Comprehensive analysis of brain injury parameters in a preclinical porcine model of acute liver failure.

Introduction: Acute liver failure (ALF) is defined as acute loss of liver function leading to hepatic encephalopathy associated with a high risk of patient death. Brain injury markers in serum and tissue can help detect and monitor ALF-associated brain injury. This study compares different brain injury parameters in plasma and tissue along with the progression of ALF. Method: ALF was induced by performing an 85% liver resection. Following the resection, animals were recovered and monitored for up to 48 h or until reaching the predefined endpoint of receiving standard medical therapy (SMT). Blood and serum samples were taken at Tbaseline, T24, and upon reaching the endpoint (Tend). Control animals were euthanized by exsanguination following plasma sampling. Postmortem brain tissue samples were collected from the frontal cortex (FCTx) and cerebellum (Cb) of all animals. Glial fibrillary acidic protein (GFAP) and tau protein and mRNA levels were quantified using ELISA and qRT-PCR in all plasma and brain samples. Plasma neurofilament light (NFL) was also measured using ELISA. Results: All ALF animals (n = 4) were euthanized upon showing signs of brain herniation. Evaluation of brain injury biomarkers revealed that GFAP was elevated in ALF animals at T24h and Tend, while Tau and NFL concentrations were unchanged. Moreover, plasma glial fibrillary acidic protein (GFAP) levels were negatively correlated with total protein and positively correlated with both aspartate transaminase (AST) and alkaline phosphatase (AP). Additionally, lower GFAP and tau RNA expressions were observed in the FCTx of the ALF group but not in the CB tissue. Conclusion: The current large animal study has identified a strong correlation between GFAP concentration in the blood and markers of ALF. Additionally, the protein and gene expression analyses in the FCTx revealed that this area appears to be susceptible, while the CB is protected from the detrimental impacts of ALF-associated brain swelling. These results warrant further studies to investigate the mechanisms behind this process.

Biodegradable biliary stents coated with mesenchymal stromal cells in a porcine choledochojejunostomy model.

BACKGROUND AIMS: Roux en y anastomosis is a preferred method of biliary reconstruction in liver transplantation that involves living donors or pediatric patients. However, biliary stricture is a frequent and serious complication, accounting for up to 40% of biliary complications in these patients. Previously, we demonstrated that extraluminal delivery of adipose-derived (AD) mesenchymal stromal cells (MSCs) decreased peri-biliary fibrosis and increased neo-angiogenesis in a porcine model of duct-to-duct biliary anastomosis. In this study, we used a porcine model of Roux en y anastomosis to evaluate the beneficial impact of a novel intraluminal MSC delivery system. METHODS: Nine animals were divided into three groups: no stent (group 1), bare stent (group 2) and stent coated with AD-MSCs (group 3). All animals underwent cholecystectomy with roux en y choledochojejunostomy. Two animals per group were followed for 4 weeks and one animal per group was followed for 8 weeks. Cholangiograms and blood were sampled at baseline and the end of study. Biliary tissue was collected and examined by Masson trichrome staining and immunohistochemical staining for MSC markers (CD34 and CD44) and for neo-angiogenesis (CD31). RESULTS: Two of three animals in group 1 developed an anastomotic site stricture. No strictures were observed in the animals of group 2 or group 3. CD34 and CD44 staining showed that AD-MSCs engrafted successfully at the anastomotic site by intraluminal delivery (group 3). Furthermore, biliary tissue from group 3 showed significantly less fibrosis and increased angiogenesis compared with the other groups. CONCLUSIONS: Intraluminal delivery of AD-MSCs resulted in successful biliary engraftment of AD-MSCs as well as reduced peri-biliary fibrosis and increased neo-angiogenesis.

Clinical characterization of a hypersensitivity mixed bacterial and fungal dermatitis in a translational model of porcine NASH.

Introduction: The development of animal models of chronic liver disease via diet modification is a promising avenue for translational research but can lead to unexpected side effects that impact model adoption. While these side effects are well characterized in rodent models of nonalcoholic steatohepatitis (NASH), limited knowledge of these effects exists for novel porcine models of NASH. To close this gap, the present study investigates the side effects of diet-based NASH induction in pigs, with a systematic analysis of the pathologic mechanisms underlying dermatitis development and evaluation of treatment approaches. Method: Twelve pigs (10 large domestic pigs, 2 Goettingen minipigs) were fed a methionine- and choline-deficient, high-fat diet for 8 weeks to induce NASH. A retrospective review of each animal's clinical record was performed to identify the side effects of the diet. Following the identification of diet-associated dermatitis, severity was judged by using a novel gradation system that characterized the individual lesions and body regions resulting in a cumulative evaluation. In addition to this clinical assessment, the etiology of the dermatitis was investigated via histopathologic and microbiologic testing. Furthermore, the success of prophylactic and therapeutic treatment approaches was evaluated by considering dermatitis development and clinical course. Results: All study animals demonstrated unexpected side effects of the methionine- and choline-deficient, high fat diet. In addition to marked dermatitis, study pigs showed impaired weight gain and developed steatorrhea and anemia. Based on the skin gradation system, five animals developed severe dermatitis, four animals moderate dermatitis, and three animals mild diet-associated dermatitis. Histological and microbiological evaluation of the affected skin showed signs of a hypersensitivity reaction with secondary infection by bacteria and fungi. The analysis showed that preemptive bathing extended the lesion-free duration by nearly 20 days. Furthermore, bathing in combination with a targeted antibiotic treatment represented a helpful treatment approach for diet-associated dermatitis. Conclusion: The provision of a methionine- and choline-deficient, high fat diet represents an effective approach for inducing NASH liver disease in pigs but predisposes study animals to multiple side effects. These side effects are universal to animals on study but can be adequately managed and do not represent a significant limitation of this model.

A non-human primate model of acute liver failure suitable for testing liver support systems.

Acute hepatic failure is associated with high morbidity and mortality for which the only definitive therapy is liver transplantation. Some fraction of those who undergo emergency transplantation have been shown to recover native liver function when transplanted with an auxiliary hepatic graft that leaves part of the native liver intact. Thus, transplantation could have been averted with the development and use of some form of hepatic support. The costs of developing and testing liver support systems could be dramatically reduced by the availability of a reliable large animal model of hepatic failure with a large therapeutic window that allows the assessment of efficacy and timing of intervention. Non-lethal forms of hepatic injury were examined in combination with liver-directed radiation in non-human primates (NHPs) to develop a model of acute hepatic failure that mimics the human condition. Porcine hepatocyte transplantation was then tested as a potential therapy for acute hepatic failure. After liver-directed radiation therapy, delivery of a non-lethal hepatic ischemia-reperfusion injury reliably and rapidly generated liver failure providing conditions that can enable pre-clinical testing of liver support or replacement therapies. Unfortunately, in preliminary studies, low hepatocyte engraftment and over-immune suppression interfered with the ability to assess the efficacy of transplanted porcine hepatocytes in the model. A model of acute liver failure in NHPs was created that recapitulates the pathophysiology and pathology of the clinical condition, does so with reasonably predictable kinetics, and results in 100% mortality. The model allowed preliminary testing of xenogeneic hepatocyte transplantation as a potential therapy.

Re-Endothelialization of Decellularized Liver Scaffolds: A Step for Bioengineered Liver Transplantation.

Bioengineered livers (BELs) are an attractive therapeutic alternative to address the donor organ shortage for liver transplantation. The goal of BELs technology aims at replacement or regeneration of the native human liver. A variety of approaches have been proposed for tissue engineering of transplantable livers; the current review will highlight the decellularization-recellularization approach to BELs. For example, vascular patency and appropriate cell distribution and expansion are critical components in the production of successful BELs. Proper solutions to these components of BELs have challenged its development. Several strategies, such as heparin immobilization, heparin-gelatin, REDV peptide, and anti-CD31 aptamer have been developed to extend the vascular patency of revascularized bioengineered livers (rBELs). Other novel methods have been developed to enhance cell seeding of parenchymal cells and to increase graft functionality during both bench and in vivo perfusion. These enhanced methods have been associated with up to 15 days of survival in large animal (porcine) models of heterotopic transplantation but have not yet permitted extended survival after implantation of BELs in the orthotopic position. This review will highlight both the remaining challenges and the potential for clinical application of functional bioengineered grafts.

Induced Pluripotent Stem Cells for the Treatment of Liver Diseases: Novel Concepts.

Millions of people worldwide with incurable liver disease die because of inadequate treatment options and limited availability of donor organs for liver transplantation. Regenerative medicine as an innovative approach to repairing and replacing cells, tissues, and organs is undergoing a major revolution due to the unprecedented need for organs for patients around the world. Induced pluripotent stem cells (iPSCs) have been widely studied in the field of liver regeneration and are considered to be the most promising candidate therapies. This review will conclude the current state of efforts to derive human iPSCs for potential use in the modeling and treatment of liver disease.

Limited Expansion of Human Hepatocytes in FAH/RAG2-Deficient Swine.

The mammalian liver's regenerative ability has led researchers to engineer animals as incubators for expansion of human hepatocytes. The expansion properties of human hepatocytes in immunodeficient mice are well known. However, little has been reported about larger animals that are more scalable and practical for clinical purposes. Therefore, we engineered immunodeficient swine to support expansion of human hepatocytes and identify barriers to their clinical application. Immunodeficient swine were engineered by knockout of the recombinase-activating gene 2 (RAG2) and fumarylacetoacetate hydrolase (FAH). Immature human hepatocytes (ihHCs) were injected into fetal swine by intrauterine cell transplantation (IUCT) at day 40 of gestation. Human albumin was measured as a marker of engraftment. Cytotoxicity against ihHCs was measured in transplanted piglets and control swine. We initially detected higher levels of human albumin in cord blood of newborn FAH/RAG2-deficient (FR) pigs compared with immunocompetent controls (196.26 ng/dL vs. 39.29 ng/dL, p = 0.008), indicating successful engraftment of ihHCs after IUCT and adaptive immunity in the fetus. Although rare hepatocytes staining positive for human albumin were observed, levels of human albumin did not rise after birth, but declined, suggesting rejection of xenografted ihHCs. Cytotoxicity against ihHCs increased after birth by 3.8% (95% CI: [2.1%-5.4%], p < 0.001) and inversely correlated with declining levels of human albumin (p = 2.1 × 10-5, R2 = 0.17). Circulating numbers of T cells and B cells were negligible in FR pigs. However, circulating natural killer (NK) cells exerted cytotoxicity against ihHCs. NK cell activity was lower in immunodeficient piglets after IUCT than in naive controls (30.4% vs. 40.1%, p = 0.011, 95% CI for difference [2.7%-16.7%]). In conclusion, ihHCs were successfully engrafted in FR swine after IUCT. NK cells were a significant barrier to expansion of hepatocytes. New approaches are needed to overcome this hurdle and allow large-scale expansion of human hepatocytes in immunodeficient swine. Impact statement There is currently a need for robust expansion of human hepatocytes. We describe an immunodeficient swine model into which we engrafted immature human hepatocytes (ihHCs). We identified the mechanism of the eventual graft rejection by the intact NK cell population, which has not been previously shown to have a significant role in xenograft rejection. By both improving engraftment and reducing NK cell-mediated cytotoxicity toward the graft through intrauterine cell transfer, we confirmed the presence of residual adaptive immunity in this model of immunodeficiency and the ability to induce hyposensitization in the NK cell population by taking advantage of the fetal microenvironment.

Functional characterization of a bioengineered liver after heterotopic implantation in pigs.

Organ bioengineering offers a promising solution to the persistent shortage of donor organs. However, the progression of this technology toward clinical use has been hindered by the challenges of reconstituting a functional vascular network, directing the engraftment of specific functional cell types, and defining appropriate culture conditions to concurrently support the health and phenotypic stability of diverse cell lineages. We previously demonstrated the ability to functionally reendothelialize the vasculature of a clinically scaled decellularized liver scaffold with human umbilical vein endothelial cells (HUVECs) and to sustain continuous perfusion in a large animal recovery model. We now report a method for seeding and engrafting primary porcine hepatocytes into a bioengineered liver (BEL) scaffold previously reendothelialized with HUVECs. The resulting BELs were competent for albumin production, ammonia detoxification and urea synthesis, indicating the presence of a functional hepatocyte compartment. BELs additionally slowed ammonia accumulation during in vivo perfusion in a porcine model of surgically induced acute liver failure. Following explant of the graft, BEL parenchyma showed maintenance of canonical endothelial and hepatocyte markers. Taken together, these results support the feasibility of engineering a clinically scaled functional BEL and establish a platform for optimizing the seeding and engraftment of additional liver specific cells.

Fumarylacetoacetate hydrolase gene as a knockout target for hepatic chimerism and donor liver production.

A reliable source of human hepatocytes and transplantable livers is needed. Interspecies embryo complementation, which involves implanting donor human stem cells into early morula/blastocyst stage animal embryos, is an emerging solution to the shortage of transplantable livers. We review proposed mutations in the recipient embryo to disable hepatogenesis, and discuss the advantages of using fumarylacetoacetate hydrolase knockouts and other genetic modifications to disable hepatogenesis. Interspecies blastocyst complementation using porcine recipients for primate donors has been achieved, although percentages of chimerism remain persistently low. Recent investigation into the dynamic transcriptomes of pigs and primates have created new opportunities to intimately match the stage of developing animal embryos with one of the many varieties of human induced pluripotent stem cell. We discuss techniques for decreasing donor cell apoptosis, targeting donor tissue to endodermal structures to avoid neural or germline chimerism, and decreasing the immunogenicity of chimeric organs by generating donor endothelium.

Cell-Mediated Therapies to Facilitate Operational Tolerance in Liver Transplantation.

Cell therapies using immune cells or non-parenchymal cells of the liver have emerged as potential treatments to facilitate immunosuppression withdrawal and to induce operational tolerance in liver transplant (LT) recipients. Recent pre-clinical and clinical trials of cellular therapies including regulatory T cells, regulatory dendritic cells, and mesenchymal cells have shown promising results. Here we briefly summarize current concepts of cellular therapy for induction of operational tolerance in LT recipients.

Long-term Outcomes of Sequential Hematopoietic Stem Cell Transplantation and Kidney Transplantation: Single-center Experience.

BACKGROUND: Experience with sequential hematopoietic stem cell transplant (HSCT) and kidney transplant (KT) is limited. METHODS: We conducted a retrospective observational study of adult patients who underwent both HSCT and KT at our center, with a median follow-up of 11 y. RESULTS: In our 54 patients cohort (94% autologous HSCT), 36 (67%) patients received HSCT first followed by KT, while 18 (33%) received KT before HSCT. In both groups, AL amyloidosis represented 50% of hematologic diagnosis. Only 4 patients expired due to hematologic disease relapse (2 patients in each group) and only 3 allografts were lost due to hematologic disease recurrence (HSCT first n = 1 and KT first n = 2). Overall 1, 5, and 10 y death-censored graft survival rates were 94%, 94%, and 94%, respectively, for the HSCT first group and 89%, 89%, and 75%, respectively, for the KT first group. Overall 1, 5, and 10 y patients survival rates were 100%, 97% and 90%, respectively, for the HSCT first group and 100%, 76%, and 63%, respectively, for the KT first group. CONCLUSIONS: Our study supports safety of sequential KT and HSCT, with improved overall patient survival compared to recipients of HSCT remaining on dialysis and good long-term kidney allograft outcome.

Outcomes of Liver Transplantation in Treated Portopulmonary Hypertension Patients With a Mean Pulmonary Arterial Pressure ≥35 mm Hg.

Portopulmonary hypertension (POPH), pulmonary arterial hypertension (PAH) that develops in the setting of portal hypertension, affects 5%-6% of patients with liver disease and is associated with significant morbidity and mortality. A mean pulmonary arterial pressure (mPAP) threshold of 35 mm Hg is used to stratify perioperative risk and liver transplant eligibility in treated POPH patients but does not take into account the specific factors that contribute to the pressure elevation. METHODS: In this case series, we describe the characteristics and posttransplant outcomes of patients with treated POPH and an mPAP ≥35 mm Hg and pulmonary vascular resistance (PVR) <250 dynes-s-cm-5 at or just before liver transplantation (LT). We also describe the effect of PAH therapy on pulmonary hemodynamics in patients with POPH. RESULTS: Sixteen patients were included. All patients were on PAH therapy at the time of LT. PAH therapy resulted in a decrease of mPAP (median 18.4%; interquartile range [IQR] 8.9%-27.0%) with a reduction in PVR (median 50.5%; IQR, 45.4%-70.7%), and an increase in both cardiac output (CO) (median 28.1%; IQR 5.7%-63.8%) and PAWP (median 50.0%; IQR 16.7%-108.3%) before LT. One year posttransplant survival was 69% (11/16); however, only 1 death was attributed to POPH. At 1-year posttransplant, 63.6% (7/11) of patients were weaned off all PAH therapy with clinical and echocardiographic resolution of POPH. CONCLUSIONS: In treated POPH patients with an mPAP ≥35 mm Hg and PVR < 250 dynes-s-cm-5 before LT, 1-year posttransplant survival was 69% and the majority of patients were able to discontinue PAH therapy.

Ex Vivo Cell Therapy by Ectopic Hepatocyte Transplantation Treats the Porcine Tyrosinemia Model of Acute Liver Failure.

The effectiveness of cell-based therapies to treat liver failure is often limited by the diseased liver environment. Here, we provide preclinical proof of concept for hepatocyte transplantation into lymph nodes as a cure for liver failure in a large-animal model with hereditary tyrosinemia type 1 (HT1), a metabolic liver disease caused by deficiency of fumarylacetoacetate hydrolase (FAH) enzyme. Autologous porcine hepatocytes were transduced ex vivo with a lentiviral vector carrying the pig Fah gene and transplanted into mesenteric lymph nodes. Hepatocytes showed early (6 h) and durable (8 months) engraftment in lymph nodes, with reproduction of vascular and hepatic microarchitecture. Subsequently, hepatocytes migrated to and repopulated the native diseased liver. The corrected cells generated sufficient liver mass to clinically ameliorate the acute liver failure and HT1 disease as early as 97 days post-transplantation. Integration site analysis defined the corrected hepatocytes in the liver as a subpopulation of hepatocytes from lymph nodes, indicating that the lymph nodes served as a source for healthy hepatocytes to repopulate a diseased liver. Therefore, ectopic transplantation of healthy hepatocytes cures this pig model of liver failure and presents a promising approach for the development of cures for liver disease in patients.

Hormonal Contribution to Liver Regeneration.

An understanding of the molecular basis of liver regeneration will open new horizons for the development of novel therapies for chronic liver failure. Such therapies would solve the drawbacks associated with liver transplant, including the shortage of donor organs, long waitlist time, high medical costs, and lifelong use of immunosuppressive agents. Regeneration after partial hepatectomy has been studied in animal models, particularly fumarylacetoacetate hydrolase-deficient (FAH -/-) mice and pigs. The process of regeneration is distinctive, complex, and well coordinated, and it depends on the interplay among several signaling pathways (eg, nuclear factor κß, Notch, Hippo), cytokines (eg, tumor necrosis factor α, interleukin 6), and growth factors (eg, hepatocyte growth factor, epidermal growth factor, vascular endothelial growth factor), and other components. Furthermore, endocrinal hormones (eg, norepinephrine, growth hormone, insulin, thyroid hormones) also can influence the aforementioned pathways and factors. We believe that these endocrinal hormones are important hepatic mitogens that strongly induce and accelerate hepatocyte proliferation (regeneration) by directly and indirectly triggering the activity of the involved signaling pathways, cytokines, growth factors, and transcription factors. The subsequent induction of cyclins and associated cyclin-dependent kinase complexes allow hepatocytes to enter the cell cycle. In this review article, we comprehensively summarize the current knowledge regarding the roles and mechanisms of these hormones in liver regeneration. Articles used for this review were identified by searching MEDLINE and EMBASE databases from inception through June 1, 2019.

Sustained perfusion of revascularized bioengineered livers heterotopically transplanted into immunosuppressed pigs.

Implanted bioengineered livers have not exceeded three days of continuous perfusion. Here we show that decellularized whole porcine livers revascularized with human umbilical vein endothelial cells and implanted heterotopically into immunosuppressed pigs whose spleens had been removed can sustain perfusion for up to 15 days. We identified peak glucose consumption rate as a main predictor of the patency of the revascularized bioengineered livers (rBELs). Heterotopic implantation of rBELs into pigs in the absence of anticoagulation therapy led to sustained perfusion for three days, followed by a pronounced immune responses directed against the human endothelial cells. A 10 day steroid-based immunosuppression protocol and a splenectomy at the time of rBEL implantation reduced the immune responses and resulted in continuous perfusion of the rBELs for over two weeks. We also show that the human endothelial cells in the perfused rBELs colonize the liver sinusoids and express sinusoidal endothelial markers similar to those in normal liver tissue. Revascularized liver scaffolds that can maintain blood perfusion at physiological pressures might eventually help to overcome the chronic shortage of transplantable human livers.

Autologous Adipose Tissue-Derived Mesenchymal Stem Cells Introduced by Biliary Stents or Local Immersion in Porcine Bile Duct Anastomoses.

Biliary complications (strictures and leaks) represent major limitations in living donor liver transplantation. Mesenchymal stem cells (MSCs) are a promising modality to prevent biliary complications because of immunosuppressive and angiogenic properties. Our goal was to evaluate the safety of adipose-derived MSC delivery to biliary anastomoses in a porcine model. Secondary objectives were defining the optimal method of delivery (intraluminal versus extraluminal) and to investigate MSC engraftment, angiogenesis, and fibrosis. Pigs were divided into 3 groups. Animals underwent adipose collection, MSC isolation, and expansion. Two weeks later, animals underwent bile duct transection, reanastomosis, and stent insertion. Group 1 received plastic stents wrapped in unseeded Vicryl mesh. Group 2 received stents wrapped in MSC-seeded mesh. Group 3 received unwrapped stents with the anastomosis immersed in an MSC suspension. Animals were killed 1 month after stent insertion when cholangiograms and biliary tissue were obtained. Serum was collected for liver biochemistries. Tissue was used for hematoxylin-eosin and trichrome staining and immunohistochemistry for MSC markers (CD44 and CD34) and for a marker of neoangiogenesis (CD31). There were no intraoperative complications. One pig died on postoperative day 3 due to acute cholangitis. All others recovered without complications. Cholangiography demonstrated no biliary leaks and minimal luminal narrowing. Surviving animals exhibited no symptoms, abnormal liver biochemistries, or clinically significant biliary stricturing. Group 3 showed significantly greater CD44 and CD34 staining, indicating MSC engraftment. Fibrosis was reduced at the anastomotic site in group 3 based on trichrome stain. CD31 staining of group 3 was more pronounced, supporting enhanced neoangiogenesis. In conclusion, adipose-derived MSCs were safely applied to biliary anastomoses. MSCs were locally engrafted within the bile duct and may have beneficial effects in terms of fibrosis and angiogenesis.

Author Correction: Sustained perfusion of revascularized bioengineered livers heterotopically transplanted into immunosuppressed pigs.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Stem Cell-Related Studies and Stem Cell-Based Therapies in Liver Diseases.

Owing to the increasing worldwide burden of liver diseases, the crucial need for safe and effective interventions for treating end-stage liver failure has been a very productive line of inquiry in the discipline of hepatology for many years. Liver transplantation is recognized as the most effective treatment for end-stage liver disease; however, the shortage of donor organs, high medical costs, and lifelong use of immunosuppressive agents represent major drawbacks and demand exploration for alternative treatments. Stem cell-based therapies have been widely studied in the field of liver diseases and are considered to be among the most promising therapies. Herein, we review recent advances in the application of stem cell-related therapies in liver disease with the aim of providing readers with relevant knowledge in this field and inspiration to spur further inquiry.