Hepatocyte Function Research Articles

Determination of the nanoparticle- and cell-specific toxicological mechanisms in 3D liver spheroids using scRNAseq analysis

Engineered nanomaterials (ENMs) are commonly used in consumer products, allowing exposure to target organs such as the lung, liver, and skin that could lead to adverse health effects in humans. To better reflect on toxicological effects in liver cells, it is important to consider the contribution of hepatocyte morphology, function, and intercellular interactions in a dynamic 3D microenvironment. Herein, we used a 3D liver spheroid model containing hepatocyte and Kupffer cells (KCs) to study the effects of three different material compositions, namely vanadium pentoxide (V2O5), titanium dioxide (TiO2), or graphene oxide (GO). Additionally, we used single-cell RNA sequencing (scRNAseq) to determine the nanoparticle (NP) and cell-specific toxicological responses. A general finding was that hepatocytes exhibit more variation in gene expression and adaptation of signaling pathways than KCs. TNF-α production tied to the NF-κB pathway was a commonly affected pathway by all NPs while impacts on the metabolic function of hepatocytes were unique to V2O5. V2O5 NPs also showed the largest number of differentially expressed genes in both cell types, many of which are related to pro-inflammatory and apoptotic response pathways. There was also evidence of mitochondrial ROS generation and caspase-1 activation after GO and V2O5 treatment, in association with cytokine production. All considered, this study provides insight into the impact of nanoparticles on gene responses in key liver cell types, providing us with a scRNAseq platform that can be used for high-content screening of nanomaterial impact on the liver, for use in biosafety and biomedical applications.

Integrated Transcriptomic and Proteomic Analysis Identifies Plasma Biomarkers of Hepatocellular Failure in Alcohol-Associated Hepatitis

Alcohol-associated hepatitis (AH) is a form of liver failure with high short-term mortality. Recent studies have shown that defective function of hepatocyte nuclear factor 4 alpha (HNF4a) and systemic inflammation are major disease drivers of AH. Plasma biomarkers of hepatocyte function could be useful for diagnostic and prognostic purposes. Herein, an integrative analysis of hepatic RNA sequencing and liquid chromatography-tandem mass spectrometry was performed to identify plasma protein signatures for patients with mild and severe AH. Alcohol-related liver disease cirrhosis, nonalcoholic fatty liver disease, and healthy subjects were used as comparator groups. Levels of identified proteins primarily involved in hepatocellular function were decreased in patients with AH, which included hepatokines, clotting factors, complement cascade components, and hepatocyte growth activators. A protein signature of AH disease severity was identified, including thrombin, hepatocyte growth factor α, clusterin, human serum factor H-related protein, and kallistatin, which exhibited large abundance shifts between severe and nonsevere AH. The combination of thrombin and hepatocyte growth factor α discriminated between severe and nonsevere AH with high sensitivity and specificity. These findings were correlated with the liver expression of genes encoding secreted proteins in a similar cohort, finding a highly consistent plasma protein signature reflecting HNF4A and HNF1A functions. This unbiased proteomic-transcriptome analysis identified plasma protein signatures and pathways associated with disease severity, reflecting HNF4A/1A activity useful for diagnostic assessment in AH.

RIPK3 dampens mitochondrial bioenergetics and lipid droplet dynamics in metabolic liver disease.

Receptor-interacting protein kinase 3 (RIPK3) mediates NAFLD progression, but its metabolic function is unclear. Here, we aimed to investigate the role of RIPK3 in modulating mitochondria function, coupled with lipid droplet (LD) architecture in NAFLD. Functional studies evaluating mitochondria and LD biology were performed in wild-type (WT) and Ripk3-/- mice fed a choline-deficient, amino acid-defined (CDAA) diet for 32 and 66 weeks and in CRISPR-Cas9 Ripk3 -null fat-loaded immortalized hepatocytes. The association between hepatic perilipin (PLIN) 1 and 5, RIPK3, and disease severity was also addressed in a cohort of patients with NAFLD and in PLIN1 -associated familial partial lipodystrophy. Ripk3 deficiency rescued impairment in mitochondrial biogenesis, bioenergetics, and function in CDAA diet-fed mice and fat-loaded hepatocytes. Ripk3 deficiency was accompanied by a strong upregulation of antioxidant systems, leading to diminished oxidative stress upon fat loading both in vivo and in vitro. Strikingly, Ripk3-/- hepatocytes displayed smaller size LD in higher numbers than WT cells after incubation with free fatty acids. Ripk3 deficiency upregulated adipocyte and hepatic levels of LD-associated proteins PLIN1 and PLIN5. PLIN1 upregulation controlled LD structure and diminished mitochondrial stress upon free fatty acid overload in Ripk3-/- hepatocytes and was associated with diminished human NAFLD severity. Conversely, a pathogenic PLIN1 frameshift variant was associated with NAFLD and fibrosis, as well as with increased hepatic RIPK3 levels in familial partial lipodystrophy. Ripk3 deficiency restores mitochondria bioenergetics and impacts LD dynamics. RIPK3 inhibition is promising in ameliorating NAFLD.

Association of non-alcoholic fatty liver disease with cardiovascular disease according to autopsy data

Rpose of the study. To study the relationship between fatal cardiovascular events and non-alcoholic fatty liver disease (NAFLD) among the population of the Irkutsk region. Materials and methods. The study was conducted on the basis of the Irkutsk Regional Bureau of Forensic Medicine. The object of the study was the medical documentation - “The autopsy report”. Results. To study the possible relationship, a retrospective analysis of the results of 2220 autopsies performed over 3 years was carried out: 2010-2012. in the pathological department. Signs of non-alcoholic fatty liver disease according to morphological studies were identified in 271 cases. Conclusions. 1.In patients with NAFLD, body weight can be equally normal and increased. Among patients with increased body weight, women predominate. 2. In men, there is a moderate positive correlation between the thickness of the subcutaneous fat and the mass of the liver; in women, this connection was not found. 3. Morphologically, coarse fatty degeneration of the liver is more common. 4. Every second patient with NAFLD (49%) died of CVD. Among the dead, men reliably predominate. The dominant cause of death is cardiovascular disease (AMI), followed by impaired cerebral circulation. Mortality peaks in middle and old age. 5. The total mortality from CVD for 3 years according to the pathology department of the Irkutsk Regional Clinical Hospital was 423 cases (19%) (n = 2220). Among all deaths from CVD, one in three (133 cases (31.4%)) had fatty changes in the liver. Conclusion: the results obtained confirm the hypothesis of the existence of a relationship between NAFLD and CVD, which is based on disorders of fat metabolism that develop as a result of impaired normal function of hepatocytes against the background of their fatty lesions. In accordance with this statement, it is necessary to think about considering NAFLD as a component of metabolic syndrome.

Cryopreservation of Whole Rat Livers by Vitrification and Nanowarming.

Liver cryopreservation has the potential to enable indefinite organ banking. This study investigated vitrification-the ice-free cryopreservation of livers in a glass-like state-as a promising alternative to conventional cryopreservation, which uniformly fails due to damage from ice formation or cracking. Our unique "nanowarming" technology, which involves perfusing biospecimens with cryoprotective agents (CPAs) and silica-coated iron oxide nanoparticles (sIONPs) and then, after vitrification, exciting the nanoparticles via radiofrequency waves, enables rewarming of vitrified specimens fast enough to avoid ice formation and uniformly enough to prevent cracking from thermal stresses, thereby addressing the two main failures of conventional cryopreservation. This study demonstrates the ability to load rat livers with both CPA and sIONPs by vascular perfusion, cool them rapidly to an ice-free vitrified state, and rapidly and homogenously rewarm them. While there was some elevation of liver enzymes (Alanine Aminotransferase) and impaired indocyanine green (ICG) excretion, the nanowarmed livers were viable, maintained normal tissue architecture, had preserved vascular endothelium, and demonstrated hepatocyte and organ-level function, including production of bile and hepatocyte uptake of ICG during normothermic reperfusion. These findings suggest that cryopreservation of whole livers via vitrification and nanowarming has the potential to achieve organ banking for transplant and other biomedical applications.

Modelling fatty liver disease with mouse liver-derived multicellular spheroids

Chronic liver disease can lead to liver fibrosis and ultimately cirrhosis, which is a significant health burden and a major cause of death worldwide. Reliable in vitro models are lacking and thus mono-cultures of cell lines are still used to study liver disease and evaluate candidate anti-fibrotic drugs. We established functional multicellular liver spheroid (MCLS) cultures using primary mouse hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells and Kupffer cells. Cell-aggregation and spheroid formation was enhanced with 96-well U-bottom plates generating over ±700 spheroids from one mouse. Extensive characterization showed that MCLS cultures contain functional hepatocytes, quiescent stellate cells, fenestrated sinusoidal endothelium and responsive Kupffer cells that can be maintained for 17 days. MCLS cultures display a fibrotic response upon chronic exposure to acetaminophen, and present steatosis and fibrosis when challenged with free fatty acid and lipopolysaccharides, reminiscent of non-alcoholic fatty liver disease (NAFLD) stages. Treatment of MCLS cultures with potential anti-NAFLD drugs such as Elafibranor, Lanifibranor, Pioglitazone and Obeticholic acid shows that all can inhibit steatosis, but only Elafibranor and especially Lanifibranor inhibit fibrosis. Therefore, primary mouse MCLS cultures can be used to model acute and chronic liver disease and are suitable for the assessment of anti-NAFLD drugs.

Physiologically relevant microsystems to study viral infection in the human liver.

Viral hepatitis is a leading cause of liver disease and mortality. Infection can occur acutely or chronically, but the mechanisms that govern the clearance of virus or lack thereof are poorly understood and merit further investigation. Though cures for viral hepatitis have been developed, they are expensive, not readily accessible in vulnerable populations and some patients may remain at an increased risk of developing hepatocellular carcinoma (HCC) even after viral clearance. To sustain infection in vitro, hepatocytes must be fully mature and remain in a differentiated state. However, primary hepatocytes rapidly dedifferentiate in conventional 2D in vitro platforms. Physiologically relevant or physiomimetic microsystems, are increasingly popular alternatives to traditional two-dimensional (2D) monocultures for in vitro studies. Physiomimetic systems reconstruct and incorporate elements of the native cellular microenvironment to improve biologic functionality in vitro. Multiple elements contribute to these models including ancillary tissue architecture, cell co-cultures, matrix proteins, chemical gradients and mechanical forces that contribute to increased viability, longevity and physiologic function for the tissue of interest. These microsystems are used in a wide variety of applications to study biological phenomena. Here, we explore the use of physiomimetic microsystems as tools for studying viral hepatitis infection in the liver and how the design of these platforms is tailored for enhanced investigation of the viral lifecycle when compared to conventional 2D cell culture models. Although liver-based physiomimetic microsystems are typically applied in the context of drug studies, the platforms developed for drug discovery purposes offer a solid foundation to support studies on viral hepatitis. Physiomimetic platforms may help prolong hepatocyte functionality in order to sustain chronic viral hepatitis infection in vitro for studying virus-host interactions for prolonged periods.

Essential phospholipids decrease apoptosis and increase membrane transport in human hepatocyte cell lines

BackgroundEssential phospholipids (EPL) have hepatoprotective effects across many liver diseases/conditions. The impact of EPL on hepatocyte function in vitro was investigated.MethodsEffects of noncytotoxic concentrations of EPL (0.1 and 0.25 mg/ml), and its constituents, polyenylphosphatidylcholine (PPC) and phosphatidylinositol (PI) (both at 0.1 and 1 mg/ml), on membrane fluidity, apoptosis and extracellular transport versus controls were investigated in human hepatocyte cell lines (HepG2, HepaRG, steatotic HepaRG). ResultsSignificantly increased membrane fluidity occurred with all 3 phospholipids (PLs) in HepG2 cultures, and with PI (1 mg/ml) in steatotic HepaRG cells. Significantly decreased tamoxifen-induced apoptosis was observed in HepG2 cells with EPL, PPC and PI. Breast cancer resistance protein (BCRP) activity was significantly increased by EPL and PI in HepG2 cells. Multidrug resistance-associated protein 2 (MRP-2) activity was unaffected by any PL in HepG2 cells, and significantly increased by EPL, PI and PPC (1 mg/ml) in HepaRG cells, and by PI (1 mg/ml) in steatotic HepaRG cells. Bile salt export protein (BSEP) activity in HepG2 cells and steatotic HepaRG cells was significantly increased by EPL (0.25 mg/ml), and PPC (both concentrations), but not by PI. The PLs had no effects on HepaRG cell BSEP activity. P-glycoprotein (P-GP) activity was significantly increased by all compounds in HepG2 cells. PI (1 mg/ml) significantly increased P-GP activity in HepaRG and steatotic HepaRG cells.ConclusionsEPL, PPC, and PI increased hepatocyte membrane fluidity, decreased apoptosis and increased hepatocellular export, all of which may improve liver function. These in-vitro investigations provide valuable insights into the mechanism of action of EPL.

Can next-generation humanized mice that reconstituted with both functional human immune system and hepatocytes model the progression of viral hepatitis to hepatocarcinogenesis?

Hepatitis B virus (HBV) and Hepatitis C virus (HCV) chronic infections cause liver immunopathological diseases such as hepatitis, fibrosis, cirrhosis, and hepatocellular carcinomas, which are difficult to treat and continue to be major health problems globally. Due to the species-specific hepato-tropism of HBV and HCV, conventional rodent models are limited in their utility for studying the infection and associated liver immunopathogenesis. Humanized mice reconstituted with both functional human immune system and hepatocytes (HIS-HuHEP mice) have been extremely instrumental for in vivo studies of HBV or HCV infection and human-specific aspects of the progression of liver immunopathogenesis. However, none of the current HIS-HuHEP mice can model the progression of viral hepatitis to hepatocarcinogenesis which may be a notorious result of HBV or HCV chronic infection in patients, suggesting that they were functionally compromised and that there is still significant space to improve and establish next-generation of HIS-HuHEP mice with more sophisticated functions. In this review, we first summarize the principal requirements to establish HIS-HuHEP mice. We then discuss the respective protocols for current HIS-HuHEP mice and their applications, as well as their advantages and disadvantages. We also raise perspectives for further improving and establishing next-generation HIS-HuHEP mice.

Serum neuregulin 4 is negatively correlated with insulin sensitivity in humans and impairs mitochondrial respiration in HepG2 cells.

Neuregulin 4 (NRG4) has been described to improve metabolic disturbances linked to obesity status in rodent models. The findings in humans are controversial. We aimed to investigate circulating NRG4 in association with insulin action in humans and the possible mechanisms involved. Insulin sensitivity (euglycemic hyperinsulinemic clamp) and serum NRG4 concentration (ELISA) were analysed in subjects with a wide range of adiposity (n = 89). In vitro experiments with human HepG2 cell line were also performed. Serum NRG4 was negatively correlated with insulin sensitivity (r = −0.25, p = 0.02) and positively with the inflammatory marker high-sensitivity C reative protein (hsCRP). In fact, multivariant linear regression analyses showed that insulin sensitivity contributed to BMI-, age-, sex-, and hsCRP-adjusted 7.2% of the variance in serum NRG4 (p = 0.01). No significant associations were found with adiposity measures (BMI, waist circumference or fat mass), plasma lipids (HDL-, LDL-cholesterol, or fasting triglycerides) or markers of liver injury. Cultured hepatocyte HepG2 treated with human recombinant NRG4 had an impact on hepatocyte metabolism, leading to decreased gluconeogenic- and mitochondrial biogenesis-related gene expression, and reduced mitochondrial respiration, without effects on expression of lipid metabolism-related genes. Similar but more pronounced effects were found after neuregulin 1 administration. In conclusion, sustained higher serum levels of neuregulin-4, observed in insulin resistant patients may have deleterious effects on metabolic and mitochondrial function in hepatocytes. However, findings from in vitro experiments should be confirmed in human primary hepatocytes.

MiR-22 Deficiency Fosters Hepatocellular Carcinoma Development in Fatty Liver.

MiR-22 is mostly considered as a hepatic tumor-suppressor microRNA based on in vitro analyses. Yet, whether miR-22 exerts a tumor-suppressive function in the liver has not been investigated in vivo. Herein, in silico analyses of miR-22 expression were performed in hepatocellular carcinomas from human patient cohorts and different mouse models. Diethylnitrosamine-induced hepatocellular carcinomas were then investigated in lean and diet-induced obese miR-22-deficient mice. The proteome of liver tissues from miR-22-deficient mice prior to hepatocellular carcinoma development was further analyzed to uncover miR-22 regulated factors that impact hepatocarcinogenesis with miR-22 deficiency. MiR-22 downregulation was consistently observed in hepatocellular carcinomas from all human cohorts and mouse models investigated. The time of appearance of the first tumors was decreased and the number of tumoral foci induced by diethylnitrosamine was significantly increased by miR-22-deficiency in vivo, two features which were further drastically exacerbated with diet-induced obesity. At the molecular level, we provide evidence that the loss of miR-22 significantly affects the energetic metabolism and mitochondrial functions of hepatocytes, and the expression of tumor-promoting factors such as thrombospondin-1. Our study demonstrates that miR-22 acts as a hepatic tumor suppressor in vivo by restraining pro-carcinogenic metabolic deregulations through pleiotropic mechanisms and the overexpression of relevant oncogenes.

Influence of biologically active substances on synthesis function and cellular destruction of hepatocytes in vitro

The aim of the study was to reveal the effect of biologically active substances on the synthesis activity and cellular destruction of hepatocytes in vitro. Liver sections were prepared for investigation and placed in culture vials with DMEM nutrient medium with 15% calf serum, glucose, and antibiotics (streptomycin and penicillin). Liver sections were incubated for 14 days with interleukin-2 (roncoleukin) at a concentration of 5000 IU/ml and 7500 IU/ml, and erythropoietin (epobiocrine, “Biopharma”, USA) at a concentration of 13 IU/ml (high concentration), 6.5 IU/ml (medium concentration) and 1.3 IU/ml (low concentration) and without stimulation (control cultures). Synthesis activity and cellular destruction of hepatocytes were studied by determining the protein content, alanine aminotransferase and aspartate aminotransferase activity in the supernatant of liver organ cultures on the 7th and 14th days of incubation. It was found that culturing organotypic cultures with IL-2 did not affect the synthesis function of hepatocytes, but reduced aspartate aminotransferase activity throughout the culture period. At a concentration of 7500 IU/ml IL-2 showed a weak hepatotoxic effect. It was found that erythropoietin at a medium concentration had a hepatoprotective effect, at a high concentration it suppressed the synthesis activity of hepatocytes and contributed to the destruction of the cytoplasmic membrane of cells. At low concentrations, erythropoietin increased the synthesis activity of liver cells but caused an increase in the activity of aminotransferases, this may indicate both mass cell death and intensification of amino acid transamination processes. It was established that interleukin and its inhibitor cause biological effects when incubated with organotypic cultures.

Comparative Transcriptome Analysis Provides Novel Molecular Events for the Differentiation and Maturation of Hepatocytes during the Liver Development of Zebrafish.

The liver plays an essential role in multiple biological functions including metabolism, detoxification, digestion, coagulation, and homeostasis in vertebrates. The specification and differentiation of embryonic hepatoblasts, the proliferation of hepatocytes, and the hepatic tissue architecture are well documented, but molecular events governing the maturation of hepatocytes during liver development remain largely unclear. In this study, we performed a comparative transcriptome analysis of hepatocytes that were sorted by flow cytometry from developing zebrafish embryos at 60, 72, and 96 hpf. We identified 667 up-regulated and 3640 down-regulated genes in hepatocytes between 60 and 72 hpf, 606 up-regulated and 3924 down-regulated genes between 60 and 96 hpf, and 1693 up-regulated genes and 1508 down-regulated genes between 72 and 96 hpf. GO enrichment analysis revealed that key biological processes, cellular components, and molecular functions in hepatocytes between 60 to 72 hpf, such as cell cycle, DNA replication, DNA repair, RNA processing, and transcription regulation, are mainly associated with the proliferation of hepatocytes. In addition to biological processes, cellular components, and molecular functions for cell proliferation, molecular functions for carbohydrate metabolism were enriched in hepatocytes during 72 to 96 hpf. KEGG enrichment analysis identified key signaling pathways, such as cell cycle, RNA degradation, ubiquitin-mediated proteolysis, ErbB and Hedgehog signaling, basal transcription factors, Wnt signaling, and glycan degradation, which are closely associated with cell proliferation or carbohydrate metabolism in hepatocytes between 60 to 72 hpf. Newly enriched signaling pathways in hepatocytes during 72 to 96 hpf include metabolisms of pyrimidine, purine, nicotinate and nicotinamide, caffeine, glycine, serine and threonine, ABC transporters, and p53 signaling that function in metabolisms of lipid, protein and energy, cellular secretion, or detoxification, indicating the functional maturation of hepatocytes between 72 to 96 hpf. These findings provide novel clues for further understanding the functional differentiation and maturation of hepatocytes during liver development.

Mir-122 upregulation and let-7f downregulation combination: The effects on hepatic differentiation of hiPSCs on the PCL-Gel-HA nanofibrous scaffold.

Cell therapy and tissue engineering as promising candidates for the liver transplantation dilemma are of special interest. Induced pluripotent stem cells (iPSCs) are one of the best sources in this field, but their differentiation methods to hepatocytes have remained challenging. We transduced human iPSCs (hiPSCs) with miR‐122 and off‐let‐7f (hiPSCsmiR‐122 + off‐let‐7f) to evaluate how they can differentiate hiPSCs to hepatocyte‐like cells (HLCs) without any extrinsic growth factor. Additionally, we studied the effect of Poly ɛ‐caprolactone‐gelatin‐hyaluronic acid (PCL‐Gel‐HA) nanofibrous scaffold as an extracellular matrix (ECM) simulator on differentiation improvement. Definitive endoderm markers (FOXA2 and SOX17), as well as hepatic markers (AFP, Albumin, CK18, HNF4α) expression, were significantly higher in hiPSCsmiR‐122 + off‐let‐7f derived HLCs (hiPSCs‐HLCs) compared to the control group (miR‐scramble transduced hiPSCs: hiPSCsscramble). hiPSCs‐HLCs indicated hepatocyte morphological characteristics and positive immunostaining for AFP, Albumin and HNF4α. Albumin and urea secretion were significantly higher in hiPSCs‐HLCs than hiPSCsscramble. Comparing these markers in the PCL‐Gel‐HA group with the tissue culture plate (TCP) group revealed that PCL‐Gel‐HA could improve differentiation towards HLCs significantly. Regarding our results, these microRNAs can be used to differentiate hiPSCs to the functional hepatocytes for disease modelling, drug screening and cell‐based therapy in future studies.

Human duodenal submucosal glands contain a defined stem/progenitor subpopulation with liver-specific regenerative potential

Common precursors for the liver, biliary tree, and pancreas exist at an early stage of development in the definitive endoderm forming the foregut. We have identified and characterised endodermal stem/progenitor cells with regenerative potential persisting in the adult human duodenum. Human duodena were obtained from organ donors, and duodenal submucosal gland cells were isolated after removal of the mucosa layer. Cells were cultured on plastic or as organoids and were transplanted into severe combined immunodeficient (SCID) mouse livers. In situ studies of submucosal glands in the human duodenum revealed cells expressing stem/progenitor cell markers that had unique phenotypic traits distinguishable from intestinal crypt cells. Genetic signature studies indicated that the cells are closer to biliary tree stem cells and to definitive endodermal cells than to adult hepatocytes, supporting the interpretation that they are endodermal stem/progenitor cells. Invitro, human duodenal submucosal gland cells demonstrated clonal growth, capability to form organoids, and ability to acquire functional hepatocyte traits. Invivo, transplanted cells engrafted into the livers of immunocompromised mice and differentiated to mature liver cells. In an experimental model of fatty liver, human duodenal submucosal gland cells were able to rescue hosts from liver damage by supporting repopulation and regeneration of the liver. A cell population with clonal growth and organoid formation capability, which has liver differentiation potency invitro and invivo in murine experimental models, is present within adult duodenal submucosal glands. These cells can be isolated, do not require reprogramming, and thus could potentially represent a novel cell source for regenerative medicine of the liver. Cell therapies for liver disease could represent an option to support liver function, but the identification of sustainable and viable cell sources is critical. Here, we describe a cell population with organoid formation capability and liver-specific regenerative potential in submucosal glands of the human duodenum. Duodenal submucosal gland cells are isolated from adult organs, do not require reprogramming, and could rescue hepatocellular damage in preclinical models of chronic, but not acute, liver injury. Duodenal submucosal gland cells could represent a potential candidate cell source for regenerative medicine of the liver, but the determination of cell dose and toxicity is needed before clinical testing in humans.

Ebola virus infection induces a delayed type I IFN response in bystander cells and the shutdown of key liver genes in human iPSC-derived hepatocytes.

SummaryLiver damage and an exacerbated inflammatory response are hallmarks of Ebola virus (EBOV) infection. Little is known about the intrinsic response to infection in human hepatocytes and their contribution to inflammation. Here, we present an induced pluripotent stem cell (iPSC)-derived hepatocyte-like cell (HLC) platform to define the hepato-intrinsic response to EBOV infection. We used this platform to show robust EBOV infection, with characteristic ultrastructural changes and evidence for viral replication. Transcriptomics analysis revealed a delayed response with minimal early transcriptomic changes, followed by a general downregulation of hepatic function and upregulation of interferon signaling, providing a potential mechanism by which hepatocytes participate in disease severity and liver damage. Using RNA-fluorescence in situ hybridization (FISH), we showed that IFNB1 and CXCL10 were mainly expressed in non-infected bystander cells. We did not observe an inflammatory signature during infection. In conclusion, iPSC-HLCs are an immune competent platform to study responses to EBOV infection.

Space microgravity improves proliferation of human iPSC-derived cardiomyocytes.

SummaryIn microgravity, cells undergo profound changes in their properties. However, how human cardiac progenitors respond to space microgravity is unknown. In this study, we evaluated the effect of space microgravity on differentiation of human induced pluripotent stem cell (hiPSC)-derived cardiac progenitors compared with 1G cultures on the International Space Station (ISS). Cryopreserved 3D cardiac progenitors were cultured for 3 weeks on the ISS. Compared with 1G cultures, the microgravity cultures had 3-fold larger sphere sizes, 20-fold higher counts of nuclei, and increased expression of proliferation markers. Highly enriched cardiomyocytes generated in space microgravity showed improved Ca2+ handling and increased expression of contraction-associated genes. Short-term exposure (3 days) of cardiac progenitors to space microgravity upregulated genes involved in cell proliferation, survival, cardiac differentiation, and contraction, consistent with improved microgravity cultures at the late stage. These results indicate that space microgravity increased proliferation of hiPSC-cardiomyocytes, which had appropriate structure and function.

Prognostic Capabilities of Diagnosing Non-Alcoholic Hepatic Steatosis at Early Stages of Its Development.

Patients with excessive body mass (n=26) and obesity (n=28) having different concomitant pathologies of the digestive organs have undergone a comprehensive examination including assessment of biochemical blood indices, ultrasound examination of the abdominal organs, dynamic hepatobiliscintigraphy, assessment of the comorbidity level as a whole (calculation of CIRS) and by the available pathology of the digestive organs. The ultrasound signs of hepatic steatosis have been found in all patients with obesity stage II and III, in 69.2% of the examined patients (95% CI: 42.37-87.32) with obesity stage I, in 30.8% (95% CI: 16.50-49.99) with excessive body mass, and in 13.9% of patients (95% CI: 6.08-28.66) with normal body mass. According to the ROC analysis, the predictors of hepatic steatosis development in patients with excessive body mass and obesity are BMI (>31, р<0.0001) and the number of digestive organ illnesses (more than 4 diseases, p<0.0001). On the basis of the data obtained, a logistic model has been developed in the form of the regression equation permitting us to predict the groups of patients with a low or high degree of risk of hepatic steatosis. The analysis of dynamic hepatobiliscintigraphy has revealed deceleration of the absorbing function of hepatocytes in patients with normal ultrasound images of the liver even in case of excessive body mass. The obtained results made it possible to develop a method of diagnosing fatty hepatosis with subsequent calculation of the functional hepatocyte activity index (FHAI) which helps not only establish functional disorders but identify the group of patients having the risk of developing these disorders. Normal ultrasound imaging of the liver and absence of the biochemical changes in the blood are not indicators of preservation of its functional activity, since the risk of functional disorders was found in 32.3% of cases (95% CI: 18.57-49.86) and reversible disorders in 19.3% (95% CI: 9.19-36.28) when FHAI was calculated for patients with normal body mass. With the increase of body mass, irreversible functional disorders of hepatocytes are observed in 80-100% of patients.Thus, the results obtained in the course of the investigation have confirmed the possibility of using new prognostic markers of hepatic steatosis for early diagnosis of non-alcoholic fatty liver disease.

2D and 3D liver models

2D and 3D liver models

Karaciğer hastalıklarında kullanılan skorlamalar malign-benign kolestaz ayrımında kullanılabilir mi?

Giriş ve Amaç: Kronik safra yolu hastalıkları hepatositlerde bilirübin ve karaciğerden ekskrete edilen toksinlere daha uzun süre maruziyete neden olarak hepatosit fonksiyonlarını bozmaktadır. Ayrıca karaciğerin primer maligniteleri gibi safra yolu malignitelerinin de fibrotik karakterde olduğunu öne süren yayınlar vardır. Bu bilgiler ışığında kronik ve malign safra yolu tıkanıklığı oluşturan kolestatik hastalıkların, akut ve benign safra yolu tıkanıklığı oluşturan hastalıklara göre karaciğer fonksiyonlarını daha fazla bozduğu ve karaciğer fibrozisini artırdığı düşünülebilir. Çalışmamızda rutin testlerle ölçülebilen ve karaciğer hastalıklarında kullanılan skorlardan; R-faktör, son dönem karaciğer hastalığı modeli/sodyum, albümin-bilirübin skoru, albümin-alkalen fosfataz oranı, aspartat aminotransferaz-trombosit oran indeksi, dört faktörü temel alan fibrozis indeksi (fibrozis-4) ve gama glutamil transpeptidaz-trombosit oranı’ndan hangilerinin malign nedenli kolestaz tanısını koymakta yardımcı bir yöntem olarak kullanılabileceğinin araştırılması hedeflenmiştir. Gereç ve Yöntem: Ondokuz Mayıs Üniversitesi Tıp Fakültesi Gastroenteroloji Kliniği’nde Temmuz 2019- Temmuz 2021 tarihleri arasında obstrüktif kolestaz tanısı alan hastaların dosyaları retrospektif olarak tarandı. Akut ve benign nedenli kolestaz tanısı alan hastalar Grup 1, kronik ve malign nedenli kolestaz tanılı hastalar Grup 2 olarak sınıflandırıldı. Hastaların ilk başvuru zamanındaki laboratuvar değerleri kullanılarak; R-faktör, son dönem karaciğer hastalığı modeli/sodyum, albümin-bilirübin skoru, albümin-alkalen fosfataz oranı, aspartat aminotransferaz-trombosit oran indeksi, fibrozis-4 indeksi ve gama glutamil transpeptidaz-trombosit oranı skorları hesaplandı, tanıları ile tanı anındaki skorları arasındaki ilişki değerlendirildi. Bulgular: Çalışmaya 116’sı erkek (%57.4), 86’sı kadın (%42.6) toplam 202 hasta dahil edildi. Grup 1’de 92 (%45.5), Grup 2’de 110 (%54.5) hasta yer almaktaydı. Grup 1 ve Grup 2 arasında demografik özellikler açısından anlamlı fark görülmedi. Grup 1’de sık görülen hastalıklar kolanjit, koledokolitiazis ve safra çamuru ile biliyer pankreatit iken Grup 2’de ise pankreas başı karsinomuydu. Değerlendirilen laboratuvar parametrelerinden alkalen fosfataz, total ve direkt bilirübin maligniteye işaret eden en önemli testlerdi (p &amp;lt; 0.001). Karaciğer hastalığı skorlarından R-faktör (p &amp;lt; 0.001), son dönem karaciğer hastalığı modeli/sodyum (p &amp;lt; 0.001) ve albümin-bilirübin skoru (p = 0.023) Grup 2, albümin-alkalen fosfataz oranı (p &amp;lt; 0.001), aspartat aminotransferaz-trombosit oran indeksi (p &amp;lt; 0.001) ve fibrozis-4 indeksi (p &amp;lt; 0.001) Grup 1 lehine anlamlıydı ancak gama glutamil transpeptidaz-trombosit oranı için her iki grup arasında anlamlı fark yoktu (p = 0.242). Tanı konma süreçleri içinde Grup 1’deki hastaların nihai tanıları çoğunlukla ultrasonografi ile konmakta iken (p &amp;lt; 0.001), Grup 2’deki hastalar bilgisayarlı tomografi ile tanı almaktaydı (p &amp;lt; 0.001). Sonuç: Çalışmamız kronik karaciğer hastalıklarında kullanılan skorlardan R-faktör, son dönem karaciğer hastalığı modeli/sodyum ve albümin-bilirübin skoru malign nedenli kolestatik hastalıkların tanısında, albümin-alkalen fosfataz oranı, aspartat aminotransferaz-trombosit oran indeksi ve fibrozis-4 indeksinin benign nedenli kolestatik hastalıkların tanısında yardımcı bir yöntem olarak kullanılabileceğini, gama glutamil transpeptidaz-trombosit oranının ise bu ayırımda kullanılamayacağını göstermektedir. Bu tip öngörücü skorlar, anamnez ve muayene bulguları ile birlikte yorumlanarak hastaların tetkik ve tedavi planlarının daha hızlı sağlanmasını kolaylaştırabilir.