[Metabolomic interference induced by short-chain chlorinated paraffins in human normal hepatic cells].

Short-chain chlorinated paraffins (SCCPs) are an emerging class of persistent organic pollutants (POPs) that are widely detected in environmental matrices and human samples. Because of their environmental persistence, long-range transport potential, bioaccumulation potential, and biotoxicity, SCCPs pose a significant threat to human health. In this study, metabolomics technology was applied to reveal the metabolomic interference in human normal hepatic (L02) cells after exposure to low (1 µg/L), moderate (10 µg/L), and high (100 µg/L) doses of SCCPs. Principal component analysis (PCA) and metabolic effect level index (MELI) values showed that all three SCCP doses caused notable metabolic perturbations in L02 cells. A total of 72 metabolites that were annotated by MS/MS and matched with the experimental spectra in the Human Metabolome Database (HMDB) or validated by commercially available standards were selected as differential metabolites (DMs) across all groups. The low-dose exposure group shared 33 and 36 DMs with the moderate- and high-dose exposure groups, respectively. The moderate-dose exposure group shared 46 DMs with the high-dose exposure group. In addition, 33 DMs were shared among the three exposure groups. Among the 72 DMs, 9, 9, and 45 metabolites participated in the amino acid, nucleotide, and lipid metabolism pathways, respectively. The results of pathway enrichment analysis showed that the most relevant metabolic pathways affected by SCCPs were the lipid metabolism, fatty acid ß-oxidation, and nucleotide metabolism pathways, and that compared with low-dose exposure, moderate- and high-dose SCCP exposures caused more notable perturbations of these metabolic pathways in L02 cells. Exposure to SCCPs perturbed glycerophospholipid and sphingolipid metabolism. Significant alterations in the levels of phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins indicated SCCP-induced biomembrane damage. SCCPs inhibited fatty acid ß-oxidation by decreasing the levels of short- and medium-chain acylcarnitines in L02 cells, indicating that the energy supplied by fatty acid oxidation was reduced in these cells. Furthermore, compared with low- and moderate-dose SCCPs, high-dose SCCPs produced a significantly stronger inhibition of fatty acid ß-oxidation. In addition, SCCPs perturbed nucleotide metabolism. The higher hypoxanthine levels observed in L02 cells after SCCP exposures indicate that SCCPs may induce several adverse effects, including hypoxia, reactive oxygen species production, and mutagenesis in L02 cells.

Morphological parameters of hepatocytes in the European mole (Talpa europaea) and herb field mouse (Sylvaemus uralensis) under industrial pollution: Qualitative and quantitative assessment.

Morphological alterations of cells and tissues usually occur in biological organisms exposed to environmental contaminants, there by acting as a biomarker of environmental pollution, thus, making this study highly pertinent. The effect of industrial pollution on the qualitative and quantitative morphological parameters of hepatocytes (through histological analysis and cytomorphometry) was studied in two contrasting species of small mammals (Talpa europaea and Sylvaemus uralensis), taking into account the animal age (young and adult groups) and liver concentrations of heavy metals (Cu, Zn, Cd, Pb). Studies were performed in the regions exposed to emissions from two currently operating copper smelters: Middle Ural Copper Smelter (Middle Urals, T. europaea catching area) and Karabash Copper Smelter (Southern Urals, S. uralensis catching area). Seven morphometric parameters of hepatocytes were measured, of which two key parameters were selected by the method of principal components-the cell packing density and nuclear-cytoplasmic ratio (N/C). It was found that cell packing density in T. europaea from the impact zone decreased relative to the background area in young animals. At the same time, the differences in this parameter between the age groups from the background zone were leveled in the impact area of catching. The N/C ratio in T. europaea hepatocytes showed no correlation with either animal age or site of capture (background or impact area). In S. uralensis, both parameters, even taking into account the age, were found to be insensitive to indicate an effect of industrial pollution. Dystrophic changes (tested through histological analysis) in the liver tissue were revealed in all animal groups, but their frequency did not depend on any of the factors (age, zone) as well as the level of accumulation of toxic heavy metals (Cd, Pb). Morphometric parameters of hepatocytes have proved to be more reliable indicators of pollution, compared to the frequency of liver histopathology, due to lower subjectivity in their evaluation.

Effect of Antiviral Treatment on Hepatitis B Virus Integration and Hepatocyte Clonal Expansion.

BACKGROUND: This study investigated the effect of nucleos(t)ide analogue (NUC) treatment on hepatitis B virus (HBV) DNA integration and hepatocyte clonal expansion, both of which are implicated in hepatocellular carcinoma (HCC) in chronic hepatitis B. METHODS: Twenty-eight patients receiving NUCs (11 lamivudine, 7 telbivudine, 10 entecavir) were included. All had liver biopsies at baseline and year 1, and 7 had a third biopsy at year 10. HBV DNA integration and hepatocyte clone size were assessed by inverse polymerase chain reaction. RESULTS: All patients had detectable HBV integration at baseline, with a median integration frequency of 1.01 × 109 per liver and hepatocyte clone size of 2.41 × 105. Neither integration frequency nor hepatocyte clone size correlated with age and HBV virologic parameters. After 1 year of treatment, HBV integration was still detectable in all patients, with a median of 5.74 × 108 integration per liver (0.22 log reduction; P = .008) and hepatocyte clone size of 1.22 × 105 (0.40 log reduction; P = .002). HBV integration remained detectable at year 10 of treatment, with a median integration frequency of 4.84 × 107 integration per liver (0.93 log reduction from baseline) and hepatocyte clone size of 2.55 × 104 (1.02 log reduction from baseline). From baseline through year 1 to year 10, there was a decreasing trend in both integration frequency and hepatocyte clone size (P = .066 and.018, respectively). CONCLUSIONS: NUCs reduced both HBV DNA integration and hepatocyte clonal expansion, suggesting another alternative pathway besides direct viral suppression to reduce HCC risk. Our findings supported the notion for a long-term NUC treatment to prevent HCC.

Quantitative proteomics reveals common and unique molecular mechanisms underlying beneficial effects of caffeine and trigonelline on human hepatocytes.

Caffeine and trigonelline are the major bioactive compounds in coffee. Caffeine alone or combined with other coffee compounds shows hepatoprotective effects. However, molecular mechanisms underlying such hepatoprotective effects remain unclear. We therefore addressed molecular effects of caffeine and trigonelline on human hepatocytes using quantitative proteomics followed by bioinformatic analyses to obtain topological and functional significance. HepG2 cells were treated with 100 µM caffeine or trigonelline for 24-h and evaluated by quantitative proteomics using nanoLC-ESI-LTQ-Orbitrap MS/MS. A total of 26 and 25 significantly altered proteins were identified in caffeine-treated and trigonelline-treated cells, respectively, compared with control cells. Topological analyses revealed that ribosomal and translation regulatory proteins predominantly served as the hub proteins associated with protein clusters. Functional analyses also revealed that these two bioactive compounds shared some molecular mechanisms via induction of translational processes. There were also other unique molecular functions and biological processes triggered or suppressed by either caffeine or trigonelline. These data highlight common and unique molecular mechanisms underlying the hepatoprotective effects of caffeine and trigonelline that may be useful for future clinical applications.

Hepatocyte cholesterol content modulates glucagon receptor signalling.

OBJECTIVE: To determine whether glucagon receptor (GCGR) actions are modulated by cellular cholesterol levels. METHODS: We determined the effects of experimental cholesterol depletion and loading on glucagon-mediated cAMP production, ligand internalisation and glucose production in human hepatoma cells, mouse and human hepatocytes. GCGR interactions with lipid bilayers were explored using coarse-grained molecular dynamic simulations. Glucagon responsiveness was measured in mice fed a high cholesterol diet with or without simvastatin to modulate hepatocyte cholesterol content. RESULTS: GCGR cAMP signalling was reduced by higher cholesterol levels across different cellular models. Ex vivo glucagon-induced glucose output from mouse hepatocytes was enhanced by simvastatin treatment. Mice fed a high cholesterol diet had increased hepatic cholesterol and a blunted hyperglycaemic response to glucagon, both of which were partially reversed by simvastatin. Simulations identified likely membrane-exposed cholesterol binding sites on the GCGR, including a site where cholesterol is a putative negative allosteric modulator. CONCLUSIONS: Our results indicate that cellular cholesterol content influences glucagon sensitivity and indicate a potential molecular basis for this phenomenon. This could be relevant to the pathogenesis of non-alcoholic fatty liver disease, which is associated with both hepatic cholesterol accumulation and glucagon resistance.

Isolation of Echimidine and Its C-7 Isomers from Echium plantagineum L. and Their Hepatotoxic Effect on Rat Hepatocytes.

Echimidine is the main pyrrolizidine alkaloid of Echium plantagineum L., a plant domesticated in many countries. Because of echimidine's toxicity, this alkaloid has become a target of the European Food Safety Authority regulations, especially in regard to honey contamination. In this study, we determined by NMR spectroscopy that the main HPLC peak purified from zinc reduced plant extract with an MS [M + H]+ signal at m/z 398 corresponding to echimidine (1), and in fact also represents an isomeric echihumiline (2). A third isomer present in the smallest amount and barely resolved by HPLC from co-eluting (1) and (2) was identified as hydroxymyoscorpine (3). Before the zinc reduction, alkaloids (1) and (2) were present mostly (90%) in the form of an N-oxide, which formed a single peak in HPLC. This is the first report of finding echihumiline and hydroxymyoscorpine in E. plantagineum. Retroanalysis of our samples of E. plantagineum collected in New Zealand, Argentina and the USA confirmed similar co-occurrence of the three isomeric alkaloids. In rat hepatocyte primary culture cells, the alkaloids at 3 to 300 µg/mL caused concentration-dependent inhibition of hepatocyte viability with mean IC50 values ranging from 9.26 to 14.14 µg/mL. Our discovery revealed that under standard HPLC acidic conditions, echimidine co-elutes with its isomers, echihumiline and to a lesser degree with hydroxymyoscorpine, obscuring real alkaloidal composition, which may have implications for human toxicity.

Determination of microRNA-122 in hepatocytes by two-step amplification of duplex-specific nuclease with laser-induced fluorescence detection.

MicroRNAs (miRNAs) play important roles in physiological and pathological processes of cells. To develop a fast, simple and sensitive method to determine miRNAs is significant for miRNA studies. In this work, determination of microRNA-122 (miR-122) was achieved by laser-induced fluorescence (LIF) detection. A vial-LIF interface was first applied for sample analysis. A two-step amplification of the fluorescence signal for miR-122 was designed and realized by applying duplex-specific nuclease in the cleaving of two sensing probes. Under optimized conditions, the analysis of a miR-122 sample could be completed in less than 50 min. Only 10 µL sample was required for each test and the detection limit for the method was 0.60 pM equal to 1.2 amol of miR-122 in 10 µL solution. Lastly, the developed method was successfully applied to determine miR-122 in chicken and duck liver. The developed method was fast, selective, sensitive and sample-saving for the determination of miRNAs.

Toxicity and Metabolomic Impact of Cobalt, Chromium, and Nickel Exposure on HepaRG Hepatocytes.

Cobalt, chromium, and nickel are used in orthopedic prostheses. They can be released, accumulate in many organs, and be toxic. The aim of this study is to evaluate the cytotoxicity of these metals on human hepatocytes and to improve our knowledge of their cellular toxicity mechanisms by metabolomic analysis. HepaRG cells were incubated for 48 h with increasing concentrations of metals to determine their IC50. Then, a nontargeted metabolomic study using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) was done at IC50 and at a lower concentration (100 nM), near to those found in the blood and liver of patients with prostheses. IC50 were defined at 940, 2, and 1380 µM for Co, Cr, and Ni, respectively. In vitro, Cr appears to be much more toxic than Co and Ni. Metabolomic analysis revealed the disruption of metabolic pathways from the low concentration of 100 nM, in particular tryptophan metabolism and lipid metabolism illustrated by an increase in phenylacetylglycine, a marker of phospholipidosis, for all three metals. They also appear to be responsible for oxidative stress. Dysregulation of these pathways impacts hepatocyte metabolism and may result in hepatotoxicity. Further investigations on accessible biological matrices should be conducted to correlate our in vitro results with the clinical data of prostheses-bearing patients.

Hepatic small extracellular vesicles promote microvascular endothelial hyperpermeability during NAFLD via novel-miRNA-7.

BACKGROUND: A recent study has reported that patients with nonalcoholic fatty liver disease (NAFLD) are more susceptible to coronary microvascular dysfunction (CMD), which may predict major adverse cardiac events. However, little is known regarding the causes of CMD during NAFLD. In this study, we aimed to explore the role of hepatic small extracellular vesicles (sEVs) in regulating the endothelial dysfunction of coronary microvessels during NAFLD. RESULTS: We established two murine NAFLD models by feeding mice a methionine-choline-deficient (MCD) diet for 4 weeks or a high-fat diet (HFD) for 16 weeks. We found that the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome-dependent endothelial hyperpermeability occurred in coronary microvessels during both MCD diet and HFD-induced NAFLD. The in vivo and in vitro experiments proved that novel-microRNA(miR)-7-abundant hepatic sEVs were responsible for NLRP3 inflammasome-dependent endothelial barrier dysfunction. Mechanistically, novel-miR-7 directly targeted lysosomal associated membrane protein 1 (LAMP1) and promotes lysosomal membrane permeability (LMP), which in turn induced Cathepsin B-dependent NLRP3 inflammasome activation and microvascular endothelial hyperpermeability. Conversely, a specific novel-miR-7 inhibitor markedly improved endothelial barrier integrity. Finally, we proved that steatotic hepatocyte was a significant source of novel-miR-7-contained hepatic sEVs, and steatotic hepatocyte-derived sEVs were able to promote NLRP3 inflammasome-dependent microvascular endothelial hyperpermeability through novel-miR-7. CONCLUSIONS: Hepatic sEVs contribute to endothelial hyperpermeability in coronary microvessels by delivering novel-miR-7 and targeting the LAMP1/Cathepsin B/NLRP3 inflammasome axis during NAFLD. Our study brings new insights into the liver-to-microvessel cross-talk and may provide a new diagnostic biomarker and treatment target for microvascular complications of NAFLD.

Rapid detection and structural characterization of methysticin metabolites generated from rat and human liver microsomes and hepatocytes using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry.

RATIONALE: Methysticin is a naturally occurring ingredient isolated from Piper methysticum Forst. The metabolic profile of methysticin is unknown. The goal of this study was to elucidate the metabolism of methysticin using rat and human liver microsomes and hepatocytes. METHODS: The incubation samples were analyzed using ultra-high-performance liquid chromatography coupled with quadrupole/orbitrap high-resolution mass spectrometry (UHPLC-HRMS). The structures of the metabolites were characterized based on the elemental composition, exact mass, and product ions. RESULTS: A total of 10 metabolites were detected and identified. Among these metabolites, M4 (ring opening of 1,3-benzodioxole) was the predominant metabolite in rat and human liver microsomes. M4 and its glucuronide conjugate (M2) were the major metabolites in rat and human hepatocytes. The metabolic pathways of methysticin are summarized as follows: (a) oxidative ring opening of 1,3-benzodioxole forms the catechol derivative (M4), which subsequently undergoes glucuronidation (M1 and M2), methylation (M8), and sulfation (M7). (b) Demethylation to yield desmethyl methysticin (M6), followed by glucuronidation (M3 and M5). (c) Hydroxylation (M9 and M10). CONCLUSIONS: For the first time, this study provides new information on the in vitro metabolic profiles of methysticin, which facilitates an understanding of the disposition of this bioactive ingredient.

Pyrotinib in vitro metabolite profiling via rat, dog and human hepatocytes using liquid chromatography-quadrupole/orbitrap mass spectrometry.

RATIONALE: Pyrotinib is an irreversible EGFR/HER2 inhibitor that has shown antitumor activity and tolerance in the treatment of breast cancer. Studies focused on its metabolic pathways and major metabolites are insufficient. In the evaluation of drug safety and therapeutic use, metabolite characterization is critical. The metabolism of pyrotinib in vitro was studied utilizing rat, dog and human hepatocytes in this study. METHODS: Pyrotinib (10 µM) was incubated with hepatocytes in Williams' E medium. The metabolites were examined and profiled using ultrahigh-performance liquid chromatography coupled with quadrupole/orbitrap high-resolution mass spectrometry. The metabolite structures were deduced by comparing their precise molecular weights, fragment ions and retention times with those of the parent drug. RESULTS: A total of 16 metabolites, including 6 novel ones, were discovered and structurally described under the present conditions. Oxidation, demethylation, dehydrogenation, O-dealkylation and glutathione (GSH) conjugation were all involved in the metabolism of pyrotinib in hepatocytes. The most predominant metabolic route was identified as GSH conjugation (M5). CONCLUSIONS: This study generated valuable metabolite profiles of pyrotinib in several species, which will aid in the understanding of the drug's disposition in various species and in evaluating the contribution of metabolites to overall effectiveness and toxicity of pyrotinib.

Identification of the metabolites of rhapontigenin in rat and human by ultra-high-performance liquid chromatography-high-resolution mass spectrometry.

RATIONALE: Rhapontigenin, a stilbene compound isolated from the medicinal plant of rhubarb rhizomes, has shown a variety of biological activities. The purpose of this study was to identify and characterize the metabolites of rhapontigenin in rat liver microsomes, hepatocytes, urine, and human liver microsomes and hepatocytes. METHODS: The samples were analyzed by ultra-high-performance liquid chromatography combined with electrospray ionization quadrupole/orbitrap high-resolution mass spectrometry (UPLC-Q/Orbitrap-HRMS). The structures of the metabolites were interpreted by MS, MS/MS data, and elemental compositions. RESULTS: A total of 11 metabolites were detected and tentatively identified. M1, identified as piceatannol, was unambiguously identified using reference standard. Our results suggested that rhapontigenin was metabolized through the following pathways: (a) demethylation to produce piceatannol (M1), which further underwent oxidation to form ortho-quinone intermediate. This intermediate was reactive and conjugated with GSH (M10 and M11), which were further converted into N-acetyl-cysteine and excreted in urine. M1 also underwent sulfation (M8) and glucuronidation (M5); (b) direct sulfation, forming M6 and M7; and (c) direct glucuronidation to form M2, M3, and M4. Glucuronidation was a major metabolic pathway in hepatocytes and urine. CONCLUSIONS: The current study provides an overview of the metabolism of rhapontigenin, which is of great importance for us to understand the disposition of this compound.

Prediction of Drug Clearance from Enzyme and Transporter Kinetics.

Accurate estimation of in vivo clearance in human is pivotal to determine the dose and dosing regimen for drug development. In vitro-in vivo extrapolation (IVIVE) has been performed to predict drug clearance using empirical and physiological scalars. Multiple in vitro systems and mathematical modeling techniques have been employed to estimate in vivo clearance. The models for predicting clearance have significantly improved and have evolved to become more complex by integrating multiple processes such as drug metabolism and transport as well as passive diffusion. This chapter covers the use of conventional as well as recently developed methods to predict metabolic and transporter-mediated clearance along with the advantages and disadvantages of using these methods and the associated experimental considerations. The general approaches to improve IVIVE by use of appropriate scalars, incorporation of extrahepatic metabolism and transport and application of physiologically based pharmacokinetic (PBPK) models with proteomics data are also discussed. The chapter also provides an overview of the advantages of using such dynamic mechanistic models over static models for clearance predictions to improve IVIVE.

Mn(II) Complex of Lipophilic Group-Modified Ethylenediaminetetraacetic Acid (EDTA) as a New Hepatobiliary MRI Contrast Agent.

Liver-specific contrast agents (CAs) can improve the Magnetic resonance imaging (MRI) detection of focal and diffuse liver lesions by increasing the lesion-to-liver contrast. A novel Mn(II) complex, Mn-BnO-TyrEDTA, with a lipophilic group-modified ethylenediaminetetraacetic acid (EDTA) structure as a ligand to regulate its behavior in vivo, is superior to Gd-EOB-DTPA in terms of a liver-specific MRI contrast agent. An MRI study on mice demonstrated that Mn-BnO-TyrEDTA can be rapidly taken up by hepatocytes with a combination of hepatobiliary and renal clearance pathways. Bromosulfophthalein (BSP) inhibition imaging, biodistribution, and cellular uptake studies confirmed that the mechanism of hepatic targeting of Mn-BnO-TyrEDTA is the hepatic uptake of the amphiphilic anion contrast agent mediated by organic anion transporting polypeptides (OATPs) expressed by functional hepatocytes.

Circulating microRNA­135a­3p in serum extracellular vesicles as a potential biological marker of non­alcoholic fatty liver disease.

Non­alcoholic fatty liver disease (NAFLD) is a widespread threat to human health. However, the present screening methods for NAFLD are time­consuming or invasive. The present study aimed to assess the potential of microRNAs (miRNAs/miRs) in serum extracellular vesicles (EVs) as a biomarker of NAFLD. C57BL/6J mice were fed either a 12­week high­fat diet (HFD) or standard chow to establish NAFLD and control groups, respectively. Serum samples were obtained from the mouse model of NAFLD, as well as 50 patients with NAFLD and 50 healthy individuals, and EVs were extracted and verified. Using reverse transcription­quantitative PCR, the mRNA expression level of selected miRNAs in the serum and EVs was analyzed. In order to determine the diagnostic value, receiver operating characteristic (ROC) curves were used. The mice treated with HFD showed notable hepatic steatosis and higher concentrations of serum alanine aminotransferase (ALT). There was also a significant decrease in the expression levels of miR­135a­3p, miR­129b­5p and miR­504­3p, and an increase in miR­122­5p expression levels in circulating EVs in mice treated with HFD and patients with NAFLD. There were also similar miR­135a­3p and miR­122­5p expression patterns in the serum. ROC analysis demonstrated that miR­135a­3p in circulating EVs was highly accurate in diagnosing NAFLD, with the area under the curve value being 0.849 (95% CI, 0.777­0.921; P<0.0001). Bioinformatics analysis indicated that dysregulated miR­135a­3p was associated with 'platelet­derived growth factor receptor signaling pathway' and 'AMP­activated protein kinase signaling pathway'. In summary, circulating miR­135a­3p in EVs may serve as a potential non­invasive biomarker to diagnose NAFLD. This miRNA was a more sensitive and specific biological marker for NAFLD compared with ALT.

3D bioprinting of hepatocytes: core-shell structured co-cultures with fibroblasts for enhanced functionality.

With the aim of understanding and recapitulating cellular interactions of hepatocytes in their physiological microenvironment and to generate an artificial 3D in vitro model, a co-culture system using 3D extrusion bioprinting was developed. A bioink based on alginate and methylcellulose (algMC) was first shown to be suitable for bioprinting of hepatocytes; the addition of Matrigel to algMC enhanced proliferation and morphology of them in monophasic scaffolds. Towards a more complex system that allows studying cellular interactions, we applied core-shell bioprinting to establish tailored 3D co-culture models for hepatocytes. The bioinks were specifically functionalized with natural matrix components (based on human plasma, fibrin or Matrigel) and used to co-print fibroblasts and hepatocytes in a spatially defined, coaxial manner. Fibroblasts acted as supportive cells for co-cultured hepatocytes, stimulating the expression of certain biomarkers of hepatocytes like albumin. Furthermore, matrix functionalization positively influenced both cell types in their respective compartments by enhancing their adhesion, viability, proliferation and function. In conclusion, we established a functional co-culture model with independently tunable compartments for different cell types via core-shell bioprinting. This provides the basis for more complex in vitro models allowing co-cultivation of hepatocytes with other liver-specific cell types to closely resemble the liver microenvironment.

Correction of a urea cycle defect after ex vivo gene editing of human hepatocytes.

Ornithine transcarbamylase deficiency (OTCD) is a monogenic disease of ammonia metabolism in hepatocytes. Severe disease is frequently treated by orthotopic liver transplantation. An attractive approach is the correction of a patient's own cells to regenerate the liver with gene-repaired hepatocytes. This study investigates the efficacy and safety of ex vivo correction of primary human hepatocytes. Hepatocytes isolated from an OTCD patient were genetically corrected ex vivo, through the deletion of a mutant intronic splicing site achieving editing efficiencies >60% and the restoration of the urea cycle in vitro. The corrected hepatocytes were transplanted into the liver of FRGN mice and repopulated to high levels (>80%). Animals transplanted and liver repopulated with genetically edited patient hepatocytes displayed normal ammonia, enhanced clearance of an ammonia challenge and OTC enzyme activity, as well as lower urinary orotic acid when compared to mice repopulated with unedited patient hepatocytes. Gene expression was shown to be similar between mice transplanted with unedited or edited patient hepatocytes. Finally, a genome-wide screening by performing CIRCLE-seq and deep sequencing of >70 potential off-targets revealed no unspecific editing. Overall analysis of disease phenotype, gene expression, and possible off-target editing indicated that the gene editing of a severe genetic liver disease was safe and effective.

Dual-mode imaging of copper transporter 1 in HepG2 cells by hyphenating confocal laser scanning microscopy with laser ablation ICPMS.

Copper transporter 1 (CTR1) is a transport protein involved in copper and cisplatin uptake. The visualization of cellular CTR1 migration and its redistribution is highly important in copper/cisplatin exposure/transport. However, to the best of our knowledge, this is a highly challenging task. Herein, a dual-mode imaging strategy for CTR1 is developed by hyphenating confocal laser scanning microscopy (CLSM) and laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) with a fluorescent/elemental bifunctional tag conjugated with anti-CTR1 antibody. The tag consists of rhodamine B and zirconium metal-organic frameworks (Zr-MOF) for CLSM fluorescence imaging and LA-ICPMS element imaging for a same group of HepG2 cells in a designated visual zone. This dual-mode imaging strategy facilitates visualization of CTR1 migration and meanwhile provides information of CTR1 redistribution in HepG2 cells by uptake of divalent copper or cisplatin. The present dual-mode imaging strategy provides in-depth information for the elucidation of CTR1 involved biological processes. Graphical abstract.

Humanized liver mouse model with transplanted human hepatocytes from patients with ornithine transcarbamylase deficiency.

Ornithine transcarbamylase deficiency (OTCD) is a metabolic and genetic disease caused by dysfunction of the hepatocytic urea cycle. To develop new drugs or therapies for OTCD, it is ideal to use models that are more closely related to human metabolism and pathology. Primary human hepatocytes (HHs) isolated from two patients (a 6-month-old boy and a 5-year-old girl) and a healthy donor were transplanted into host mice (hemi-, hetero-OTCD mice, and control mice, respectively). HHs were isolated from these mice and used for serial transplantation into the next host mouse or for in vitro experiments. Histological, biochemical, and enzyme activity analyses were performed. Cultured HHs were treated with ammonium chloride or therapeutic drugs. Replacement rates exceeded 80% after serial transplantation in both OTCD mice. These highly humanized OTCD mice showed characteristics similar to OTCD patients that included increased blood ammonia levels and urine orotic acid levels enhanced by allopurinol. Hemi-OTCD mice showed defects in OTC expression and significantly low enzymatic activities, while hetero-OTCD mice showed residual OTC expression and activities. A reduction in ammonium metabolism was observed in cultured HHs from OTCD mice, and treatment with the therapeutic drug reduced the ammonia levels in the culture medium. In conclusion, we established in vivo OTC mouse models with hemi- and hetero-patient HHs. HHs isolated from the mice were useful as an in vitro model of OTCD. These OTC models could be a source of valuable patient-derived hepatocytes that would enable large scale and reproducible experiments using the same donor.

Hepatic cell-sheet fabrication of differentiated mesenchymal stem cells using decellularized extracellular matrix and thermoresponsive polymer.

PURPOSE: Liver tissue engineering via cell sheet technology would open new doors for treatment of patients with liver failure. Decellularized tissues could provide sufficient extracellular matrix (ECM) to support development of hepatocytes in in vivo niches. Besides, with the potential of temperature responsive polymer (pNIPAAm) as an intelligent surface for controlling the attachment/detachment of cell, we set out to generate three in vitro microenvironments models including I: pNIPAAm hydrogel (pN hydrogel), II: decellularized ECM incorporated into pNIPAAm hydrogel (dECM + pN hydrogel) and III: decellularized ECM scaffold (dECM scaffold) to investigate the structural and function cues of hepatocyte-like cells after differentiation of adipose tissue-derived mesenchymal stem cells (AT-MSCs) on the surface of these models. METHOD: dECM scaffold was obtained after decellularization of rat liver, and its efficiency was analyzed. pN hydrogel and dECM + pN hydrogel (1:3 and 2:3 ratios) of were fabricated, and scaffold architecture was characterized. Each well of culturing plates was coated separately with these three constructs and AT-MSCs were instructed to differentiate into hepatocyte-like cells (HLCs). After recellularization, patterns of differentiation, and expression of hepatogenic markers were investigated via biochemical assays and qRT-PCR at different time points. RESULTS: Multipotency of AT-MSCs, after their ability for osteogenesis and adipogenesis was documented. Production of dense and intact cell sheets was reported in dECM + pN hydrogel, as opposed to pN hydrogel and dECM scaffold. Also, statistically significant difference of HLCs functionality in dECM + pN hydrogel was confirmed after evaluation of the expression of hepatocyte markers including, alpha-fetoprotein, cytokeratin 18, cytochrome P450-2E1 and phosphoenolpyruvate carboxykinase. CONCLUSION: Our results proved dECM + pN hydrogel were able to preserve hepatocyte function in cell sheets owing to the high level of albumin, urea, hepatogenic markers, and glycogenesis potential of HLCs. Accordingly, dECM incorporated in pN hydrogel could remodel microenvironments to guide the AT-MSCs into conducive differentiation and proliferation to give rise to multilayer sheets of cells in their own ECM.