Effects of polyphenol supplementation on hepatic steatosis, intima-media thickness and non-invasive vascular elastography in obese adolescents: a pilot study protocol.

INTRODUCTION: Non-alcoholic fatty liver disease (NAFLD) is increasingly prevalent in obese adolescents. Increased systemic inflammation and decreased gut microbial diversity linked to obesity affect the liver and are also associated with cardiovascular diseases in adulthood. However, NAFLD and vascular alterations are reversible. METHODS AND ANALYSIS: This pilot study evaluated the feasibility of a prospective open-label randomised controlled trial evaluating the effects of polyphenols on NAFLD and vascular parameters in obese adolescents. Children aged 12-18 years with hepatic steatosis (n=60) will be recruited. The participants will be randomised with a 1:1 allocation ratio to receive polyphenol supplementation one time per day for 8 weeks along with the clinician-prescribed treatment (group B, n=30) or to continue the prescribed treatment without taking any polyphenols (group A, n=30). The outcome measures will be collected from both the groups at day 1 before starting polyphenol supplementation, at day 60 after 8 weeks of supplementation and at day 120, that is, 60 days after supplementation. The changes in hepatic steatosis and vascular parameters will be measured using liver and vascular imaging. Furthermore, anthropometric measures, blood tests and stool samples for gut microbiome analysis will be collected. After evaluating the study's feasibility, we hypothesise that, as a secondary outcome, compared with group A, the adolescents in group B will have improved NAFLD, vascular parameters, systemic inflammation and gut microbiome. ETHICS AND DISSEMINATION: This study is approved by Health Canada and the hospital ethics. Participants and their parents/tutors will both provide consent. Trial results will be communicated to the collaborating gastroenterologists who follow the enrolled participants. Abstracts and scientific articles will be submitted to high-impact radiological societies and journals. CLINICALTRIALS: gov ID: NCT03994029. Health Canada authorisation referral number: 250 811. Protocole version 13, 2 June 2023. TRIAL REGISTRATION NUMBER: NCT03994029.

Early predictors of unfavourable outcome in progressive cholestasis of northwestern Quebec.

Background: Progressive cholestasis of northwestern Quebec (PCNQ) is a rare and severe form of cirrhosis affecting children from Quebec's First Nations. First described by our group in 1981 and historically named North American Indian childhood cirrhosis, such a condition often requires liver transplantation during the pediatric age. This study aimed at suggesting a more culturally sensitive name for the disease and identifying early prognostic factors for an unfavourable outcome. Methods: We retrospectively collected data of all 14 consecutive patients diagnosed with PCNQ over the last 20 years and compared children listed for liver transplant before 18 years of age (LT, n = 7) to those with milder disease progression (no-LT, n = 7). Results: Compared with the no-LT group, LT children developed serious complications with an unusually high incidence of gastrointestinal bleeding. Over the first 12 months from presentation, a greater increase of alanine aminotransferase plasma levels, decrease of total bilirubin, and increase of alanine aminotransferase-to-total bilirubin ratio was observed in the LT group. Bone mineral density was lower in LT children independently of vitamin D levels. Patients with PCNQ showed poorer bone health than age-matched children with other cholestatic disorders. Conclusions: In the name of cultural sensitivity, PCNQ should be the preferred name for this condition. Variation of alanine aminotransferase and total bilirubin plasma levels over the first 12 months from presentation might be used for the early identification of children with PCNQ who are at higher risk of unfavourable outcomes. This might help optimize clinical management to populations that are underserved by health care services.

Autologous humanized mouse models of iPSC-derived tumors enable characterization and modulation of cancer-immune cell interactions.

Modeling the tumor-immune cell interactions in humanized mice is complex and limits drug development. Here, we generated easily accessible tumor models by transforming either primary skin fibroblasts or induced pluripotent stem cell-derived cell lines injected in immune-deficient mice reconstituted with human autologous immune cells. Our results showed that fibroblastic, hepatic, or neural tumors were all efficiently infiltrated and partially or totally rejected by autologous immune cells in humanized mice. Characterization of tumor-immune infiltrates revealed high expression levels of the dysfunction markers Tim3 and PD-1 in T cells and an enrichment in regulatory T cells, suggesting rapid establishment of immunomodulatory phenotypes. Inhibition of PD-1 by Nivolumab in humanized mice resulted in increased immune cell infiltration and a slight decrease in tumor growth. We expect that these versatile and accessible cancer models will facilitate preclinical studies and the evaluation of autologous cancer immunotherapies across a range of different tumor cell types.

Generation of Complex Syngeneic Liver Organoids from Induced Pluripotent Stem Cells to Model Human Liver Pathophysiology.

The study of human liver pathophysiology has been hampered for decades by the lack of easily accessible, robust, and representative in vitro models. The discovery of induced pluripotent stem cells (iPSCs)-which can be generated from patients' somatic cells, engineered to harbor specific mutations, and differentiated into hepatocyte-like cells-opened the way to more meaningful modeling of liver development and disease. Nevertheless, representative modeling of many complex liver conditions requires the recreation of the interplay between hepatocytes and nonparenchymal liver cells. Here we describe protocols we developed to generate and characterize complex human liver organoids composed of iPSC-derived hepatic, endothelial, and mesenchymal cells. With all cell types derived from the same iPSC population, such organoids reproduce the liver niche, allowing for the study of liver development and the modeling of complex inflammatory and fibrotic conditions. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Differentiation of human iPSCs into hepatic progenitor cells (hepatoblasts) Basic Protocol 2: Differentiation of human iPSCs into endothelial progenitor cells Support Protocol 1: Characterization of iPSC-derived endothelial progenitor cells Basic Protocol 3: Differentiation of human iPSCs into mesenchymal progenitor cells Support Protocol 2: Characterization of iPSC-derived mesenchymal progenitor cells Basic Protocol 4: Generation of complex syngeneic liver organoids.

Leveraging interacting signaling pathways to robustly improve the quality and yield of human pluripotent stem cell-derived hepatoblasts and hepatocytes.

Pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs) have shown great potential as an alternative to primary human hepatocytes (PHHs) for in vitro modeling. Several differentiation protocols have been described to direct PSCs toward the hepatic fate. Here, by leveraging recent knowledge of the signaling pathways involved in liver development, we describe a robust, scalable protocol that allowed us to consistently generate high-quality bipotent human hepatoblasts and HLCs from both embryonic stem cells and induced PSC (iPSCs). Although not yet fully mature, such HLCs were more similar to adult PHHs than were cells obtained with previously described protocols, showing good potential as a physiologically representative alternative to PHHs for in vitro modeling. PSC-derived hepatoblasts effectively generated with this protocol could differentiate into mature hepatocytes and cholangiocytes within syngeneic liver organoids, thus opening the way for representative human 3D in vitro modeling of liver development and pathophysiology.

Congenital absence of the portal vein: Define the portosystemic shunt, avoid liver transplantation.

Liver transplantation has been historically recommended for patients with congenital absence of the portal vein associated with extrahepatic congenital portosystemic shunts. Here, based on a case report of a 2-year-old girl and a thorough review of all published cases from 1974 to 2020, we show that such a diagnosis most often conceals a hypoplastic portal vein, which can be successfully re-permeabilized through the closure of the shunt in order to re-establish a physiological vascular anatomy. This highlights the importance of achieving a detailed anatomical description of extrahepatic congenital portosystemic shunts with a balloon occlusion test in order to plan the best surgical approach and avoid unnecessary liver transplantation.

High-throughput assessment of mutations generated by genome editing in induced pluripotent stem cells by high-resolution melting analysis.

BACKGROUND AND AIMS: Genome editing of induced pluripotent stem cells (iPSCs) holds great potential for both disease modeling and regenerative medicine. Although clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 provides an efficient and precise genome editing tool, iPSCs are especially difficult to transfect, resulting in a small percentage of cells carrying the desired correction. A high-throughput method to identify edited clones is required to reduce the time and costs of such an approach. METHODS: Here we assess high-resolution melting analysis (HRMA), a simple and efficient real-time polymerase chain reaction-based method, and compare it with more commonly used assays. RESULTS AND CONCLUSIONS: Our data show that HRMA is a robust and highly sensitive method, allowing the cost-effective and time-saving screening of genome-edited iPSCs. Samples can be prepared directly from 96-well microtiter plates for high-throughput analysis, and amplicons can be further analyzed with downstream techniques for further confirmation, if needed.

Non-alcoholic fatty liver disease severity and metabolic complications in obese children: impact of omega-3 fatty acids.

Although n-3 polyunsaturated fatty acids (PUFA) revealed promising therapeutic results in non-alcoholic fatty liver disease (NAFLD), which is considered as the most prevalent cause of chronic hepatic disease, inconsistencies are calling for further confirmatory trials to demonstrate therapeutic efficacy and safety. The study, registered as NCT02201160 on www.clinicaltrials.gov, was designed to compare two groups of NAFLD with a different severity, and to evaluate the efficacy of n-3 PUFA supplementation. Twenty young male participants of French Canadian origin with NAFLD were enrolled and classified into moderate (mNAFLD) and severe (sNAFLD) fatty liver groups, according to transaminase levels, ultrasonography, NAFLD Activity Score and Fatty Liver Index (FLI). The sNAFLD patients were assigned to consume 2 g of n-3 PUFA for 6 months. sNAFLD patients displayed higher insulinemia, insulin resistance (IR), oxidative stress (OxS), systolic blood pressure and the risk lipid indicators of cardiovascular diseases. Supplementation of n-3 PUFA for 6 months resulted in a significant increase in concentrations of eicosapentaenoic and docosahexaenoic acids in red blood cells along with an attenuation of hepatic steatosis as reflected by the reduction of the FLI, ALT and ALT/AST ratio. Moreover, the n-3 PUFA improved the lipid profile and carotid intima-media thickness, while reducing metabolic and OxS markers as well as raising adiponectin. In conclusion, NAFLD severity was essentially related to IR. Treatment with n-3 PUFA has an evidently beneficial effect on liver steatosis and related metabolic abnormalities. Furthermore, the cross association of omega-3 index with cardiometabolic markers may serve as a predictor for cardiovascular risk disorders in NAFLD.

Successful Liver Transplants for Liver Failure Associated With Pyruvate Kinase Deficiency.

Pyruvate kinase deficiency (PKD) is the most common cause of congenital nonspherocytic chronic hemolytic anemia, and patients normally present with mild to severe anemia, unconjugated hyperbilirubinemia, and splenomegaly. Only a few reports of PKD have documented its association with severe, progressive liver failure. In all those cases, the patients died before liver transplant (LT) or immediately after transplant. We report 2 case patients with liver failure associated with PKD who successfully underwent LT and splenectomy: an infant who presented with neonatal cholestasis and a young adult with a severe form of PKD and having been transfusion dependent during childhood. After transplant, both patients have normal liver function test results and have considerably decreased their need for blood transfusion despite ongoing, mild hemolysis. We suggest that PKD can lead to severe liver dysfunction and that LT and splenectomy can be life-saving procedures in such cases.

Predisposing Factors for Spontaneous Closure of Congenital Portosystemic Shunts.

In a review of 382 cases of congenital portosystemic shunt, we found that presentation with neonatal cholestasis strongly predicts spontaneous closure of intrahepatic shunts (OR 8.3, 95% CI 3.4-20.2). Spontaneous closure before the 24th month of age is more likely for distal or multiple shunts, but rare for patent ductus venosus.

Anti-CD20 treatment of giant cell hepatitis with autoimmune hemolytic anemia.

Giant cell hepatitis with autoimmune hemolytic anemia (GCH-AHA) is a rare autoimmune disease of infancy characterized by severe liver disease associated with Coombs-positive hemolytic anemia. We recently showed that GCH-AHA is probably caused by a humoral immune mechanism. Such data support the use of rituximab, an anti-CD-20 monoclonal antibody specifically targeting B lymphocytes, as a treatment for GCH-AHA. We describe here the detailed clinical evolution of 4 children with GCH-AHA who showed a complete response to rituximab. All patients shared a severe course of the disease with poor control on standard and aggressive immunosuppression. Rituximab was well tolerated, and no side effects or infections were registered. Several doses were needed to induce remission, and 5 to 11 additional maintenance injections were necessary in the 2 more severe cases. Weaning from corticosteroids was achieved in all subjects. A steroid-sparing effect was noted in the 3 children who started rituximab early in the course of the disease. Overall, we show here that there is a strong rationale for treating GCH-AHA with rituximab. Early treatment could reduce the use of corticosteroids. Nevertheless, short-term steroids should be initially associated with rituximab to account for autoantibodies' half-life. Repeated injections are needed to treat and prevent relapses, but the best frequency and duration of treatment remain to be defined.

Downregulation of Sox9 expression associates with hepatogenic differentiation of human liver mesenchymal stem/progenitor cells.

Understanding the mechanisms triggering hepatogenic differentiation of stem/progenitor cells would be useful for studying postnatal liver regeneration and development of liver cell therapies. Many evidences support the involvement of Sox9 transcription factor in liver development. Here, we investigate the possibility of liver mesenchymal stem/progenitor cells to constitutively express Sox9 by using reverse transcription-quantitative polymerase chain reaction, immunocytochemistry, and western blotting. The involvement of Sox9 in hepatogenic differentiation was assessed by following its expression at different steps of the process, evaluating the impact of its altered expression, and analyzing its expression in human liver disease specimen. Liver mesenchymal stem/progenitor cells constitutively express Sox9 at both the mRNA and protein levels. Upon hepatogenic differentiation, Sox9 expression is downregulated mainly in the maturation step after oncostatin M treatment. Induction of Sox9 expression using transforming growth factor beta is accompanied with a decrease of the quality of hepatogenic differentiation. Blunting Sox9 expression using specific ShRNA clearly alters the levels of several hepatic markers, an effect confirmed in HepG2 cells. In human liver disease specimen, Sox9 expression is enhanced at both the mRNA and protein levels compared with healthy donors. The current data demonstrate that Sox9 may play a pivotal role in hepatocyte lineage development, including adult liver mesenchymal stem/progenitor cells. Further studies on the identification of pathways regulated by or regulating Sox9 will certainly gain insight into the molecular networks controlling hepatogenic differentiation.

Human umbilical cord matrix stem cells maintain multilineage differentiation abilities and do not transform during long-term culture.

Umbilical cord matrix stem cells (UCMSC) have generated great interest in various therapeutic approaches, including liver regeneration. This article aims to analyze the specific characteristics and the potential occurrence of premalignant alterations of UCMSC during long-term expansion, which are important issues for clinical applications. UCMSC were isolated from the umbilical cord of 14 full-term newborns and expanded in vitro until senescence. We examined the long-term growth potential, senescence characteristics, immunophenotype and multilineage differentiation capacity of these cells. In addition, their genetic stability was assessed through karyotyping, telomerase maintenance mechanisms and analysis of expression and functionality of cell cycle regulation genes. The tumorigenic potential was also studied in immunocompromised mice. In vitro, UCMSC reached up to 33.7 ± 2.1 cumulative population doublings before entering replicative senescence. Their immunophenotype and differentiation potential, notably into hepatocyte-like cells, remained stable over time. Cytogenetic analyses did not reveal any chromosomal abnormality and the expression of oncogenes was not induced. Telomere maintenance mechanisms were not activated. Just as UCMSC lacked transformed features in vitro, they could not give rise to tumors in vivo. UCMSC could be expanded in long-term cultures while maintaining stable genetic features and endodermal differentiation potential. UCMSC therefore represent safe candidates for liver regenerative medicine.

Differentiated umbilical cord matrix stem cells as a new in vitro model to study early events during hepatitis B virus infection.

UNLABELLED: The role of cell differentiation state on hepatitis B virus (HBV) replication has been well demonstrated, whereas how it determines cell susceptibility to HBV entry is far less understood. We previously showed that umbilical cord matrix stem cells (UCMSC) can be differentiated towards hepatocyte-like cells in vitro. In this study we infected undifferentiated (UD-) and differentiated (D-) UCMSCs with HBV and studied the infection kinetics, comparing them to primary human hepatocytes (PHHs). UD-UCMSCs, although permissive to viral binding, had a very limited uptake capacity, whereas D-UCMSCs showed binding and uptake capabilities similar to PHHs. Likewise, asialoglycoprotein receptor (ASGPR) was up-regulated in UCMSCs upon differentiation. In D-UCMSCs, a dose-dependent inhibition of HBV binding and uptake was observed when ASGPR was saturated with known specific ligands. Subsequent viral replication was shown in D-UCMSCs but not in UD-UCMSCs. Susceptibility of UCMSCs to viral replication correlated with the degree of differentiation. Replication efficiency was low compared to PHHs, but was confirmed by (1) a dose-dependent inhibition by specific antiviral treatment using tenofovir; (2) the increase of viral RNAs along time; (3) de novo synthesis of viral proteins; and (4) secretion of infectious viral progeny. CONCLUSION: UCMSCs become supportive of the entire HBV life cycle upon in vitro hepatic differentiation. Despite low replication efficiency, D-UCMSCs proved to be fully capable of HBV uptake. Overall, UCMSCs are a unique human, easily available, nontransformed, in vitro model of HBV infection that could prove useful to study early infection events and the role of the cell differentiation state on such events.