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Unravelling the therapeutic effect of hnMSCs in an in vitro hepatic disease model of insulin resistance using hepatocyte-like cells and Kupffer cells

Publication . Rodrigues, Joana S.; Miranda, Joana Paiva Gomes; Ruas, Jorge Manuel Lira Gonçalves

Chronic liver diseases represent one of the leading causes of mortality worldwide. In particular, metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common chronic liver disease in the Western world, fuelled by the increasing prevalence of obesity and diabetes. However, an effective therapy able to treat most patients is not yet available. Therefore, understanding disease development to identify potential drug targets is a pressing need. In this context, human stem cell-derived hepatocyte-like cells (HLCs) constitute a promising alternative to human primary hepatocytes and animal models for studying the mechanisms underlying liver disease and for testing the efficacy and safety of drugs or new therapies. However, the use of HLCs as a preclinical tool has been hampered by the immature phenotype obtained with current differentiation protocols. To address these problems, this thesis tested approaches to obtain a functional human-based stem cell-derived HLC model, able to mimic MASLD pathophysiological mechanisms and to evaluate therapeutic candidates. Accordingly, by modulating the concentration of differentiation-promoting compounds, such as glucose, insulin and dexamethasone, we could achieve a more physiologically relevant model with improved HLC biotransformation capacity and insulin responsiveness. Furthermore, HLC adaptation to a fit-for-purpose microfluidic culture system, constituting a microphysiological system, enhanced the degree of differentiation and drug metabolism capacity, demonstrated by the increased presence of hepatic-specific markers and production of diclofenac glucuronidation metabolites. Moreover, the establishment of insulin responsive HLCs set up the grounds for the development of a MASLD model. Thus, by exposing HLCs to oleic and palmitic acids, we could observe lipid accumulation, activation of gluconeogenesis, synthesis of lipotoxic species, such as ceramides and diacylglycerol, increased reactive oxygen species production and mitochondrial dysfunction along with induction of lipogenesis and gradual decrease in the antioxidant response. Additionally, considering that MASLD onset is correlated with liver zonation, in particular, perivenous (PV) hepatocytes, we explored the role of the hypoxia mimetic dimethyloxalylglycine (DMOG) in creating a PV-like HLC subpopulation. Of note, DMOG was more effective in inducing the expression of genes associated with PV-specific functions than exposing HLCs to 5% pO2. In addition, HLCs treated with DMOG presented signs of faster MASLD development, namely lower expression of genes involved in fatty acid oxidation and antioxidant response along with higher ceramide production levels. Thus, this platform not only presents itself as an alternative for the implementation of PV hepatocytes, but it could be further explored for the identification of biomarkers enabling earlier MASLD diagnosis. Lastly, the incorporation of the immune response in this hepatic model, by co-culturing HLCs and THP-1-derived macrophages, provided evidence on the therapeutic effect of mesenchymal stem cells’ secretome at the metabolic and immunomodulatory levels. Overall, this thesis demonstrated that recapitulating liver microenvironment is fundamental for unravelling the full potential of stem cell-derived HLCs for metabolic disease modelling, drug screening and drug metabolism assessment. Importantly, the therapeutic properties of the MSC secretome in MASLD setting should be evaluated in more detail.

2024-10Doctoral thesis

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Improving human mesenchymal stem cell-derived hepatic cell energy metabolism by manipulating glucose homeostasis and glucocorticoid signaling

Publication . Rodrigues, Joana S.; Faria-Pereira, Andreia; Camões, Sérgio P.; Serras, Ana S.; Morais, Vanessa A.; Ruas, Jorge Lira; Miranda, Joana P

Introduction: The 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. Methods: We 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. Results: Using 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. Conclusion: HLCs 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.

2023Journal article

Open access