Hepatoblastomas exhibit marked NNMT downregulation driven by promoter DNA hypermethylation.

Hepatoblastomas exhibit the lowest mutational burden among pediatric tumors. We previously showed that epigenetic disruption is crucial for hepatoblastoma carcinogenesis. Our data revealed hypermethylation of nicotinamide N-methyltransferase, a highly expressed gene in adipocytes and hepatocytes. The expression pattern and the role of nicotinamide N-methyltransferase in pediatric liver tumors have not yet been explored, and this study aimed to evaluate the effect of nicotinamide N-methyltransferase hypermethylation in hepatoblastomas. We evaluated 45 hepatoblastomas and 26 non-tumoral liver samples. We examined in hepatoblastomas if the observed nicotinamide N-methyltransferase promoter hypermethylation could lead to dysregulation of expression by measuring mRNA and protein levels by real-time quantitative polymerase chain reaction, immunohistochemistry, and Western blot assays. The potential impact of nicotinamide N-methyltransferase changes was evaluated on the metabolic profile by high-resolution magic angle spinning nuclear magnetic resonance spectroscopy. Significant nicotinamide N-methyltransferase downregulation was revealed in hepatoblastomas, with two orders of magnitude lower nicotinamide N-methyltransferase expression in tumor samples and hepatoblastoma cell lines than in hepatocellular carcinoma cell lines. A specific TSS1500 CpG site (cg02094283) of nicotinamide N-methyltransferase was hypermethylated in tumors, with an inverse correlation between its methylation level and nicotinamide N-methyltransferase expression. A marked global reduction of the nicotinamide N-methyltransferase protein was validated in tumors, with strong correlation between gene and protein expression. Of note, higher nicotinamide N-methyltransferase expression was statistically associated with late hepatoblastoma diagnosis, a known clinical variable of worse prognosis. In addition, untargeted metabolomics analysis detected aberrant lipid metabolism in hepatoblastomas. Data presented here showed the first evidence that nicotinamide N-methyltransferase reduction occurs in hepatoblastomas, providing further support that the nicotinamide N-methyltransferase downregulation is a wide phenomenon in liver cancer. Furthermore, this study unraveled the role of DNA methylation in the regulation of nicotinamide N-methyltransferase expression in hepatoblastomas, in addition to evaluate the potential effect of nicotinamide N-methyltransferase reduction in the metabolism of these tumors. These preliminary findings also suggested that nicotinamide N-methyltransferase level may be a potential prognostic biomarker for hepatoblastoma.

iPSC-derived cells for whole liver bioengineering.

Liver bioengineering stands as a prominent alternative to conventional hepatic transplantation. Through liver decellularization and/or bioprinting, researchers can generate acellular scaffolds to overcome immune rejection, genetic manipulation, and ethical concerns that often accompany traditional transplantation methods, in vivo regeneration, and xenotransplantation. Hepatic cell lines derived from induced pluripotent stem cells (iPSCs) can repopulate decellularized and bioprinted scaffolds, producing an increasingly functional organ potentially suitable for autologous use. In this mini-review, we overview recent advancements in vitro hepatocyte differentiation protocols, shedding light on their pivotal role in liver recellularization and bioprinting, thereby offering a novel source for hepatic transplantation. Finally, we identify future directions for liver bioengineering research that may allow the implementation of these systems for diverse applications, including drug screening and liver disease modeling.

Applied Hepatic Bioengineering: Modeling the Human Liver Using Organoid and Liver-on-a-Chip Technologies.

The liver is the most important metabolic hub of endo and xenobiotic compounds. Pre-clinical studies using rodents to evaluate the toxicity of new drugs and cosmetics may produce inconclusive results for predicting clinical outcomes in humans, moreover being banned in the European Union. Human liver modeling using primary hepatocytes presents low reproducibility due to batch-to-batch variability, while iPSC-derived hepatocytes in monolayer cultures (2D) show reduced cellular functionality. Here we review the current status of the two most robust in vitro approaches in improving hepatocyte phenotype and metabolism while mimicking the hepatic physiological microenvironment: organoids and liver-on-chip. Both technologies are reviewed in design and manufacturing techniques, following cellular composition and functionality. Furthermore, drug screening and liver diseases modeling efficiencies are summarized. Finally, organoid and liver-on-chip technologies are compared regarding advantages and limitations, aiming to guide the selection of appropriate models for translational research and the development of such technologies.

Pre-coating decellularized liver with HepG2-conditioned medium improves hepatic recellularization.

Liver transplantation from compatible donors has been the main therapy available for patients with irreversible hepatic injuries. Due to the increasing shortage of organs suitable for transplantation, tissue engineering technologies are important alternatives or surrogate approaches for the future of human organ transplantations. New bioengineering tools have been designed to produce decellularized organs (i.e. scaffolds) which could be recellularized with human cells. Specifically, there is an unmet need for developing reproducible protocols for inducing better cellular spreading in decellularized liver scaffolds. The aim of the present work was to investigate the possibility to improve liver scaffold recellularization by pre-coating decellularized tissue scaffolds with HepG2-conditioned medium (CM). Furthermore, we evaluated the capability of commercial human liver cells (HepG2) to adhere to several types of extracellular matrices (ECM) as well as CM components. Wistar rat livers were decellularized and analyzed by histology, scanning electron microscopy (SEM), immunohistochemistry and residual DNA-content analysis. Human induced pluripotent stem cells (hiPSCs)-derived mesenchymal cells (hiMSCs), and human commercial hepatic (HepG2) and endothelial (HAEC) cells were used for liver scaffold recellularization with or without CM pre-coating. Recellularization occurred for up to 5 weeks. Hepatic tissues and CM were analyzed by proteomic assays. We show that integrity and anatomical organization of the hepatic ECM were maintained after decellularization, and proteomic analysis suggested that pre-coating with CM enriched the decellularized liver ECM. Pre-coating with HepG2-CM highly improved liver recellularization and revealed the positive effects of liver ECM and CM components association.

Adult and iPS-derived non-parenchymal cells regulate liver organoid development through differential modulation of Wnt and TGF-ß.

BACKGROUND: Liver organoid technology holds great promises to be used in large-scale population-based drug screening and in future regenerative medicine strategies. Recently, some studies reported robust protocols for generating isogenic liver organoids using liver parenchymal and non-parenchymal cells derived from induced pluripotent stem cells (iPS) or using isogenic adult primary non-parenchymal cells. However, the use of whole iPS-derived cells could represent great challenges for a translational perspective. METHODS: Here, we evaluated the influence of isogenic versus heterogenic non-parenchymal cells, using iPS-derived or adult primary cell lines, in the liver organoid development. We tested four groups comprised of all different combinations of non-parenchymal cells for the liver functionality in vitro. Gene expression and protein secretion of important hepatic function markers were evaluated. Additionally, liver development-associated signaling pathways were tested. Finally, organoid label-free proteomic analysis and non-parenchymal cell secretome were performed in all groups at day 12. RESULTS: We show that liver organoids generated using primary mesenchymal stromal cells and iPS-derived endothelial cells expressed and produced significantly more albumin and showed increased expression of CYP1A1, CYP1A2, and TDO2 while presented reduced TGF-ß and Wnt signaling activity. Proteomics analysis revealed that major shifts in protein expression induced by this specific combination of non-parenchymal cells are related to integrin profile and TGF-ß/Wnt signaling activity. CONCLUSION: Aiming the translation of this technology bench-to-bedside, this work highlights the role of important developmental pathways that are modulated by non-parenchymal cells enhancing the liver organoid maturation.

3D bioprinting of liver spheroids derived from human induced pluripotent stem cells sustain liver function and viability in vitro.

The liver is responsible for many metabolic, endocrine and exocrine functions. Approximately 2 million deaths per year are associated with liver failure. Modern 3D bioprinting technologies allied with autologous induced pluripotent stem cells (iPS)-derived grafts could represent a relevant tissue engineering approach to treat end stage liver disease patients. However, protocols that accurately recapitulates liver's epithelial parenchyma through bioprinting are still underdeveloped. Here we evaluated the impacts of using single cell dispersion (i.e. obtained from conventional bidimensional differentiation) of iPS-derived parenchymal (i.e. hepatocyte-like cells) versus using iPS-derived hepatocyte-like cells spheroids (i.e. three-dimensional cell culture), both in combination with non-parenchymal cells (e.g. mesenchymal and endothelial cells), into final liver tissue functionality. Single cell constructs showed reduced cell survival and hepatic function and unbalanced protein/amino acid metabolism when compared to spheroid printed constructs after 18 days in culture. In addition, single cell printed constructs revealed epithelial-mesenchymal transition, resulting in rapid loss of hepatocyte phenotype. These results indicates the advantage of using spheroid-based bioprinting, contributing to improve current liver bioprinting technology towards future regenerative medicine applications and liver physiology and disease modeling.

TET Upregulation Leads to 5-Hydroxymethylation Enrichment in Hepatoblastoma.

Hepatoblastoma is an embryonal liver tumor carrying few genetic alterations. We previously disclosed in hepatoblastomas a genome-wide methylation dysfunction, characterized by hypermethylation at specific CpG islands, in addition to a low-level hypomethylation pattern in non-repetitive intergenic sequences, in comparison to non-tumoral liver tissues, shedding light into a crucial role for epigenetic dysregulation in this type of cancer. To explore the underlying mechanisms possibly related to aberrant epigenetic modifications, we evaluated the expression profile of a set of genes engaged in the epigenetic machinery related to DNA methylation (DNMT1, DNMT3A, DNMT3B, DNMT3L, UHRF1, TET1, TET2, and TET3), as well as the 5-hydroxymethylcytosine (5hmC) global level. We observed in hepatoblastomas a general disrupted expression of these genes from the epigenetic machinery, mainly UHRF1, TET1, and TET2 upregulation, in association with an enrichment of 5hmC content. Our findings support a model of active demethylation by TETs in hepatoblastoma, probably during early stages of liver development, which in combination with UHRF1 overexpression would lead to DNA hypomethylation and an increase in overall 5hmC content. Furthermore, our data suggest that decreased 5hmC content might be associated with poor survival rate, highlighting a pivotal role of epigenetics in hepatoblastoma development and progression.

Publisher Correction: Discordant congenital Zika syndrome twins show differential in vitro viral susceptibility of neural progenitor cells.

The original PDF version of this Article contained errors in the spelling of Luiz Carlos Caires-Júnior, Uirá Souto Melo, Bruno Henrique Silva Araujo, Alessandra Soares-Schanoski, Murilo Sena Amaral, Kayque Alves Telles-Silva, Vanessa van der Linden, Helio van der Linden, João Ricardo Mendes de Oliveira, Nivia Maria Rodrigues Arrais, Joanna Goes Castro Meira, Ana Jovina Barreto Bispo, Esper Abrão Cavalheiro, and Robert Andreata-Santos, which were incorrectly given as Luiz Carlos de Caires Jr., UiráSouto Melo, Bruno Silva Henrique Araujo, Alessandra Soares Schanoski, MuriloSena Amaral, Kayque Telles Alves Silva, Vanessa Van der Linden, Helio Van der Linden, João Mendes Ricardo de Oliveira, Nivia Rodrigues Maria Arrais, Joanna Castro Goes Meira, Ana JovinaBarreto Bispo, EsperAbrão Cavalheiro, and Robert Andreata Santos. Furthermore, in both the PDF and HTML versions of the Article, the top panel of Fig. 3e was incorrectly labeled '10608-1' and should have been '10608-4', and financial support from CAPES and DECIT-MS was inadvertently omitted from the Acknowledgements section. These errors have now been corrected in both the PDF and HTML versions of the Article.

Discordant congenital Zika syndrome twins show differential in vitro viral susceptibility of neural progenitor cells.

Congenital Zika syndrome (CZS) causes early brain development impairment by affecting neural progenitor cells (NPCs). Here, we analyze NPCs from three pairs of dizygotic twins discordant for CZS. We compare by RNA-Seq the NPCs derived from CZS-affected and CZS-unaffected twins. Prior to Zika virus (ZIKV) infection the NPCs from CZS babies show a significantly different gene expression signature of mTOR and Wnt pathway regulators, key to a neurodevelopmental program. Following ZIKV in vitro infection, cells from affected individuals have significantly higher ZIKV replication and reduced cell growth. Whole-exome analysis in 18 affected CZS babies as compared to 5 unaffected twins and 609 controls excludes a monogenic model to explain resistance or increased susceptibility to CZS development. Overall, our results indicate that CZS is not a stochastic event and depends on NPC intrinsic susceptibility, possibly related to oligogenic and/or epigenetic mechanisms.