Isolated hepatocytes in a bioartificial liver: A single group view and experience

Despite recent advances in medical supportive therapy, patients with severe fulminant hepatic failure (FHF) have mortality rate approaching 90%. Investigators have attempted to improve survival by using various extracorporeal liver support systems loaded with sorbents and liver tissue preparations. None of them succeeded in gaining clinical acceptance and orthotopic liver transplantation (OLT) remains a primary therapeutic option for patients with FHF. In this study, authors discuss the systems which utilize isolated hepatocytes. Most of these devices were tested in vitro and in animals with chemically and surgically induced liver failure. In some studies, signficant levels of detoxification and liver functions were achieved. The authors describe their own hepatocyte-based artificial liver (BAL). It is based on plasma perfusion through a hollow-fiber module seeded with matrix-anchored porcine hepatocytes. The BAL was used 14 times to treat 9 patients with acute liver failure. On 10 occasions, a charcoal column was included in the plasma circuit. Each treatment lasted 7 +/- 1 h. All procedures were tolerated well and 8 patients (including 6 patients with FHF) underwent OLT. Five patients with increased intracranial pressure (ICP) and evidence of decerebration had normalization of ICP and enjoyed full neurologic recovery after OLT. Laboratory data showed evidence for bilirubin conjugation, decrease in blood ammonia, maintenance of low lactic acid levels, and increase in the ration between the branched chain and aromatic amino acids. No allergic reactions to xenogeneic hepatocytes were observed. The authors conclude that BAL treatment with porcine hepatocytes appears to be safe and can help maintain patients alive and neurologically intact until a liver becomes available for transplantation. (c) 1994 John Wiley & Sons, Inc.     

Artificial and Bioartificial Liver Support

The liver is a complex organ with various vital functions in synthesis, detoxification and regulation; its failure therefore constitutes a life threatening condition1. Liver failure (LF) can either occur without preceding liver disease (acute liver failure, ALF), usually caused either by intoxication (Amanita phalloides, acetaminophen, methylendioxymethamphteamine) or as acute decompensation of chronic liver-related illness (acute-on-chronic liver failure, AoCLF). In both cases, its symptoms include icterus, hepatic encephalopathy and impairment of coagulation status and may result in multi organ failure. Exceptionally, liver failure may also be triggered by certain diseases (Budd-Chiari-syndrome, Morbus Wilson) or pregnancy. The only long-term therapy in most cases is orthotopic liver transplantation, unless the liver is able to regenerate. Many patients, especially those who are not listed for high urgency transplantation, may not survive until a suitable donor organ is available, since donor organs are rare. In other cases, contraindications do not permit liver transplantation. For these indications, extracorporeal liver assist devices have been developed in order to either bridge the patient to transplantation or temporarily support the failing organ until it is able to regenerate.     

Liver tissue engineering: Recent advances in the development of a bio-artificial liver

Orthotopic liver transplantation is the most common treatment for patients with end-stage liver failure. However, liver transplantation is greatly limited by a donor shortage. Liver tissue engineering may offer a promising strategy to solve this problem by providing transplantable, bioartificial livers. Diverse types of cells, biomaterials, and growth factor delivery systems have been tested for efficient regeneration of liver tissues that possess hepatic functions comparable to native livers. This article reviews recent advances in liver tissue engineering and describes cell sources, biomaterial scaffolds, and growth factor delivery systems that are currently being used to improve the regenerative potential of tissue-engineered livers.     

The role of cytokines in liver failure and regeneration: potential new molecular therapies

The liver is a unique organ, and first in line, the hepatocytes encounter the potential to proliferate during cell mass loss. This phenomenon is tightly controlled and resembles in some way the embryonal co-inhabitant cell lineage of the liver, the embryonic hematopoietic system. Interestingly, both the liver and hematopoietic cell proliferation and growth are controlled by various growth factors and cytokines. IL-6 and its signaling cascade inside the cells through STAT3 are both significantly important for liver regeneration as well as for hematopoietic cell proliferation. The process of liver regeneration is very complex and is dependent on the etiology and extent of liver damage and the genetic background. In this review we will initially describe the clinical relevant condition, portraying a number of available animal models with an emphasis on the relevance of each one to the human condition of fulminant hepatic failure (FHF). The discussion will then be focused on the role of cytokines in liver failure and regeneration, and suggest potential new therapeutic modalities for FHF. The recent findings on the role of IL-6 in liver regeneration and the activity of the designer IL-6/sIL-6R fusion protein, hyper-IL-6, in particular, suggest that this molecule could significantly enhance liver regeneration in humans, and as such could be a useful treatment for FHF in patients.     

Amniotic-fluid-derived mesenchymal stem cells overexpressing interleukin-1 receptor antagonist improve fulminant hepatic failure

Uncontrolled hepatic immunoactivation is regarded as the primary pathological mechanism of fulminant hepatic failure (FHF). The major acute-phase mediators associated with FHF, including IL-1β, IL-6, and TNF-α, impair the regeneration of liver cells and stem cell grafts. Amniotic-fluid-derived mesenchymal stem cells (AF-MSCs) have the capacity, under specific conditions, to differentiate into hepatocytes. Interleukin-1-receptor antagonist (IL-1Ra) plays an anti-inflammatory and anti-apoptotic role in acute and chronic inflammation, and has been used in many experimental and clinical applications. In the present study, we implanted IL-1Ra-expressing AF-MSCs into injured liver via the portal vein, using D-galactosamine-induced FHF in a rat model. IL-1Ra expression, hepatic injury, liver regeneration, cytokines (IL-1β, IL-6), and animal survival were assessed after cell transplantation. Our results showed that AF-MSCs over-expressing IL-1Ra prevented liver failure and reduced mortality in rats with FHF. These animals also exhibited improved liver function and increased survival rates after injection with these cells. Using green fluorescent protein as a marker, we demonstrated that the engrafted cells and their progeny were incorporated into injured livers and produced albumin. This study suggests that AF-MSCs genetically modified to over-express IL-1Ra can be implanted into the injured liver to provide a novel therapeutic approach to the treatment of FHF.     

Reduced hepatic injury in Toll-like receptor 4-deficient mice following D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure

Liver transplantation is the only therapy of proven benefit in fulminant hepatic failure (FHF). Lipopolysaccharide (LPS), D-galactosamine (GalN)-induced FHF is a well established model of liver injury in mice. Toll-Like Receptor 4 (TLR4) has been identified as a receptor for LPS. The aim of this study was to investigate the role of TLR4 in FHF induced by D-GalN/LPS administration in mice. Wild type (WT) and TLR4 deficient (TLR4ko) mice were studied in vivo in a fulminant model induced by GalN/LPS. Hepatic TLR4 expression, serum liver enzymes, hepatic and serum TNF-α and interleukin-1β levels were determined. Apoptotic cells were identified by immunohistochemistry for caspase-3. Nuclear factor-kappaβ (NF-κ β) and phosphorylated c-Jun hepatic expression were studied using Western blot analysis. All WT mice died within 24 hours after administration of GalN/LPS while all TLR4ko mice survived. Serum liver enzymes, interleukin-1β, TNF-α level, TLR4 mRNA expression, hepatic injury and hepatocyte apoptosis all significantly decreased in TLR4ko mice compared with WT mice. A significant decrease in hepatic c-Jun and IκB signaling pathway was noted in TLR4ko mice compared with WT mice. In conclusion, following induction of FHF, the inflammatory response and the liver injury in TLR4ko mice was significantly attenuated through decreased hepatic c-Jun and NF-κB expression and thus decreased TNF-α level. Down-regulation of TLR4 expression plays a pivotal role in GalN/LPS induced FHF. These findings might have important implications for the use of the anti TLR4 protein signaling as a potential target for therapeutic intervention in FHF.     

Current understanding of mesenchymal stem cells in liver diseases

Liver diseases caused by various factors have become a significant threat to public health worldwide. Liver transplantation has been considered as the only effective treatment for end-stage liver diseases; however, it is limited by the shortage of donor organs, postoperative complications, long-term immunosuppression, and high cost of treatment. Thus, it is not available for all patients. Recently, mesenchymal stem cells (MSCs) transplantation has been extensively explored for repairing hepatic injury in various liver diseases. MSCs are multipotent adult progenitor cells originated from the embryonic mesoderm, and can be found in mesenchymal tissues including the bone marrow, umbilical cord blood, adipose tissue, liver, lung, and others. Although the precise mechanisms of MSC transplantation remain mysterious, MSCs have been demonstrated to be able to prevent the progression of liver injury and improve liver function. MSCs can self-renew by dividing, migrating to injury sites and differentiating into multiple cell types including hepatocytes. Additionally, MSCs have immune-modulatory properties and release paracrine soluble factors. Indeed, the safety and effectiveness of MSC therapy for liver diseases have been demonstrated in animals. However, pre-clinical and clinical trials are largely required to confirm its safety and efficacy before large scale clinical application. In this review, we will explore the molecular mechanisms underlying therapeutic effects of MSCs on liver diseases. We also summarize clinical advances in MSC-based therapies.     

Cell-based therapy of acute liver failure: The extracorporeal bioartificial liver

The need for an alternative ttreatment to orthotopic liver transplantation for acute liver failure is a major issue, and systems capable of temporalily providing liver functions are being actively tested. Liver assist devices based on detoxication by dialysis or hemoperfusion through various membranes or cartridges proved to be inefficient because of their lack of metabolic function. An extracorporeal hybrid bioartificial liver might be an appropriate treatment, since it can provide liver-specific functions, maintain the patient alive, and allow spontaneous recovery of the patient's own liver or act as a bridge toward liver transplantation. Many devices have been proposed, including flat culture substrates, hollow-fiber bioreactors, or microcarriers, using xenogenic hepatocytes or hepatoma cell lines. Various drawbacks of these devices led us to attempt to develop a reliable extracorporeal bioartificial liver based on alginate bead-entrapped hepatocytes. This system was used successfully for the correction of the Gunn rat genetic defect, which results in lack of bilirubin conjugation. The development of this system for clinical purposes requires large yields of functional hepatocytes. We have isolated normal porcine hepatocytes by collagenase perfusion of the liver. Cells were immobilized in membrane-coated alginate gel beads, which were subsequently inoculated into a bioreactor. Porcine hepatocytes expreessed liver-specific functions at high levels, particularly protein neosysnthesis and enzymatic activities involved in detoxication and biotransformation processes. In addition, hepatocytes entrapped in coated alginate beads were isolated from immunoglobulins. This system represents a promising tool for the design of anoartificial liver in human beings.Abbreviations ALF acute liver failure - EBAL extracorporeal bioartificial liver - OLT orthotopic liver transplantation     

Liver tissue engineering: promises and prospects of new technology

Today, many patients suffer from acute liver failure and hepatoma. This is an area of high unmet clinical need as these conditions are associated with very high mortality. There is an urgent need to develop techniques that will enable liver tissue engineering or generate a bioartificial liver, which will maintain or improve liver function or offer the possibility of liver replacement. Liver tissue engineering is an innovative way of constructing an implantable liver and has the potential to alleviate the shortage of organ donors for orthotopic liver transplantation. In this review we describe, from an engineering perspective, progress in the field of liver tissue engineering, including three main aspects involving cell sources, scaffolds and vascularization.     

Ethics of the heart: ethical and policy challenges in the treatment of advanced heart failure

Heart failure is a major cause of morbidity and mortality in the United States and worldwide, accounting for immense health-care costs. Advanced therapies such as transplantation, ventricular assist devices, and implantable cardioverter defibrillators have had great success in significantly improving life expectancy and morbidity, however these advances have contributed substantially to the economic burden associated with this epidemic. Concomitantly, the accessibility of these advanced therapies is limited, due to a finite number of available organs for heart transplantation and, in the future, the economic costs associated with both transplant and device therapy. This article discusses ethical and policy challenges in the treatment of advanced heart failure, including decisions regarding procurement of hearts for transplant and allocation to recipients; and the complex issues surrounding the use of implantable cardioverter defibrillators and ventricular assist devices, including quality of life, advanced directive planning in the context of these devices, and resource utilization. Based on these challenges, we recommend that a discussion of these complex matters be incorporated into cardiovascular training programs.     

Wilson disease: Current status and the future

The focus of this minireview is on the current status and new advances in diagnosis and treatment of Wilson disease, an autosomal recessive disorder of copper metabolism. Molecular diagnostics have improved and complements current biochemical and clinical methods for screening for Wilson disease. Screening for Wilson disease in newborns is feasible and has been tested in limited populations, but is not yet widely performed. Identification of patients with Wilson disease as the cause of acute liver failure is possible using standard biochemical tests. Treatments for Wilson disease include chelating agents and zinc salts and liver transplantation. Future therapies may include hepatocyte transplantation and gene therapy, both of which have been tested and shown to work in animal models of Wilson disease. Future human studies await advances in these areas.     

Scoring Systems for Predicting Mortality after Liver Transplantation

Background

Liver transplantation can prolong survival in patients with end-stage liver disease. We have proposed that the Sequential Organ Failure Assessment (SOFA) score calculated on post-transplant day 7 has a great discriminative power for predicting 1-year mortality after liver transplantation. The Chronic Liver Failure - Sequential Organ Failure Assessment (CLIF-SOFA) score, a modified SOFA score, is a newly developed scoring system exclusively for patients with end-stage liver disease. This study was designed to compare the CLIF-SOFA score with other main scoring systems in outcome prediction for liver transplant patients.

Methods

We retrospectively reviewed medical records of 323 patients who had received liver transplants in a tertiary care university hospital from October 2002 to December 2010. Demographic parameters and clinical characteristic variables were recorded on the first day of admission before transplantation and on post-transplantation days 1, 3, 7, and 14.

Results

The overall 1-year survival rate was 78.3% (253/323). Liver diseases were mostly attributed to hepatitis B virus infection (34%). The CLIF-SOFA score had better discriminatory power than the Child-Pugh points, Model for End-Stage Liver Disease (MELD) score, RIFLE (risk of renal dysfunction, injury to the kidney, failure of the kidney, loss of kidney function, and end-stage kidney disease) criteria, and SOFA score. The AUROC curves were highest for CLIF-SOFA score on post-liver transplant day 7 for predicting 1-year mortality. The cumulative survival rates differed significantly for patients with a CLIF-SOFA score ≤8 and those with a CLIF-SOFA score >8 on post-liver transplant day 7.

Conclusion

The CLIF-SOFA score can increase the prediction accuracy of prognosis after transplantation. Moreover, the CLIF-SOFA score on post-transplantation day 7 had the best discriminative power for predicting 1-year mortality after liver transplantation.     

Third-generation continuous flow left ventricular assist devices

Tremendous advances have been made in the treatment of end-stage heart failure patients with left ventricular assist devices (LVADs). An important factor playing a role in the improved clinical outcomes is the development of continuous flow, rotary LVADs. New technology using magnetic levitation and hydrodynamic suspension to eliminate contact bearings offers the potential of more durable and efficacious mechanical circulatory blood pumps. Clinical trials evaluating these novel "third-generation" LVADs are in progress.     

The therapeutic potential of bone marrow-derived mesenchymal stem cells on hepatic cirrhosis

Hepatic cirrhosis is the end-stage of chronic liver diseases. The majority of patients with hepatic cirrhosis die from life-threatening complications occurring at their earlier ages. Liver transplantation has been the most effective treatment for these patients. Since liver transplantation is critically limited by the shortage of available donor livers, searching for an effective alternative therapy has attracted great interest in preclinical studies. The transplantation of autologous bone marrow-derived mesenchymal stem cells holds great potential for treating hepatic cirrhosis. Mesenchymal stem cells can differentiate to hepatocytes, stimulate the regeneration of endogenous parenchymal cells, and enhance fibrous matrix degradation. Experimental and clinical studies have shown promising beneficial effects. This review is intended to translate the bench study results to the patients' bedside. The potential interventions of mesenchymal stem cells on cirrhosis are illustrated in terms of the cellular and molecular mechanisms of hepatic fibrogenesis.     

Liver diseases in the dish: iPSC and organoids as a new approach to modeling liver diseases

Liver diseases negatively impact the quality of life and survival of patients, and often require liver transplantation in cases that progress to organ failure. Understanding the cellular and molecular mechanisms of liver development and pathogenesis has been a challenging task, in part for the lack of adequate cellular models directly relevant to the human diseases.Recent technological advances in the stem cell field have shown the potentiality of induced pluripotent stem cells (iPSC) and liver organoids as the next generation tool to model in vitro liver diseases. Hepatocyte-like cells and cholangiocyte are currently being generated from skin fibroblasts and mononuclear blood cells reprogrammed into iPSC and have been successfully used for disease modeling, drug testing and gene editing, with the hope to be able to find application also in regenerative medicine. Protocols to generate other liver cell types are still under development, but the field is advancing rapidly. On the other end, liver cells can now be isolated from liver specimens (liver explants or liver biopsies) and cultured in specific conditions to form polarized 3D organoids. The purpose of this review is to summarize all these recent technological advances and their potential applications but also to analyze the current issues to be addressed before the technology can reach its full potential.     

Hepatic progenitor cells

Liver diseases are associated with a marked reduction in the viable mass of hepatocytes. The most severe cases of liver disease (liver failure) are treated by orthotopic liver transplantation. One alternative to whole organ transplantation for patients with hepatic failure (and hereditary liver disease) is hepatocyte transplantation. However, there is a serious limitation to the treatment of liver diseases either by whole organ or hepatocyte transplantation, and that is the shortage of organ donors. Therefore, to overcome the problem of organ shortage, additional sources of hepatocytes must be found. Alternative sources of cells for transplantation have been proposed including embryonic stem cells, immortalised liver cells and differentiated cells. One other source of cells for transplantation found in the adult liver is the progeny of stem cells. These cells are termed hepatic progenitor cells (HPCs). The therapeutic potential of HPCs lies in their ability to proliferate and differentiate into hepatocytes and cholangiocytes. However, using HPCs as a cell therapy cannot be exploited fully until the mechanisms governing hepatocyte differentiation are elucidated. Here, we discuss the fundamental cellular and molecular elements required for HPC differentiation to hepatocytes.     

Dynamic Patterns of serum metabolites in fulminant hepatic failure pigs

Fulminant hepatic failure (FHF) is still an intractable disease associated with serious metabolic disorder. Investigating the dynamic changes of serum metabolites during the development of FHF would facilitate revealing the pathogenesis and also promote its treatment. Therefore, this study characterized the dynamic metabonome of serum from FHF Pigs using ultra performance liquid chromatography?Cmass spectrometry. Based on multiple statistical analysis of the resulting dataset, three types of up-regulated and one type of down-regulated patterns were delineated. Each pattern demonstrated distinct trends at different stages during the whole process of FHF, implying the differential clinical significance of them. Specifically, aromatic amino acids (Pattern 1) and lysophosphatidylcholines (LPCs) (Pattern 4) might be good markers for evaluating the severity of FHF, while some conjugated bile acids, long chain acylcarnitines (Pattern 2) and Glycocholic acid (Pattern 3) could indicate liver injury in the early stage. Inspired from the PCA plot that the pathogenetic condition of FHF aggravated with sampling time, a linear discriminant analysis (LDA) model based on phenylalanine and LPC 18:1 were further constructed for evaluating the severity of FHF. The leave-one-out cross-validation accuracy of 91.67% for the training set and the prediction accuracy of 92.31% for the external validation set confirmed its excellent performance. In conclusion, findings obtained from the present study, including four types of Dynamic Patterns of serum metabolites during FHF development and an LDA model for evaluating the severity of FHF, will be of great help to the research and management of FHF in the future.     

Mammalian hepatocytes as a foundation for treatment in human liver failure.

Technological advances in the separation and culture of mammalian hepatocytes have facilitated the use of these cells as the foundation for either hepatocyte transplantation or hepatocyte-seeded hollow fiber liver assist devices (LAD). To fully appreciate the practical applications of these tissue engineering solutions, it is necessary to understand the types of human liver failure as well as the corresponding animal models. The most immediate application of this type of technology is the treatment of hepatic encephalopathy (HE), an acute and highly fatal complication of fulminant hepatic failure. Although the pathogenesis of HE is unknown, failure of the detoxification function of the liver is accepted as playing an important role in this disorder. Consequently, the assaying and preservation of P450 activity in the grafted cells or in the LAD must be among the main targets of this research. This review explores the problems in hepatocyte transplantation and culture that deserve special consideration and emphasizes the conditions contributing to the in vitro maintenance of phenotypic expression of these cells.     

Establishing a metabolomic model for the prognosis of hepatitis B virus-induced acute-on-chronic liver failure treated with different liver support systems

Liver failure induced by hepatitis B virus (HBV) is a severe disease with a high mortality rate. Liver support treatment is a powerful method for treating liver failure and is a bridge to liver transplantation. Patients with similar liver function indices, however, have different outcomes following treatment, and no satisfying prediction parameters exist. In this study, we used ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) to investigate plasma metabolites in groups of acute-on-chronic liver failure patients with different prognosis. An orthogonal partial least squares (OPLS) model, with satisfactory explanatory and predictive ability (R ² Y?=?0.943, Q ²?=?0.913) was established using SIMCA-P?+?12.0. The model was based on samples collected just before the first artificial treatment for comparable model efficacy. The concordance statistics of our model was 0.968 (95% CI [0.951, 0.985]) which is superior to that of the MELD (the Model for End-stage Liver Diseases) score (0.737, 95% CI [0.578, 0.896]). Three groups of markers were identified: lysophosphatidylcholine, primary fatty acid amides and conjugated bile acids. Lysophosphatidylcholine and conjugated bile acids were protection factors for survival and primary fatty acid amides were risk factors. The cut-off point for the predictive value of our model was greater than or equal to 0.196, at which the model showed the best discrimination between the recovery and non-recovery groups, with a sensitivity of 95% and a specificity of 87%. This metabolomic model, based on plasma UPLC-MS profiles, provides not only excellent discrimination and prognostic ability for HBV-induced acute-on-chronic liver failure but also early and precise warning of possible liver transplantation.