Macro-encapsulation of mesenchymal stem cells in acute and chronic liver injury animal models.

BACKGROUND AND AIM: Stem cell treatments using scaffolds for liver disease have been well studied. However, macro-encapsulation of mesenchymal stem cells (MSCs) to minimize or inhibit stem cell homing has not been evaluated. Here, we conducted a proof-of-concept study using MSCs macro-encapsulated in poly lactic-co-glycolic acid in liver disease models. METHODS: Poly lactic-co-glycolic acid semipermeable membranes (surface pore size up to 40 µm) were used as the macro-encapsulation system. Macro-encapsulated pouches were loaded with MSCs and sealed. Each pouch was implanted in the subcutaneous region of the dorsum or interlobular space of the liver. Acute liver injury was induced using thioacetamide intraperitoneal injection thrice a week. For the chronic liver fibrosis model, thioacetamide dose was gradually increased, starting from 100 to 400 mg/kg over 16 weeks (thrice a week). RESULTS: In the acute liver injury model, the treated groups showed decreased liver inflammation and necrosis compared with the control. Hepatic fibrosis decreased in the treated group in the chronic liver fibrosis model compared with that in the control group. Encapsulated MSCs exhibited changed cell morphology and characteristics after implantation, showing increased periodic acid-Schiff staining and CYP2E1 expression. Migration and homing of MSCs into the liver was not observed. Under hypoxic conditions, macro-encapsulated MSCs secreted more growth hormones, including vascular endothelial growth factor, platelet-derived growth factor, angiopoietin-2, and placental growth factor, than monolayered MSCs in vitro. CONCLUSIONS: Macro-encapsulated MSCs attenuate hepatic inflammation and fibrosis by upregulating hypoxia-induced growth hormone secretion in liver disease models.

Decrease in fat de novo synthesis and chemokine ligand expression in non-alcoholic fatty liver disease caused by inhibition of mixed lineage kinase domain-like pseudokinase.

BACKGROUND AND AIM: Receptor-interacting serine/threonine kinase 3 and mixed lineage kinase domain-like pseudokinase (MLKL) have gained attention as apoptosis alternate cell death signaling molecules. We aimed to evaluate the role of MLKL in non-alcoholic fatty liver disease (NAFLD). METHODS: Hepatic tissue MLKL expression was compared between NAFLD patients and healthy controls. High-fat diet was fed to wild-type and MLKL-knockout (KO) mice for 12 weeks. Brown adipose fat tissue was measured by [18 F]-fluorodeoxyglucose positron emission tomography. Energy expenditure was measured by indirect calorimetry. Anti-MLKL effects were also evaluated in in vitro setting using U937 and HepG2 cells. RESULTS: Hepatic tissue MLKL expression increased in NAFLD patients compared with healthy controls. MLKL expression increased according to the degree of steatosis, ballooning, and inflammation. High-fat diet-fed MLKL-KO mice displayed decreased alanine aminotransferase, triglycerides, liver weight, NAFLD activity score (6.3 vs 3.5, P < 0.001), steatosis score (3.0 vs 1.8, P < 0.001), inflammation, and ballooning degeneration compared with wild-type mice. SREBP1c, fatty acid synthase, and SCD-1 expressions decreased in MLKL-KO mice. Adipose tissue F4/80-positive crown-like structures were also reduced in MLKL-KO mice. HepG2 cells treated with necrosulfonamide (an MLKL inhibitor) showed reduced Nile red staining and reduced SREBP1c and SCD-1 expressions. Stimulation of necroptosis using lipopolysaccharide + caspase inhibitor (zVAD) increased CXCL1/2 expressions in U937 monocyte cells. Lipopolysaccharide + zVAD-induced increased expressions of CXCL1/2 were reduced with necrosulfonamide treatment. CONCLUSIONS: Mixed lineage kinase domain-like pseudokinase inhibition has protective effects in non-alcoholic steatohepatitis by decreasing hepatic de novo fat synthesis and chemokine (C-X-C motif) ligand expressions.

Effect of Stem Cell Treatment on Acute Liver Failure Model Using Scaffold.

BACKGROUND: Injecting MSCs via blood vessel is most commonly used method, which has a major drawback of safety. The aim of our study was to evaluate efficacy using scaffold-loaded MSCs in acute liver failure model. METHOD: Acute liver failure was induced in mice using thioacetamide (TAA) (200 mg/kg, i.p) once a day for two consecutive days. The animals were divided in four acute liver failure groups: (1) TAA; (2) empty scaffold; (3) MSCs injected through tail vein; (4) MSC + Scaffold, scaffold loaded with MSCs, to evaluate the mortality and changes in liver function. Polylactic-co-glycolic acid scaffold alone and loaded with human MSCs was implanted on mice dorsum. RESULTS: TAA dose was titrated until one-third mortality rate was achieved. TAA (200 mg/kg) once daily for two consecutive days was injected to establish the acute liver failure model. The mortality of TAA and scaffold groups was 55.9% and 63.2%, respectively. Although, mortality of MSC-TV group decreased 14.7% as compared to TAA group (p = 0.200), MSC + Scaffold group had the lowest mortality (31.4%) (p = 0.013). Cells implanted in PLGA biomaterial were survived until 3 weeks, and their function was increased. Area of hepatic inflammation and necrosis was significantly reduced in MSC-TV and MSC + Scaffold groups; but there was no difference between the two groups. Gene expressions related to inflammation were significantly decreased in MSC-TV and MSC + Scaffold groups compared to TAA group. In MSC + Scaffold group, no migration of stem cells to liver tissue was observed. Although, not all cells in scaffold were stained, some of them were differentiated into hepatocyte-like cells which stained positive for PAS and CYP2E1 antibody. CONCLUSION: Scaffold loaded with MSCs showed protective effects via paracrine signaling on acute liver failure model.

Feasibility and Stability of Liver Biopsy before Treatment for Preclinical Nonalcoholic Fatty Liver Studies.

BACKGROUND: The heterogeneity of histological findings in preclinical diet-induced nonalcoholic fatty liver disease (NAFLD) animal models is highly challenging. Here, we aimed to evaluate the feasibility and stability of repeated liver biopsy in NAFLD animal models. METHODS: Heterogeneity of diet-induced NAFLD was evaluated at different time points in 52 high-fat diet (HFD), 35 methionine choline-deficiency diet (MCD), and 166 western diet (WD) induced NAFLD mice. Serial liver biopsies (left lateral, right medial, and left medial lobes) were performed monthly for up to 3 months. Mortality rates and changes in food intake, body weight, and liver enzymes were assessed. RESULTS: At 12 weeks, of the HFD animals, 14% and 30% did not develop steatosis and lobular inflammation, respectively; of the MCD animals, 7% did not develop lobular inflammation; and of the WD animals, 14% and 51% did not develop steatosis and lobular inflammation, respectively. The mortality rate of repeated liver biopsy was 1.62% (2/123 mice died). Repeated liver biopsy can be used to trace disease progression. Although body weight, food intake, and liver enzymes slightly changed after biopsy, all recovered within a week. Repeated liver biopsy did not affect the degrees of inflammation and steatosis of the other liver lobes. CONCLUSION: The diet-induced NAFLD models were quite heterogeneous. Our results suggest that the repeated liver biopsy before treatment was applicable and stable in this NAFLD animal study.

Auranofin attenuates hepatic steatosis and fibrosis in nonalcoholic fatty liver disease via NRF2 and NF- κB signaling pathways.

BACKGROUND/AIMS: We aim to evaluate the effects of auranofin, a known antioxidant, on hepatic steatosis, inflammation, and fibrosis, contributing to non-alcoholic steatohepatitis (NASH) development in vivo and in vitro. METHODS: Transcriptome analysis of LX-2 cells was that expression patterns of genes changed by auranofin, and their related pathways were estimated. We used the gene set enrichment analysis (GSEA) program to determine the pathway involved in overall genetic change. In vitro, LX-2 and HepG2 cells were treated with transforming growth factor (TGF)-ß1 and palmitic acid (PA), respectively, and the antifibrotic and antiadipogenic effect function of auranofin was evaluated. RESULTS: Transcriptome analysis revealed that auranofin decreased the expression of 15 genes, including thrombospondin 1, endothelin 1 (ET-1), fibronectin 1, and LOX. The molecular functions of these genes are involved in collagen binding. GSEA of the overall gene expression pattern revealed that many genes increased in the reactive oxygen species pathway and decreased in the inflammatory response. Auranofin decreased nuclear factor kappa B (NF-κB) and IκBα in TGF-ß1-induced LX-2 cells, thereby reducing ET-1 and fibrosis. Furthermore, increased pNRF2 in PA-induced HepG2 cells led to increased antioxidant marker expression and decreased lipid accumulation. In the bile duct ligation model mice, auranofin reduced the fibrosis area and increased the survival rate. Auranofin reduced liver fibrosis and lipid accumulation in NASH model mice fed on a Western diet. CONCLUSION: Auranofin inhibits lipogenesis and fibrosis formation and is a proposed candidate for NASH treatment.

Optimal Hypoxic Preconditioning of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells (hES-MSCs) and Their Characteristics.

BACKGROUND AND OBJECTIVES: Hypoxia is frequently used to enhance stem cell function. However, the optimal level of hypoxia for growth and function of human embryonic stem cell-derived mesenchymal stem cells (hES-MSCs) is yet to be determined. The purpose of this study was to find the optimal level of hypoxia for hES-MSCs and characteristics of hES-MSCs cultured under these optimal hypoxic conditions. METHODS AND RESULTS: Cell viability and changes in the morphology of hES-MSCs were determined through cell proliferation and CCK-8 assay. The hES-MSCs were preconditioned under various hypoxic conditions (0.5∼5% O2 and 24∼72 h). The expression of cytokines in each culture condition was compared using cytokine array analysis. The morphology of hES-MSCs did not change under various hypoxic culture conditions. hES-MSCs viability after 48 h incubation in 2% O2 condition was higher than that in normoxic condition. HIF1α expression was increased up to six folds after 48 h of hypoxic preconditioning. HIF1α expression in hES-MSCs peaked after 48 h of incubation in 1% O2 condition. The expressions of PDGF-BB, IGFBP-6, VEGF-A, and angiogenin were increased after hES-MSCs were incubated for 48 h in 2% O2 condition. CONCLUSIONS: The hES-MSCs viability and expressions of PDGF-BB, IGFBP-6, VEGF-A, and angiogenin increased after 48 h incubation in 2% O2 condition.

The Use of Foxa2-Overexpressing Adipose Tissue-Derived Stem Cells in a Scaffold System Attenuates Acute Liver Injury.

Background/Aims: For the clinical application of stem cell therapy, functional enhancement is needed to increase the survival rate and the engraftment rate. The purpose of this study was to investigate functional enhancement of the paracrine effect using stem cells and hepatocyte-like cells and to minimize stem cell homing by using a scaffold system in a liver disease model. Methods: A microporator was used to overexpress Foxa2 in adipose tissue-derived stem cells (ADSCs), which were cultured in a poly(lactic-co-glycolic acid) (PLGA) scaffold. Later, the ADSCs were cultured in hepatic differentiation medium for 2 weeks by a 3-step method. For in vivo experiments, Foxa2-overexpressing ADSCs were loaded in the scaffold, cultured in hepatic differentiation medium and later were implanted in the dorsa of nude mice subjected to acute liver injury (thioacetamide intraperitoneal injection). Results: Foxa2-overexpressing ADSCs showed greater increases in hepatocyte-specific gene markers (alpha fetoprotein [AFP], cytokeratin 18 [CK18], and albumin), cytoplasmic glycogen storage, and cytochrome P450 expression than cells that underwent the conventional differentiation method. In vivo experiments using the nude mouse model showed that 2 weeks after scaffold implantation, the mRNA expression of AFP, CK18, dipeptidyl peptidase 4 (CD26), and connexin 32 (CX32) was higher in the Foxa2-overexpressing ADSCs group than in the ADSCs group. The Foxa2-overexpressing ADSCs scaffold treatment group showed attenuated liver injury without stem cell homing in the thioacetamide-induced acute liver injury model. Conclusions: Foxa2-overexpressing ADSCs applied in a scaffold system enhanced hepatocyte-like differentiation and attenuated acute liver damage in an acute liver injury model without homing effects.

Mismatched effects of receptor interacting protein kinase-3 on hepatic steatosis and inflammation in non-alcoholic fatty liver disease.

AIM: To validate the effects of receptor interacting protein kinase-3 (RIP3) deletion in non-alcoholic fatty liver disease (NAFLD) and to clarify the mechanism of action. METHODS: Wild-type (WT) and RIP3 knockout (KO) mice were fed normal chow and high fat (HF) diets for 12 wk. The body weight was assessed once weekly. After 12 wk, the liver and serum samples were extracted. The liver tissue expression levels of RIP3, microsomal triglyceride transfer protein, protein disulfide isomerase, apolipoprotein-B, X-box binding protein-1, sterol regulatory element-binding protein-1c, fatty acid synthase, cluster of differentiation-36, diglyceride acyltransferase, peroxisome proliferator-activated receptor alpha, tumor necrosis factor-alpha (TNF-α), and interleukin-6 were assessed. Oleic acid treated primary hepatocytes from WT and RIP3KO mice were stained with Nile red. The expression of inflammatory cytokines, including chemokine (C-X-C motif) ligand (CXCL) 1, CXCL2, and TNF-α, in monocytes was evaluated. RESULTS: RIP3KO HF diet fed mice showed a significant gain in body weight, and liver weight, liver to body weight ratio, and liver triglycerides were increased in HF diet fed RIP3KO mice compared to HF diet fed WT mice. RIP3KO primary hepatocytes also had increased intracellular fat droplets compared to WT primary hepatocytes after oleic acid treatment. RIP3 overexpression decreased hepatic fat content. Quantitative real-time polymerase chain reaction analysis showed that the expression of very-low-density lipoproteins secretion markers (microsomal triglyceride transfer protein, protein disulfide isomerase, and apolipoprotein-B) was significantly suppressed in RIP3KO mice. The overall NAFLD Activity Score was the same between WT and RIP3KO mice; however, RIP3KO mice had increased fatty change and decreased lobular inflammation compared to WT mice. Inflammatory signals (CXCL1/2, TNF-α, and interleukin-6) increased after lipopolysaccharide and pan-caspase inhibitor (necroptotic condition) treatment in monocytes. Neutrophil chemokines (CXCL1, and CXCL2) were decreased, and TNF-α was increased after RIP3 inhibitor treatment in monocytes. CONCLUSION: RIP3 deletion exacerbates steatosis, and partially inhibits inflammation in the HF diet induced NAFLD model.

Does necroptosis have a crucial role in hepatic ischemia-reperfusion injury?

BACKGROUND: Previous studies have demonstrated protective effects of anti-receptor interacting protein kinase 1 (RIP1), a key necroptosis molecule. However, it is uncertain whether necroptosis has a crucial role in hepatic IR injury. Therefore, we evaluated the role of necroptosis in hepatic IR injury. METHOD: The IR mice underwent 70% segmental IR injury induced by the clamping of the hepatic artery and portal vein for 1 hr followed by reperfusion for 4 hr. The key necroptosis molecules (RIP1, RIP3, and MLKL) and other key molecules of regulated necrosis (PGAM5 and caspase-1) were evaluated in the warm IR injury model. A RIP1 inhibitor (necrostain-1s) and/or an anti-mitochondrial permeability transition (MPT)-mediated necrosis mediator (cyclosporine A, CyA) were administered before clamping. Necrotic injury was quantified using Suzuki's scoring system. qRT-PCR and western blot were performed to evaluate RIP1, RIP3, MLKL and PGAM5 expressions. RESULTS: RIP1, RIP3, MLKL and PGAM5 expression did not change in the hepatic IR injury model. Moreover, Nec1s pretreatment did not improve histology or biochemical markers. The overall Suzuki score (cytoplasmic vacuolization, sinusoidal congestion and hepatocytes necrosis) was increased in the RIP3(-/-) mice compared to the IR group (3.5 vs. 5, p = 0.026). CyA pretreatment and/or RIP3(-/-) mice decreased Bax/Bcl2 expression; however, it did lead to an overall change in the levels of AST, ALT and LDH or necrotic injury. The Bax/Bcl2 ratio and the expression of caspase-1 and caspase-3 did not increase in our hepatic IR injury model. CONCLUSION: Key necroptosis molecules did not increase in the necrosis-dominant hepatic IR injury model. Anti-necroptosis and/or cyclosporine-A treatment did not have an overall protective effect on necrosis-dominant hepatic IR injury.

Granulocyte colony stimulating factor treatment in non-alcoholic fatty liver disease: beyond marrow cell mobilization.

Protective effects of granulocyte colony stimulating factor (G-CSF) in acute liver injury via marrow cell mobilization have been reported in several studies. But exact mode of action and optimal protocol of G-CSF has been still doubt in chronic disease. Here we investigated mode of action and optimization of G-CSF as a treatment for non-alcoholic fatty liver disease (NAFLD). Various doses of conventional G-CSF (30 µg/kg once weekly, once daily for 5 days, twice weekly) and long acting G-CSF (30 µg/kg once a month) were evaluated in two kinds of NAFLD animal models to optimize the G-CSF protocol. G-CSF receptor expression highest increased in NAFLD model among various liver diseases compare to control (NAFLD: 14.7 times, alcohol hepatitis: 7.1 times, cirrhosis: 2.4 times, and ischemia reperfusion: 6.8 times). G-CSF treatment reduced intrahepatic fat accumulation, and inflammation in two kinds of NAFLD animal models. G-CSF increased PI3K/Akt expression in hepatocyte as well as decreased apoptotic drive (increased Bcl-2 expression and decreased Bax expression) in animal model. Five day consecutive G-CSF treatment and once a month long acting G-CSF increased marrow derived stem cell marker in peripheral blood. But twice a week conventional G-CSF treatment did not increased CD34+ cell in peripheral blood and liver neither. Not only high dose G-CSF (once daily for 5 days) but also hepatotropic dose G-CSF (twice a week) significantly reduced hepatocyte apoptosis via PI3K and Akt pathway activation without marrow cell mobilization in NAFLD animal model.