Stem cells with decellularized liver scaffolds in liver regeneration and their potential clinical applications

End‐stage hepatic failure is a potentially life‐threatening condition for which orthotopic liver transplantation (OLT) is the only effective treatment. However, a shortage of available donor organs for transplantation each year results in the death of many patients waiting for liver transplantation. Cell‐based therapies and hepatic tissue engineering have been considered as alternatives to liver transplantation. However, primary hepatocyte transplantation has rarely produced therapeutic effects because mature hepatocytes cannot be effectively expanded in vitro, and the availability of hepatocytes is often limited by shortages of donor organs. Decellularization is an attractive technique for scaffold preparation in stem cell‐based liver engineering, as the resulting material can potentially retain the liver architecture, native vessel network and specific extracellular matrix (ECM). Thus, the reconstruction of functional and practical liver tissue using decellularized scaffolds becomes possible. This review focuses on the current understanding of liver tissue engineering, whole‐organ liver decellularization techniques, cell sources for recellularization and potential clinical applications and challenges.     

Therapeutic liver repopulation for metabolic liver diseases: Advances from bench to bedside

Metabolic liver diseases are characterized by inherited defects in hepatic enzymes or other proteins with metabolic functions. Therapeutic liver repopulation (TLR), an approach of massive liver replacement by transplanted normal hepatocytes, could be used to provide the missing metabolic function elegantly. However, partial and transient correction of the underlying metabolic defects due to very few integrated donor cell mass remains the major obstacle for the effective and widespread use of this approach. Little engraftment and proliferation insufficiency lead to the poor outcome. This article reviews the advances in the mechanisms of initial engraftment and selective proliferation and suggests some effective treatment strategies, from pharmacological preconditioning to stem cell transplantation, to optimize liver repopulation with liver cell transplantation. Enhancing cell viability and plating efficiency, increasing sinusoidal spaces, regulation of sinusoidal endothelial cell barrier and controlling inflammatory reaction may promote initial cell engraftment. Liver‐directed irradiation, reversible portal vein embolization and fetal liver stem/progenitor cell transplantation induce preferential proliferation of donor cells substantially without severe side‐effects. Furthermore, it seems better to use combined approaches to achieve a high level of liver repopulation for the management of metabolic liver diseases.     

Liver fluke‐infested graft used for living‐donor liver transplantation: case report and review of the literature

Clonorchiasis is a cholangiopathy caused by foodborne trematode parasites, also known as liver flukes. Clonorchiasis is endemic in a wide geographical area extending from Eastern Europe to Southeast Asia. Infested hosts may remain asymptomatic for decades and consequently their liver can become available as a graft. To date, 20 liver transplantations with liver fluke‐infested grafts have been reported in the literature. All of them occurred in Asian countries. We, here, report the first case to our knowledge in the Western world of living‐donor liver transplantation (LDLT) with an Opisthorchis felineus‐infested graft, and present a review of the literature. A 6‐month‐old girl with decompensated secondary biliary cirrhosis underwent an LDLT with a left lateral graft infested with O. felineus. After prompt diagnosis and adequate therapy, both donor and recipient had an uneventful postoperative course and long‐term follow‐up. Liver grafts infested with liver flukes do not pose a contraindication to liver donation from deceased or living donors, provided that a correct diagnosis and treatment are performed in a timely fashion.     

Establishment of a three-dimensional co-culture system by porcine hepatocytes and bone marrow mesenchymal stem cells in vitro

Aim: The application of porcine hepatocytes in liver support systems has been hampered by the short‐term survival. Co‐cultivation of hepatocytes with non‐parenchymal cells may be beneficial for optimizing cell functions via heterotypic interactions. In this study, we present a new cultivation system of porcine hepatocytes and mesenchymal stem cells (MSCs) in a randomly distributed co‐culture manner. Methods: Mononuclear cells were isolated from bone marrow aspirate of swines (n = 3) by density gradient centrifugation. MSCs were characterized by flow cytometry with CD29, CD44, CD45 and CD90, respectively. Then freshly isolated hepatocytes were simultaneously inoculated with MSCs in a hepatocyte dominant manner. The morphological and functional changes of heterotypic interactions were characterized. Results: Ninety percent MSCs of passage 3 were positive for CD29, CD44 and CD90, but negative for CD45. A rapid attachment and self‐organization of three‐dimensional hepatocyte aggregates were encouraged. The cell ultrastructure indicating heterotypic junctions remained similar to that of hepatocytes in vivo. Fluorescence microscopy further verified that MSCs served as a feeder layer for hepatocyte aggregates. Hepatocyte performance levels such as albumin secretion, urea synthesis and CYP3A1 induction were all significantly enhanced in co‐culture group compared with hepatocyte homo‐culture (P < 0.05). The best hepatic function levels were achieved on day 2 and moderately decreased in the following co‐culture days. Moreover, the cell cycle of hepatocytes manifested the same trend in parallel to the enhancement of hepatocyte functionality. Conclusions: A three‐dimensional co‐culture system by porcine hepatocytes and bone marrow MSCs was for the first time established in vitro. Enhanced liver‐specific functions make such a co‐culture system a promising tool for tissue engineering, cell biology, and bioartificial liver devices.     

Adipose‐derived stem cells: A candidate for liver regeneration

The scarcity of donor livers and the impracticality of hepatocyte transplantation represent the biggest obstacles for the treatment of liver failure. Adipose‐derived stem cells, with their ability to differentiate into the hepatic lineage, provide a reliable alternative cell source with clear ethical and practical advantages. Moreover, adipose‐derived stem cells can effectively repair liver damage by the dominant indirect pattern and increase the number of hepatocytes by the secondary direct pattern. In recent years, the development of the indirect pattern, which mainly includes immunomodulatory and trophic effects, has become a hot topic in the field of cell engineering. Therefore, adipose‐derived stem cells are considered to be ideal therapeutic stem cells for human liver regeneration. In this article, we reviewed the advantages of adipose‐derived stem cells in liver regeneration, and explore their underlying mechanisms.     

Morphogenesis of liver epithelial cells

The mammalian liver is a physiologically important organ performing various types of metabolism, producing serum proteins, detoxifying bilirubin and ammonia, and protecting the body from infection. Those physiological functions are achieved with the 3D tissue architecture of liver epithelial cells. The liver contains two types of epithelial cells, namely, hepatocytes and cholangiocytes. They split from hepatoblasts (embryonic liver stem cells) in mid‐gestation and differentiate into structurally and functionally mature cells. Analyses of mutant mice showing abnormal liver organogenesis have identified genes involved in liver development. In vitro culture systems have been used to examine the mechanism in which each molecule or signaling pathway regulates the morphogenesis and functional differentiation of hepatocytes and cholangiocytes. In addition, liver epithelial cells as well as mesenchymal, sinusoidal endothelial and hematopoietic cells can be purified from developing livers, which enables us to perform genome‐wide screening to identify novel genes regulating epithelial morphogenesis in the liver. By combining these in vivo and in vitro systems, the liver could be a unique and suitable model for revealing a principle, governing epithelial morphogenesis. In this review, we summarize recent progress in the understanding of the development of liver epithelial tissue structures.     

Reconstruction of hepatic organoid by hepatic stem cells

Recent advances in culture methods, stem cell research, and tissue engineering provide clues for making tissues in vitro that are functionally and structurally similar to hepatic tissues. To reconstruct hepatic organoids, two approaches to establish the methods have been proposed: the use of cells and the combination of cells and a scaffold (called tissue engineering). Recently, the coculture of hepatic cells (mature hepatocytes, small hepatocytes, hepatoblasts) and hepatic nonparenchymal cells has been reported to form hepatic organoids that possess differentiated hepatic functions. On the other hand, hepatocytes in a roller bottle were shown to form specific structures, consisting of biliary epithelial cells, connective tissue, mature hepatocytes, and endothelial cells. In this review, the studies of hepatic tissue formation in vitro will be summarized.     

Human hepatocyte transplantation: state of the art

Hepatocyte transplantation is making its transition from bench to bedside for liver‐based metabolic disorders and acute liver failure. Over eighty patients have now been transplanted world wide and the safety of the procedure together with medium‐term success has been established. A major limiting factor in the field is the availability of good quality cells as hepatocytes are derived from grafts that are deemed unsuitable for transplantation. Alternative sources of cell, including stem cells may provide a sustainable equivalent to primary hepatocytes. There is also a need to develop techniques that will improve the engraftment, survival and function of transplanted hepatocytes. Such developments may allow hepatocyte transplantation to become an accepted and practical alternative to liver transplantation in the near future.     

Bioartificial liver support

Orthotopic liver transplantation is the only definitive therapy for patients with fulminant hepatic failure (FHF). However, due to shortage of organs, a large number of patients die before a liver can be procured for transplantation. In FHF the need for a liver is particularly urgent because of rapid deterioration in the patients' condition with the onset of cerebral edema and intracranial hypertension leading to irreversible brain damage. It is thus necessary to develop an extracorporeal liver support system to help maintain patients alive and neurologically intact until an organ becomes available for transplantation. Multiple attempts have been made, ranging from the use of plasma exchange to utilization of charcoal columns and extracorporeal devices loaded with liver tissue to develop liver support systems for treating patients with acute severe liver failure. None of these systems has achieved wide clinical use, and FHF due to multiple causes continues to be associated with significant morbidity and mortality. In this paper, the authors review the history of extracorporeal liver support for acute liver failure and discuss their experience with a hollow fiber bioartificial liver support system utilizing porcine hepatocytes in the treatment of patients with acute liver failure.     

Post‐prophylaxis Toxoplasma chorioretinitis following donor–recipient mismatched liver transplantation

Toxoplasmosis may be transferred by organ transplantation. The most common clinical presentation is with multisystem disease, although isolated ocular toxoplasmosis has been described. Many centers have suggested that universal use of co‐trimoxazole prophylaxis obviates the need for specific Toxoplasma testing. We report a case of donor‐acquired ocular toxoplasmosis after liver transplantation despite co‐trimoxazole prophylaxis. The diagnosis was confirmed by Toxoplasma polymerase chain reaction assay in conjunction with seroconversion. The fact that the infection was donor acquired was confirmed by serological mismatch and the absence of sporozoite‐specific antigen antibody in the recipient.     

Asymptomatic transmission of Treponema pallidum (syphilis) through deceased donor liver transplantation

A 55‐year‐old woman underwent liver transplantation (LT) with a graft from a deceased donor. Mandatory pre‐donation investigations showed positive syphilis serology that was available only after the transplant, with high Treponema pallidum particle agglutination assay titer compatible with donor syphilis infection. Despite the institution of appropriate post‐exposure prophylaxis, the recipient demonstrated latent seroconversion; however, liver graft function improved without evidence of syphilitic hepatitis or other manifestations of the disease. Through this first reported case of asymptomatic transmission of syphilis following LT, we highlight the investigations and treatment strategies for donor‐derived syphilis in liver transplant recipients. This report supplements the existing limited evidence on safe use of infected grafts from syphilitic donors through appropriate post‐exposure prophylaxis.     

Encapsulated xenogeneic hepatocytes remain functional after peritoneal implantation despite immunization of the host

Background/Aims: Xenogeneic hepatocytes encapsulated in semipermeable membranes could be used in the future for the treatment of acute liver failure and congenital liver defects. However, host immune response could affect the viability and function of transplanted cells. The purpose of this study was to investigate the immunological consequences of intraperitoneal implantation of encapsulated xenogeneic hepatocytes and their effects.Methods: Recipient Lewis rats received 2×10⁷ human hepatocytes encapsulated in semipermeable hydrogel-based hollow fibers, 2×10⁷ free human hepatocytes or 2×10⁷ encapsulated Lewis rat hepatocytes. The presence of human albumin in rat sera was assessed by Western blot and the presence of anti-human hepatocytes and anti-human albumin antibodies by ELISA.Results: Anti-hepatocyte antibodies were detected on the 7th day, and their level increased progressively on days 21 and 28 in rats grafted with encapsulated or free human hepatocytes. Anti-albumin antibodies were detected on day 7 and increased progressively in rats grafted with encapsulated human hepatocytes, but were not detected in the other groups. No immune complexes or complement components of donor origin were detected by immunofluorescence in the recipients' tissue. Despite immunization of the host, encapsulated xenogeneic hepatocytes survived and produced albumin, whereas free hepatocytes had been lysed.Conclusion: Transplantation of encapsulated xenogeneic hepatocytes resulted in immunization of the host with production of anti-hepatocyte and anti-albumin antibodies. However, hepatocytes could be efficiently protected by the membrane and remained viable and functional during the study.     

A case of Mycobacterium mucogenicum infection in a liver transplant recipient and a review of the literature

Bacterial, viral, and fungal infections can be devastating in a postoperative liver transplant recipient on multidrug immunosuppressive therapy. Various atypical (nontuberculous mycobacteria [NTM]) mycobacterial infections have been reported in the solid organ transplant population, but to our knowledge, no cases of Mycobacterium mucogenicum infections have been reported. Here, we report a case of a patient with end‐stage liver disease secondary to primary biliary cirrhosis, model for end‐stage liver disease score of 29, who underwent deceased‐donor orthotopic liver transplantation, with her postoperative course complicated by multiple pleural effusions and peritonitis. Despite numerous courses of antibiotics, her condition did not improve. Acid‐fast bacilli cultures grew M. mucogenicum, which was then treated with appropriate antimicrobical therapy. M. mucogenicum, a rapidly growing NTM that can be present in water contamination, should be recognized as a potential source of infection, especially in the immunocompromised population.     

Bioengineering of liver assist devices

Over 30 000 patients die annually in the United States from liver failure. In fulminant hepatic failure, a clinical syndrome associated with high mortality, orthotopic liver transplantation is the primary therapeutic option for patients not responding to supportive therapy. However, the persistent scarcity of donor organs has limited this therapeutic modality, resulting in a continued increase in the number of patients who die waiting for a donor liver. An extracorporeal bioartificial liver device could provide vital support to a liver failure patient until a donor liver was available or until the patient's own liver regenerated. Although it is unclear which liver‐specific functions must be provided by such a device to be effective, a constant challenge has been to obtain stable, well‐differentiated, and normally functioning hepatocytes that can be cultured at high cell densities. Many of the devices currently undergoing clinical trials are limited by designs which are prone to substrate limitations, resulting in compromised hepatocyte function. In devices that avoid substrate limitations, hepatocyte functions can be optimized, thereby leading to increased device efficiency. In this overview, the authors describe the critical issues involved in bioartificial liver development and discuss their experiences in hepatocyte culture optimization within the context of a microchannel, flat‐plate bioartificial liver device with an internal membrane oxygenator.     

PNPLA3 as a liver steatosis risk factor following living‐donor liver transplantation for hepatitis C

Aim

Liver steatosis frequently occurs following liver transplantation (LT) and can affect patient outcome. Here, we aimed to clarify the steatosis and steatohepatitis risk factors that apply after living‐donor LT for chronic hepatitis C.

Methods

We retrospectively examined 43 transplant recipients and donors, and tested for single nucleotide polymorphisms in the PNPLA3 gene. Liver biopsies taken 1 year after transplantation and yearly thereafter, or when abnormal liver enzyme levels were detected, were examined by histopathology.

Results

Liver steatosis (>5% steatotic hepatocytes) was evident in 13 of 43 cases (30%), and steatohepatitis in 3 (7.0%). The average time to steatosis after LT was 2.74 ± 1.55 years. The PNPLA3 rs738409 GG genotype, a steatosis risk factor, was identified in 13 recipients and 10 donors. Steatosis prevalence did not differ according to recipient genotype. However, this condition was significantly more common among patients who received tissue from donors carrying the rs738409 GG genotype compared to those with grafts from donors of the CC or CG genotype (60, 7, and 26%, respectively; P < 0.05). All 3 steatohepatitis cases were associated with the GG donor genotype.

Conclusion

The PNPLA3 rs738409 GG donor genotype affects liver steatosis and steatohepatitis risk following living‐donor LT.     

Fatal Escherichia coli skin and soft tissue infections in liver transplant recipients: report of three cases

Gram‐negative bacilli are unusual agents of skin and soft tissue infections. Most previous cases have been reported in cirrhotic or immunocompromised patients, including a single case in a liver transplant recipient. The present report describes 3 cases of fatal skin or soft tissue infections caused by Escherichia coli that occurred in the postoperative course of liver transplantation. The 3 patients were profoundly immunosuppressed as a result of pre‐transplant cirrhosis and the postoperative administration of a potent immunosuppressive therapy. Skin and soft tissue infections developed within the first week after liver transplantation, while graft liver function was satisfactory. The 3 patients presented with fever and skin lesions with or without bullae. Despite prompt appropriate antibiotic therapy and surgical debridement, the outcome was rapidly fatal (24 h on average). E. coli was isolated from subcutaneous tissues in 2 cases and from several blood cultures in the third one. The 3 isolates belonged to distinct phylogenetic groups, and did not harbor most of the virulence factors usually reported in extraintestinal pathogenic E. coli isolates. Our report suggests that E. coli can cause severe skin or soft tissue infection in the postoperative course of liver transplantation. The onset of infection is very early and the outcome is extremely poor, despite prompt adapted medical and surgical treatment. Host factors, rather than E. coli bacterial virulence potential, appear to be the major determinants of severity in these patients.     

Diagnostik und Behandlung des akuten Leberversagens

Background

In Germany about 200 patients suffer from acute liver failure each year. Due to its rare occurrence and the severity of the disease course only few evidence-based therapeutic strategies are available.

Objectives

This review aims to discuss the most important developments for the diagnosis and therapy of acute liver failure and provides an outlook of their future clinical relevance.

Results

The established enzyme parameters combined with synthesis- and detoxification-related markers insufficiently predict the severity and disease course of acute liver failure. In future, levels of released microRNAs or cleaved cytokeratin 18 fragments may improve the diagnostic repertoire. Currently, only few drug-based therapeutic approaches are available, but much effort has been invested in artificial liver support devices. Based on their favorable risk assessment cell-free detoxification systems are applied sporadically during the treatment of patients with advanced liver diseases, even if to date larger studies have failed to prove a significant survival benefit. Extracorporeal liver assist devices and cell transplantation approaches rely on the availability of metabolically active donor hepatocytes and, thus, the generation of liver cells from appropriate stem cells is gaining interest.

Conclusion

Current research in stem cell biology and tissue engineering suggest in initial animal studies the feasibility of stem cell-based artificial liver support systems for future treatment of acute liver failure.     

Treatment of acetaminophen-induced acute liver failure in the mouse with conditionally immortalized human hepatocytes

BACKGROUND/AIMS: Liver failure is a life threatening condition currently treated by palliative measures and, when applicable, organ transplantation. The use of a bioartificial organ capable of fulfilling the main functions of the liver would represent an attractive alternative. However, the shortage of suitable donor cells, and their limited growth ability have impeded the development of this strategy. We investigated whether lentiviral vectors allow for conditional immortalization of human hepatocytes and whether these immortalized hepatocytes could reverse lethal acute liver failure. METHODS: We exposed primary human hepatocytes to Cre-excisable lentiviral vectors coding for SV40T Antigen, telomerase, and/or Bmi-1 and tested the functionality of the resulting cell lines. Therapeutic potential of immortalized hepatocytes were tested in a murine model of acetaminophen-induced hepatic injury. RESULTS: The immortalized hepatocytes grew continuously yet were non-tumorigenic, stopped proliferating when exposed to Cre recombinase, and conserved defining properties of primary hepatocytes, including the ability to secrete liver-specific proteins and to detoxify drugs. The implantation of encapsulated immortalized human hepatocytes rescued mice from lethal doses of acetaminophen. CONCLUSIONS: Lentiviral vectors represent tools of choice for immortalization of non-dividing primary cells, and lentivirally immortalized human hepatocytes are promising reagents for cell-based therapy of acute liver failure.     

Very early‐onset of Cryptococcus neoformans disease following liver transplantation: Report of two cases and a review of the literature

Cryptococcosis is the third most common invasive fungal infection following solid organ transplantation, and mortality is high. Most cases occur late and are due to reactivation of latent infection; however, very early reactivation and donor‐derived transmission can occur. Routine screening pre‐transplant and antifungal prophylaxis for cryptococcosis post‐transplant in solid organ transplantation are not standard practice. We present two cases of very early‐onset Cryptococcus neoformans disease following liver transplantation to highlight the need to consider individualized pre‐transplant screening and be aware that reactivation of Cryptococcosis neoformans can occur in the immediate post‐transplant period.     

Elevated concentrations of 15‐deoxy‐Δ12,14‐prostaglandin J2 in chronic liver disease propose therapeutic trials with peroxisome proliferator activated receptor γ‐inducing drugs

Background/Aims: Current knowledge confers a crucial role to connective tissue growth factor (CTGF/CCN2) in hepatic fibrogenesis. Hepatocytes are likely to be the major cellular source of CTGF in the liver in which CTGF is sensitively upregulated by TGF‐β. Recently, we demonstrated that the methylxanthine derivate caffeine leads to an upregulation of peroxisome proliferator activated receptor γ (PPARγ) expression in hepatocytes, thus sensitizing these cells to the well‐known inhibitory effect of 15‐deoxy‐Δ^12,14‐prostaglandin J₂ (15‐d‐PGJ₂) on CTGF expression. However, upregulation of the receptor alone is not sufficient per se; its physiological ligand 15‐d‐PGJ₂ is required to exert an inhibitory effect on transforming growth factor‐β (TGF‐β) target genes such as CTGF. Methods: This study compared serum concentrations of 15‐d‐PGJ₂ in Caucasian patients with fibrotic liver diseases (n=289), Caucasian controls (n=136) and Caucasian non‐liver disease (NLD) sick (n=307), as well as of Chinese patients with hepatocellular carcinoma (HCC) (n=43) and Chinese healthy controls (n=63) in order to characterize their suitability for therapeutic approaches with PPARγ‐inducing (i.e. CTGF inhibitory) drugs such as caffeine. Results: The presented data showed that Caucasian patients with ongoing hepatic fibrogenesis (mean 6.2±5.9 μg/L) displayed strikingly higher serum concentrations of 15‐d‐PGJ₂ than healthy probands (mean 2.3±1.0) and Caucasian patients with NLD (mean 2.7±1.4 μg/L). Similar results were found in Chinese patients with fully developed HCC (mean 1.3±0.7 μg/L) compared with Chinese healthy controls (mean 0.4±0.2 μg/L). Conclusions: In conclusion, our data thus proposed an increased suitability of these patient groups for therapeutic approaches with drugs inducing PPARγ expression, such as methylxanthine derivates.