The poor predictability of human liver toxicity is still causing high attrition rates of drug candidates in the pharmaceutical industry at the non-clinical, clinical, and post-marketing authorization stages. This is in part caused by animal models that fail to predict various human adverse drug reactions (ADRs), resulting in undetected hepatotoxicity at the non-clinical phase of drug development. In an effort to increase the prediction of human hepatotoxicity, different approaches to enhance the physiological relevance of hepatic in vitro systems are being pursued. Three-dimensional (3D) or microfluidic technologies allow to better recapitulate hepatocyte organization and cell-matrix contacts, to include additional cell types, to incorporate fluid flow and to create gradients of oxygen and nutrients, which have led to improved differentiated cell phenotype and functionality. This comprehensive review addresses the drug-induced hepatotoxicity mechanisms and the currently available 3D liver in vitro models, their characteristics, as well as their advantages and limitations for human hepatotoxicity assessment. In addition, since toxic responses are greatly dependent on the culture model, a comparative analysis of the toxicity studies performed using two-dimensional (2D) and 3D in vitro strategies with recognized hepatotoxic compounds, such as paracetamol, diclofenac, and troglitazone is performed, further highlighting the need for harmonization of the respective characterization methods. Finally, taking a step forward, we propose a roadmap for the assessment of drugs hepatotoxicity based on fully characterized fit-for-purpose in vitro models, taking advantage of the best of each model, which will ultimately contribute to more informed decision-making in the drug development and risk assessment fields.

Doxorubicin (Dox) is one of the most widely used treatments for breast cancer, although limited by the well-documented cardiotoxicity and other off-target effects. Mesenchymal stem cell (MSC) secretome has shown immunomodulatory and regenerative properties, further potentiated under 3D conditions. This work aimed to uncover the effect of the MSC-derived secretome from 3D (CM3D) or 2D (CM2D) cultures, in human malignant breast cells (MDA-MB-231), non-tumor breast epithelial cells (MCF10A) and differentiated AC16 cardiomyocytes, co-treated with Dox. A comprehensive proteomic analysis of CM3D/CM2D was also performed to unravel the underlying mechanism. CM3D/CM2D co-incubation with Dox revealed no significant differences in MDA-MB-231 viability when compared to Dox alone, whereas MCF10A and AC16 viability was consistently improved in Dox+CM3D-treated cells. Moreover, neither CM2D nor CM3D affected Dox anti-migratory and anti-invasive effects in MDA-MB-231. Notably, Ge-LC-MS/MS proteomic analysis revealed that CM3D displayed protective features that might be linked to the regulation of cell proliferation (CAPN1, CST1, LAMC2, RANBP3), migration (CCN3, MMP8, PDCD5), invasion (TIMP1/2), oxidative stress (COX6B1, AIFM1, CD9, GSR) and inflammation (CCN3, ANXA5, CDH13, GDF15). Overall, CM3D decreased Dox-induced cytotoxicity in non-tumor cells, without compromising Dox chemotherapeutic profile in malignant cells, suggesting its potential use as a chemotherapy adjuvant to reduce off-target side effects.

The in vitro toxicology field seeks for reliable human relevant hepatic models for predicting xenobiotics metabolism and for the safety assessment of chemicals and developing drugs. The low availability and rapid loss of the phenotype or low biotransformation activity of primary hepatocytes urged the stem cell differentiation into hepatocyte-like cells (HLCs). Umbilical cord-derived mesenchymal stem cells (UC-MSC), in particular, offer a highly available cell source, with few ethical issues and higher genetic stability. However, the dynamic and complex microenvironment of liver development, including the cell-ECM and cell-cell interactions, pressure gradients (oxygen and nutrients) and growth factor signaling that are critical for the differentiation and maturation of hepatocytes, challenges the progress of in vitro hepatic models. Promising strategies like (i) cytokine and growth factor supplementation mimicking the liver development; (ii) epigenetic modification; and (iii) bioengineering techniques to recreate the liver microphysiological environment are gaining increasing importance for the development of relevant in vitro liver models to address the need for higher predictivity and cost efficiency. In this context, this chapter reviews the existing knowledge and recent advances on the approaches for deriving HLCs from UC-MSC and their application for in vitro toxicology.

IntroductionThe development of reliable hepatic in vitro models may provide insights into disease mechanisms, linking hepatocyte dysmetabolism and related pathologies. However, several of the existing models depend on using high concentrations of hepatocyte differentiation-promoting compounds, namely glucose, insulin, and dexamethasone, which is among the reasons that have hampered their use for modeling metabolism-related diseases. This work focused on modulating glucose homeostasis and glucocorticoid concentration to improve the suitability of a mesenchymal stem-cell (MSC)-derived hepatocyte-like cell (HLC) human model for studying hepatic insulin action and disease modeling.MethodsWe have investigated the role of insulin, glucose and dexamethasone on mitochondrial function, insulin signaling and carbohydrate metabolism, namely AKT phosphorylation, glycogen storage ability, glycolysis and gluconeogenesis, as well as fatty acid oxidation and bile acid metabolism gene expression in HLCs. In addition, we evaluated cell morphological features, albumin and urea production, the presence of hepatic-specific markers, biotransformation ability and mitochondrial function.ResultsUsing glucose, insulin and dexamethasone levels close to physiological concentrations improved insulin responsiveness in HLCs, as demonstrated by AKT phosphorylation, upregulation of glycolysis and downregulation of Irs2 and gluconeogenesis and fatty acid oxidation pathways. Ammonia detoxification, EROD and UGT activities and sensitivity to paracetamol cytotoxicity were also enhanced under more physiologically relevant conditions.ConclusionHLCs kept under reduced concentrations of glucose, insulin and dexamethasone presented an improved hepatic phenotype and insulin sensitivity demonstrating superior potential as an in vitro platform for modeling energy metabolism-related disorders, namely for the investigation of the insulin signaling pathway.

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