Danhua Zhu

Efficacy of coupled low-volume plasma exchange with plasma filtration adsorption in treating pigs with acute liver failure: A randomised study

BACKGROUND & AIMS Extracorporeal blood purification systems for supportive therapy of liver failure are widely used. We developed a novel blood purification system, named Lis artificial liver system (Li-ALS), which couples low-volume plasma exchange (low-volume PE) with plasma filtration adsorption (PFA). This study aims to evaluate the efficacy of our novel system in pigs with acute liver failure (ALF). METHODS Thirty-two pigs were infused with D-galactosamine (1.3g/kg) to induce ALF. All animals were equally and randomly divided into four groups: the ALF control group received intensive care, the PFA group underwent five hour plasma recycling filtration and adsorption purification, the low-volume PE group received one hour low-volume PE, and the Li-ALS group underwent one hour low-volume PE, followed by five hour PFA. Intervention was initiated 36hours after drug administration. The efficacy of each treatment was assessed by survival time and improvement in hematological, biochemical, and immunohistological parameters. RESULTS Pigs in the Li-ALS group survived longer than those in the other groups (p<0.001, ALF control: 60±2h; PFA group: 74±2h; low-volume PE group: 75±2h; and Li-ALS group: 90±3h). Liver enzyme, bilirubin, bile acid and blood ammonia levels were decreased significantly after Li-ALS treatment, and increases in inflammatory cytokines were ameliorated. A higher hepatocyte regeneration index was also observed in the Li-ALS group. CONCLUSION Our novel Li-ALS could expedite liver regeneration and improve survival time; hence, it could be promising for treating ALF.

Establishment and characterization of an immortalized human hepatic stellate cell line for applications in co-culturing with immortalized human hepatocytes.

Background and objective. The liver-specific functions of hepatocytes are improved by co-culturing hepatocytes with primary hepatic stellate cells (HSC). However, primary HSC have a short lifespan in vitro, which is considered a major limitation for their use in various applications. This study aimed to establish immortalized human HSC using the simian virus 40 large T antigen (SV40LT) for applications in co-culturing with hepatocytes and HSC in vitro. Methods. Primary human HSC were transfected with a recombinant retrovirus containing SV40LT. The immortalized human HSC were characterized by analyzing their gene expression and functional characteristics. The liver-specific functions of hepatocytes were evaluated in a co-culture system incorporating immortalized human hepatocytes with HSC-Li cells. Results. The immortalized HSC line, HSC-Li, was obtained after infection with a recombinant retrovirus containing SV40LT. The HSC-Li cells were longitudinally spindle-like and had numerous fat droplets in their cytoplasm as shown using electron microscopy. Hepatocyte growth factor (HGF), VEGF Receptor 1(Flt-1), collagen type Iα1 and Iα2 mRNA expression levels were observed in the HSC-Li cells by RT-PCR. Immunofluorescence staining showed that the HSC-Li cells were positive for α-smooth muscle actin (α-SMA), platelet-derived growth factor receptor-beta (PDGFR-β), vimentin, and SV40LT protein expression. The HSC-Li cells produced both HGF and transforming growth factor-beta1 (TGF-β1) in a time-dependent manner. Real-time PCR showed that albumin, CYP3A5, CYP2E1, and UGT2B7 mRNA expression generally increased in the co-culture system. The enzymatic activity of CYP1A2 under the co-culture conditions also generally increased as compared to the monoculture of immortalized human hepatocytes. Conclusions. We successfully established the immortalized human HSC cell line HSC-Li. It has the specific phenotypic and functional characteristics of primary human HSC, which would be a useful tool to develop anti-fibrotic therapies. Co-culturing with the HSC-Li cells improved the liver-specific functions of hepatocytes, which may be valuable and applicable for bioartificial liver systems.

A New Fluidized Bed Bioreactor Based on Diversion-Type Microcapsule Suspension for Bioartificial Liver Systems

A fluidized bed bioreactor containing encapsulated hepatocytes may be a valuable alternative to a hollow fiber bioreactor for achieving the improved mass transfer and scale-up potential necessary for clinical use. However, a conventional fluidized bed bioreactor (FBB) operating under high perfusion velocity is incapable of providing the desired performance due to the resulting damage to cell-containing microcapsules and large void volume. In this study, we developed a novel diversion-type microcapsule-suspension fluidized bed bioreactor (DMFBB). The void volume in the bioreactor and stability of alginate/chitosan microcapsules were investigated under different flow rates. Cell viability, synthesis and metabolism functions, and expression of metabolizing enzymes at transcriptional levels in an encapsulated hepatocyte line (C3A cells) were determined. The void volume was significantly less in the novel bioreactor than in the conventional FBB. In addition, the microcapsules were less damaged in the DMFBB during the fluidization process as reflected by the results for microcapsule retention rates, swelling, and breakage. Encapsulated C3A cells exhibited greater viability and CYP1A2 and CYP3A4 activity in the DMFBB than in the FBB, although the increases in albumin and urea synthesis were less prominent. The transcription levels of several CYP450-related genes and an albumin-related gene were dramatically greater in cells in the DMFBB than in those in the FBB. Taken together, our results suggest that the DMFBB is a promising alternative for the design of a bioartificial liver system based on a fluidized bed bioreactor with encapsulated hepatocytes for treating patients with acute hepatic failure or other severe liver diseases.

A tryptophan derivative, ITE, enhances liver cell metabolic functions in vitro

Cell encapsulation provides a three-dimensional support by incorporating isolated cells into microcapsules with the goal of simultaneously maintaining cell survival and function, as well as providing active transport for a bioreactor in vitro similarly to that observed in vivo. However, the biotransformation and metabolic functions of the encapsulated cells are not satisfactory for clinical applications. For this purpose, in this study, hepatoma-derived Huh7 cells/C3A cells were treated with 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), an endogenous non-toxic ligand for aryl hydrocarbon receptor, in monolayer cultures and on microspheres. The mRNA and protein levels, as well as the metabolic activities of drug metabolizing enzymes, albumin secretion and urea synthesis were determined. When the Huh7 and C3A cells cultured in a monolayer on two-dimensional surfaces, ITE enhanced the protein levels and the metabolic activities of the major cytochrome P450 (CYP450) enzymes, CYP1A1, CYP1A2, CYP3A4 and CYP1B1, and slightly increased albumin secretion and urea synthesis. Moreover, when cultured on microspheres, ITE also substantially increased the protein levels and metabolic activities of CYP1A1, CYP1A2, CYP3A4 and CYP1B1 in both liver cell lines. On the whole, our findings indicate that ITE enhances the enzymatic activities of major CYP450 enzymes and the metabolic functions of liver cells cultured in monolayer or on microspheres, indicating that it may be utilized to improve the functions of hepatocytes. Thus, it may be used in the future for the treatment of liver diseases.

Transporting carriers for intracellular targeting delivery via non-endocytic uptake pathways

Abstract To develop novel therapies for clinical treatments, it increasingly depends on sophisticated delivery systems that facilitate the drugs entry into targeting cells. Profound understanding of cellular uptake routes for transporting carriers promotes the optimization of performance in drug delivery systems. Although endocytic pathway is the most important part of cellular uptake routes for many delivery systems, it suffers the trouble of enzymatic degradation of transporting carriers trapped in endosomes/lysosomes. Therefore, it is desirable to develop alternative transporting methods for delivery systems via non-endocytic pathways to achieve more effective intracellular delivery. In this review, we summarize the literature exploring transporting carriers that mediate intracellular delivery via non-endocytic pathways to present the current research status in this field. Cell-penetrating peptides, pH (low) insertion peptides, and nanoparticles are categorized to exhibit their ability to directly transport various cargos into cytoplasm via non-endocytic uptake in different cell lines. It is hoped that this review can spur the interesting on development of drug delivery systems via non-endocytic uptake pathway.

Proteomic Signature of Acute Liver Failure: From Discovery and Verification in a Pig Model to Confirmation in Humans

Acute liver failure (ALF) is a fatal condition hallmarked by rapid development. The present study aimed to describe the dynamic alterations of serum proteins associated with ALF development, and to seek for novel biomarkers of ALF. Miniature pigs (n = 38) were employed to establish ALF models by infusing d-galactosamine (GALN, 1.3 g/kg). A total of 1310 serum proteins were compared in pooled serum samples (n = 10) before and 36 h after GALN administration through label-free quantitation (LFQ) based shotgun proteomics. Functional analysis suggested a significant enrichment of ALF-related proteins involved in energy metabolism. Temporal changes of 20 energy metabolism related proteins were investigated in individual pigs (n = 8) via parallel reaction monitoring (PRM) based targeted proteomics. In addition, mitochondrion degeneration and gene expression alteration of aerobic metabolism genes were confirmed in GALN-insulted pig liver. In clinical validation study enrolled 34 ALF patients and 40 healthy controls, fructose-1,6-bisphosphatase 1 (FBP1) showed a prognostic value for short-term survival (30 days) equal to that of the Model of End-stage Liver Disease score (ROC-AUC = 0.778). Survival analysis suggested significantly higher death-related hazard in ALF patients with higher FBP1 levels (>16.89 μg/dL) than in those with lower FBP1 levels (p = 0.002). Additionally, serum retinol binding protein 4 (RBP4) level was found decreased prior to ALT elevation in GALN-insulted pig model. We also confirmed that serum level of RBP4 is significantly lower in ALF patients (p < 0.001) as compared with healthy controls. In summary, this translational study, displayed by multistaged proteomics techniques, unveiled underlying functional changes related to the development of ALF and facilitated the discovery of novel ALF markers.

Adipogenic placenta-derived mesenchymal stem cells are not lineage restricted by withdrawing extrinsic factors: developing a novel visual angle in stem cell biology.

Current evidence implies that differentiated bone marrow mesenchymal stem cells (BMMSCs) can act as progenitor cells and transdifferentiate across lineage boundaries. However, whether this unrestricted lineage has specificities depending on the stem cell type is unknown. Placental-derived mesenchymal stem cells (PDMSCs), an easily accessible and less invasive source, are extremely useful materials in current stem cell therapies. No studies have comprehensively analyzed the transition in morphology, surface antigens, metabolism and multilineage potency of differentiated PDMSCs after their dedifferentiation. In this study, we showed that after withdrawing extrinsic factors, adipogenic PDMSCs reverted to a primitive cell population and retained stem cell characteristics. The mitochondrial network during differentiation and dedifferentiation may serve as a marker of absent or acquired pluripotency in various stem cell models. The new population proliferated faster than unmanipulated PDMSCs and could be differentiated into adipocytes, osteocytes and hepatocytes. The cell adhesion molecules (CAMs) signaling pathway and extracellular matrix (ECM) components modulate cell behavior and enable the cells to proliferate or differentiate during the differentiation, dedifferentiation and redifferentiation processes in our study. These observations indicate that the dedifferentiated PDMSCs are distinguishable from the original PDMSCs and may serve as a novel source in stem cell biology and cell-based therapeutic strategies. Furthermore, whether PDMSCs differentiated into other lineages can be dedifferentiated to a primitive cell population needs to be investigated.

Dynamic changes of plasma metabolites in pigs with GalN-induced acute liver failure using GC–MS and UPLC–MS

Metabolomics facilitates investigation of the mechanisms of disease and screening for biomarkers. Here, a gas chromatography-mass spectrometry (GC-MS) and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS)-based metabolomics approach was employed to identify plasma biomarkers of acute liver failure (ALF) in pigs. Blood was collected from pigs at 12h intervals during ALF. Hepatic injury was quantified by determining liver function and histopathology. Based on a multivariate data matrix and pattern recognition, two upregulated metabolites, namely, amino acids and conjugated bile acids, and two downregulated metabolites, lysophosphatidylcholines (LPCs) and phosphatidylcholines (PCs), were identified. All of these metabolites showed a strong relationship with the extent of liver injury. Amino acids were biomarkers of the severity of liver impairment, conjugated bile acids were predictive of early stage liver damage, and LPCs and PCs were related to the prognosis of liver injury. In conclusion, our results demonstrated the occurrence of marked metabolic disturbances during ALF and that integrated metabolomics analysis facilitates identification of biomarkers of disease.

Efficient generation of functional hepatocyte-like cells from mouse liver progenitor cells via indirect co-culture with immortalized human hepatic stellate cells

BACKGROUND Differentiation of liver progenitor cells (LPCs) to functional hepatocytes holds great potential to develop new strategies for hepatocyte transplantation and the screening of drug-induced cytotoxicity. However, reports on the efficient and convenient hepatic differentiation of LPCs to hepatocytes are few. The present study aims to investigate the possibility of generating functional hepatocytes from LPCs in an indirect co-culture system. METHODS Mouse LPCs were co-cultured in Transwell plates with an immortalized human hepatic stellate cell line (HSC-Li) we previously established. The morphology, expression of hepatic markers, and functions of mouse LPC-derived cells were monitored and compared with those of conventionally cultured LPCs. RESULTS Co-culturing with HSC-Li cells induced differentiation of mouse LPCs into functional hepatocyte-like cells. The differentiated cells were morphologically transformed into hepatocyte-like cells 3 days after co-culture initiation. In addition, the differentiated cells expressed liver-specific genes and possessed hepatic functions, including glycogen storage, low-density lipoprotein uptake, albumin secretion, urea synthesis, and cytochrome P450 1A2 enzymatic activity. CONCLUSIONS Our method, which employs indirect co-culture with HSC-Li cells, can efficiently induce the differentiation of LPCs into functional hepatocytes. This finding suggests that this co-culture system can be a useful method for the efficient generation of functional hepatocytes from LPCs.