyte antigen molecules were identified. Novel fusion transcripts were detected. The combination of gene expressions, fusion gene transcripts, and mutation gene expressions significantly improved the classification of liver cancer cells versus benign hepatocytes. In conclusion, LOOPSeq single-cell technology may hold promise to provide a new level of precision analysis on the mammalian transcriptome.

Culture expansion of primary cells evokes highly reproducible DNA methylation (DNAm) changes. We have identified CG dinucleotides (CpGs) that become continuously hyper- or hypomethylated during long-term culture of mesenchymal stem cells (MSCs) and other cell types. Bisulfite barcoded amplicon sequencing (BBA-seq) demonstrated that DNAm patterns of neighboring CpGs become more complex without evidence of continuous pattern development and without association to oligoclonal subpopulations. Circularized chromatin conformation capture (4C) revealed reproducible changes in nuclear organization between early and late passages, while there was no enriched interaction with other genomic regions that also harbor culture-associated DNAm changes. Chromatin immunoprecipitation of CTCF did not show significant differences during long-term culture of MSCs, however culture-associated hypermethylation was enriched at CTCF binding sites and hypomethylated CpGs were devoid of CTCF. Taken together, our results support the notion that DNAm changes during culture-expansion are not directly regulated by a targeted mechanism but rather resemble epigenetic drift.Julia Franzen et al. investigate if changes in DNA methylation at specific genetic loci during cell culture expansion are due to a specific mechanism or gradual deregulation of an epigenetic state. Their results suggest that changes in CpG methylation are due to indirect epigenetic drift, rather than a consequence of targeting by DNA methyltransferases.

Culture medium of mesenchymal stromal cells (MSCs) is usually supplemented with either human platelet lysate (HPL) or fetal calf serum (FCS). Many studies have demonstrated that proliferation and cellular morphology are affected by these supplements – it is therefore important to determine if they favor outgrowth of different subpopulations and thereby impact on the heterogeneous composition of MSCs. We have isolated and expanded human bone marrow-derived MSCs in parallel with HPL or FCS and demonstrated that HPL significantly increases proliferation and leads to dramatic differences in cellular morphology. Remarkably, global DNA-methylation profiles did not reveal any significant differences. Even at the transcriptomic level, there were only moderate changes in pairwise comparison. Furthermore, the effects on proliferation, cytoskeletal organization, and focal adhesions were reversible by interchanging to opposite culture conditions. These results indicate that cultivation of MSCs with HPL or FCS has no systematic bias for specific cell types.

Background The use of mesenchymal stromal cells (MSCs) for research and clinical application is hampered by cellular heterogeneity and replicative senescence. Generation of MSC-like cells from induced pluripotent stem cells (iPSCs) may circumvent these limitations, and such iPSC-derived MSCs (iMSCs) are already tested in clinical trials. So far, a comparison of MSCs and iMSCs was particularly addressed in bulk culture. Despite the high hopes in cellular therapy, only little is known how the composition of different subclones changes in these cell preparations during culture expansion. Methods In this study, we used multicolor lentiviral genetic barcoding for the marking of individual cells within cell preparations. Based on this, we could track the clonal composition of syngenic MSCs, iPSCs, and iMSCs during culture expansion. Furthermore, we analyzed DNA methylation patterns at senescence-associated genomic regions by barcoded bisulfite amplicon sequencing. The proliferation and differentiation capacities of individual subclones within MSCs and iMSCs were investigated with limiting dilution assays. Results Overall, the clonal composition of primary MSCs and iPSCs gradually declined during expansion. In contrast, iMSCs became oligoclonal early during differentiation, indicating that they were derived from few individual iPSCs. This dominant clonal outgrowth of iMSCs was not associated with changes in chromosomal copy number variation. Furthermore, clonal dynamics were not clearly reflected by stochastically acquired DNA methylation patterns. Limiting dilution assays revealed that iMSCs are heterogeneous in colony formation and in vitro differentiation potential, while this was even more pronounced in primary MSCs. Conclusions Our results indicate that the subclonal diversity of MSCs and iPSCs declines gradually during in vitro culture, whereas derivation of iMSCs may stem from few individual iPSCs. Differentiation regimen needs to be further optimized to achieve homogeneous differentiation of iPSCs towards iMSCs.

The only definitive therapy for end‐stage liver disease is whole‐organ transplantation. The success of this intervention is severely limited by the complexity of the surgery, the cost of patient care, the need for long‐term immunosuppression, and the shortage of donor organs. In rodents and humans, end‐stage degeneration of hepatocyte function is associated with disruption of the liver‐specific transcriptional network and a nearly complete loss of promoter P1‐driven hepatocyte nuclear factor 4‐alpha (P1‐HNF4α) activity. Re‐expression of HNF4α2, the predominant P1‐HNF4α, reinstates the transcriptional network, normalizes the genes important for hepatocyte function, and reverses liver failure in rodents. In this study, we tested the effectiveness of supplementary expression of human HNF4α2 messenger RNA (mRNA) in primary human hepatocytes isolated from explanted livers of patients who underwent transplant for end‐stage irreversibly decompensated liver failure (Child‐Pugh B, C) resulting from alcohol‐mediated cirrhosis and nonalcoholic steatohepatitis. Re‐expression of HNF4α2 in decompensated cirrhotic human hepatocytes corrects the disrupted transcriptional network and normalizes the expression of genes important for hepatocyte function, improving liver‐specific protein expression. End‐stage liver disease in humans is associated with both loss of P1‐HNF4α expression and failure of its localization to the nucleus. We found that while HNF4α2 re‐expression increased the amount of P1‐HNF4α protein in hepatocytes, it did not alter the ability of hepatocytes to localize P1‐HNF4α to their nuclei. Conclusion: Re‐expression of HNF4α2 mRNA in livers of patients with end‐stage disease may be an effective therapy for terminal liver failure that would circumvent the need for organ transplantation. The efficacy of this strategy may be enhanced by discovering the cause for loss of nuclear P1‐HNF4α localization in end‐stage cirrhosis, a process not found in rodent studies. Re‐expression of HNF4α2 in decompensated cirrhotic human hepatocytes corrects the disrupted transcriptional network and normalizes the expression of genes important for hepatocyte function, improving liver‐specific protein expression. mRNA‐mediated re‐expression of HNF4α2 in the livers of patients with the end‐stage disease may be an effective therapy for terminal liver failure that would circumvent the need for organ transplantation.

The initial creation of human‐induced pluripotent stem cells (iPSCs) set the foundation for the future of regenerative medicine. Human iPSCs can be differentiated into a variety of cell types in order to study normal and pathological molecular mechanisms. Currently, there are well‐defined protocols for the differentiation, characterization, and establishment of functionality in human iPSC‐derived hepatocytes (iHep) and iPSC‐derived cholangiocytes (iCho). Electrophysiological study on chloride ion efflux channel activity in iHep and iCho cells has not been previously reported. We generated iHep and iCho cells and characterized them based on hepatocyte‐specific and cholangiocyte‐specific markers. The relevant transmembrane channels were selected: cystic fibrosis transmembrane conductance regulator, leucine rich repeat‐containing 8 subunit A, and transmembrane member 16 subunit A. To measure the activity in these channels, we used whole‐cell patch‐clamp techniques with a standard intracellular and extracellular solution. Our iHep and iCho cells demonstrated definitive activity in the selected transmembrane channels, and this approach may become an important tool for investigating human liver biology of cholestatic diseases. Transmembrane Channel Activity in Human Hepatocytes and Cholangiocytes Derived from Induced Pluripotent Stem Cells.

Hepatocyte nuclear factor 4 alpha (HNF4α) is a transcription factor that plays a critical role in hepatocyte function, and HNF4α‐based reprogramming corrects terminal liver failure in rats with chronic liver disease. In the livers of patients with advanced cirrhosis, HNF4α RNA expression levels decrease as hepatic function deteriorates, and protein expression is found in the cytoplasm. These findings could explain impaired hepatic function in patients with degenerative liver disease. In this study, we analyzed HNF4α localization and the pathways involved in post‐translational modification of HNF4α in human hepatocytes from patients with decompensated liver function. RNA‐sequencing analysis revealed that AKT‐related pathways, specifically phospho‐AKT, is down‐regulated in cirrhotic hepatocytes from patients with terminal failure, in whom nuclear levels of HNF4α were significantly reduced, and cytoplasmic expression of HNF4α was increased. cMET was also significantly reduced in failing hepatocytes. Moreover, metabolic profiling showed a glycolytic phenotype in failing human hepatocytes. The contribution of cMET and phospho‐AKT to nuclear localization of HNF4α was confirmed using Spearman's rank correlation test and pathway analysis, and further correlated with hepatic dysfunction by principal component analysis. HNF4α acetylation, a posttranslational modification important for nuclear retention, was also significantly reduced in failing human hepatocytes when compared with normal controls. Conclusion: These results suggest that the alterations in the cMET‐AKT pathway directly correlate with HNF4α localization and level of hepatocyte dysfunction. This study suggests that manipulation of HNF4α and pathways involved in HNF4α posttranslational modification may restore hepatocyte function in patients with terminal liver failure. Although drug induced liver injury (DILI) is a rare clinical event, it carries significant morbidity and mortality, leaving it as the leading cause of acute liver failure in the United States. It is one of the most challenging diagnoses encountered by gastroenterologists. DILI is also the most common single adverse event that has led to withdrawal of drugs from the marketplace, drug attrition and failure of implicated drugs to obtain FDA approval. The development of various drug injury networks have played a vital role in expanding our knowledge regarding drug, herbal and dietary supplement related liver injury. In this review, we discuss what defines liver injury, epidemiology of DILI, it's biochemical and pathologic patterns, and management.

Acute hepatic failure is associated with high morbidity and mortality for which the only definitive therapy is liver transplantation. Some fraction of those who undergo emergency transplantation have been shown to recover native liver function when transplanted with an auxiliary hepatic graft that leaves part of the native liver intact. Thus, transplantation could have been averted with the development and use of some form of hepatic support. The costs of developing and testing liver support systems could be dramatically reduced by the availability of a reliable large animal model of hepatic failure with a large therapeutic window that allows the assessment of efficacy and timing of intervention. Non-lethal forms of hepatic injury were examined in combination with liver-directed radiation in non-human primates (NHPs) to develop a model of acute hepatic failure that mimics the human condition. Porcine hepatocyte transplantation was then tested as a potential therapy for acute hepatic failure. After liver-directed radiation therapy, delivery of a non-lethal hepatic ischemia-reperfusion injury reliably and rapidly generated liver failure providing conditions that can enable pre-clinical testing of liver support or replacement therapies. Unfortunately, in preliminary studies, low hepatocyte engraftment and over-immune suppression interfered with the ability to assess the efficacy of transplanted porcine hepatocytes in the model. A model of acute liver failure in NHPs was created that recapitulates the pathophysiology and pathology of the clinical condition, does so with reasonably predictable kinetics, and results in 100% mortality. The model allowed preliminary testing of xenogeneic hepatocyte transplantation as a potential therapy.

Until now little is known about the functional integrity of human hepatocytes after hypothermic storage. In order to address this limitation, we evaluated several commercially available hypothermic preservation media for their abilities to protect freshly isolated hepatocytes during prolonged cold storage. Human hepatocytes were isolated from non-transplantable/rejected donor livers and resuspended in ice-cold University of Wisconsin solution (UW), HypoThermosol-Base (HTS-Base), or HypoThermosol-FRS (HTS-FRS) with or without the addition of fetal bovine serum. Cells were stored at 4°C for 24-72 h, and evaluated for hepatocyte viability (trypan blue exclusion, or labeling with fluorochromes), cell attachment, and function. The energy status of hepatocytes was evaluated by measurement of intracellular adenosine 5'-triphosphate. To determine whether the test cells expressed metabolic functions of freshly isolated cells, the activities of major phase I (cytochromes P450, FMO) and phase II (UGT, ST) drug-metabolizing enzymes were examined. Although hepatocytes are shown to be satisfactory after 24 h storage in all of the tested solutions, the cell viability, energy status, and xenobiotic metabolism following cold preservation in HTS-FRS was consistently and, in some cases, markedly higher when compared with other systems. The same metabolites for each of the tested substrates were detected in all groups of cells. Moreover, the use of HTS-FRS eliminates the need for serum in preservation solutions. HTS-FRS represents an improved solution compared to HTS-Base and UW for extending the shipping/storage time of human hepatocytes.