Metabolic dysfunction-associated steatotic liver disease (MASLD) is one of the most prevalent liver disorders, affecting approximately one-third of the global adult population. The disease begins with hepatic fat accumulation (steatosis) and can progress to inflammation, fibrosis, and hepatocellular carcinoma. To elucidate the complex mechanisms underlying MASLD, we have developed a novel mathematical model that integrates glucose and lipid metabolisms, oxidative stress, insulin signaling and insulin resistance, and cytokines functions. We demonstrated that variations in extracellular fatty acid and lactate levels, along with changes in the activities of key glycolytic and triglyceride-synthesizing enzymes observed in actual patients, exert a substantial impact on oxidative stress and subsequent cellular damage. Moreover, this model enabled us to evaluate daily metabolic dynamics associated with protein expression patterns specific to steatotic livers. Importantly, it also allowed simulation of cytokine release from hepatocytes into the bloodstream (autocrine and endocrine effects) and the impact of locally elevated cytokines concentrations derived from immune cells (paracrine effects). Our model revealed the dynamics of the early stages of MASLD progression in response to alterations in blood metabolite levels, hepatic enzyme activities, insulin profiles, and cytokine patterns. Furthermore, we identified specific combinations of these factors that may mitigate hepatic fat accumulation or oxidative stress, highlighting the importance of patient-specificity. This study presents the first mechanistic framework grounded in experimental data to describe the crosstalk among hepatic metabolism, insulin, and cytokines, serving as a powerful tool for elucidating disease mechanisms and developing therapeutic strategies.

Hepatic inflammation induced by lipotoxicity is a key event in the progression of liver steatosis to non-alcoholic steatohepatitis (NASH). To characterize this process, we investigated the response of HepaSH cells-a novel mature human hepatocyte derived from chimeric mice-to a weak but chronic palmitic acid (PA) exposure, a prevalent dietary fatty acid. To mimic the hepatic microphysiological environment, cells were cultured using organ-on-chip technology and exposed to PA for two weeks. Functional assays revealed no changes in glucose or lactate metabolism. Likewise, PA exposure did not alter albumin secretion, TGF-β1 or TNF-α production, bile acid synthesis, or CYP450 biotransformation activity. Furthermore, there was no lipid accumulation or change in triglyceride production. However, PA-treated cells cleared the palmitic acid and increased the levels of reactive oxygen species (H₂O₂) and interleukin-6 (IL-6) compared to controls. mRNA analysis concurrently revealed perturbations in transcriptional regulators and genes associated with lipid metabolism, NF-KB, inflammation, and mitochondrial functions. Addtional RT-qPCR confirmed the activation of the JNK1/MAPK8 gene in p38/MAPK and JAK2 in the JAK/STAT/IL-6 pathways in response to PA. We believed that our findings using an advanced mature human hepatic on-chip model would help to provide insight of the early stages of PA-induced lipotoxicity in hepatocytes.

Interactions between the liver and pancreas are key features of the carbohydrate and lipid homeostasis in healthy and pathological patients. To investigate the crosstalk between the two organs, we have developed an organ-on-chip coculture model derived from human induced pluripotent stem cells. The presence of pancreatic-derived tissue in the culture environment contributed to increase the CYP3A4 activity, the glycogen storage, and the expression of genes related to lipids, bile acids and sterol metabolism in the liver derived tissue. Concomitantly, the presence of liver cells led to increase the C-peptide secretion in pancreas. The coculture with liver modulated the pancreatic differentiation by increasing the activity of important transcription factors (REST, MAFB, PBX1) and by downregulating several hormone encoding genes (INS, GCG, TTR). The liver also stimulated the expression of genes involved in the response to inflammation in pancreas (via TGFβ/SMAD pathway). In parallel we observed a pancreatic cell reorganization coupled with the activation of the cell proliferation related transcription factor (SCRT1) and the upregulation of cellular remodeling genes (FLNA, FLNB, FN1, COL4A5). Finally, the pancreatic lipid genes were also upregulated in presence of the liver tissue. Overall, our results reflect a complex synergy between both tissues. We believe that those results are an encouraging step toward the development of relevant human model using advanced organ-on-chip technology and stem cells sources.

Correction for 'Generation of β-like cell subtypes from differentiated human induced pluripotent stem cells in 3D spheroids' by Lisa Morisseau et al., Mol. Omics, 2023, https://doi.org/10.1039/d3mo00050h.

Hepatic zonation is a physiological feature of the liver, known to be key in the regulation of the metabolism of nutrients and xenobiotics and the biotransformation of numerous substances. However, the reproduction of this phenomenon remains challenging in vitro as only part of the processes involved in the orchestration and maintenance of zonation are fully understood. The recent advances in organ‐on‐chip technologies, which allow for the integration of multicellular 3D tissues in a dynamic microenvironment, could offer solutions for the reproduction of zonation within a single culture vessel.

Hepatocyte growth factor (HGF) is the natural ligand of the MET receptor tyrosine kinase. This ligand–receptor couple is essential for the maturation process of hepatocytes. Previously, the rational design of a synthetic protein based on the assembly of two K1 domains from HGF led to the production of a potent and stable MET receptor agonist. In this study, we compared the effects of K1K1 with HGF during the differentiation of hepatocyte progenitors derived from human induced pluripotent stem cells (hiPSCs). In vitro, K1K1, in the range of 20 to 200 nM, successfully substituted for HGF and efficiently activated ERK downstream signaling. Analysis of the levels of hepatocyte markers showed typical liver mRNA and protein expression (HNF4α, albumin, alpha‐fetoprotein, CYP3A4) and phenotypes. Although full maturation was not achieved, the results suggest that K1K1 is an attractive candidate MET agonist suitable for replacing complex and expensive HGF treatments to induce hepatic differentiation of hiPSCs.

The liver zonation is an important phenomenon characterized by a gradient of several functions along the liver acinus. However, this gradient remains difficult to reproduce in in‐vitro conditions, making the obtention of an in‐vitro method to recapitulate the liver zonation a challenging issue. In this study, we evaluated the spatial evolution of the transcriptome profile of human induced pluripotent stem cells (hiPSCs) differentiated toward hepatocytes‐like cells (HLCs) phenotype in a microfluidic biochip environment. Cells collected at the inlet of the biochip, where the oxygen concentration is higher, were identified by the expression of genes involved in metabolic pathways related to cellular reorganization and cell proliferation. Cells collected in the middle and at the outlet of the biochips, where oxygen concentrations are lower, were characterized by the upregulation of genes involved in cellular detoxification processes (CYP450), PPAR signaling or arginine biosynthesis. A subset of 16 transcription factors (TFs) was extracted and identified as upstream regulators to HNF1A and PPARA. These TFs are also known as regulators to target genes engaged in the Wnt/βcatenin pathway, in the TGFβ/BMP/SMAD signaling, in the transition between epithelial mesenchymal transition (EMT) and mesenchymal epithelial transition (MET), in the homeostasis of lipids, bile acids and carbohydrates homeostasis, in drug metabolism, in the estrogen processing and in the oxidative stress response. Overall, the analysis allowed to confirm a partial zonation‐like pattern in hiPSCs‐derived HLCs in the microfluidic biochip environment. These results provide important insights into the reproduction of liver zonation in‐vitro for a better understanding of the phenomenon.

The development of an in vitro functional liver zonation model is a major issue to reproduce physiological liver features. Oxygen concentration is one of the potential explanations of a primary regulating factor of zonation. In this frame, we investigated the oxygen gradient inside a microfluidic device containing rat hepatocyte cultures. The device integrated a platinum (Pt) (II) octaethylporphyrin sensor, allowing a 2D mapping of the oxygen concentration. After 3 hr adhesion of the hepatocytes, the sensor indicated an intense oxygen depletion, leading to an oxygen shortage in the center of the device. After a 30 min perfusion of the culture medium, we monitored the formation of the oxygen gradient along the culture due to cellular respiration. The profile of the oxygen gradient was modulated and controlled by increasing either the perfusion flow rate or the device thickness. In addition, the oxygen gradient was time dependent as far as it decreased with the time of culture. Perivenous and periportal liver patterns were characterized by the immunostaining of the hepatic markers. We put in evidence a spatio temporal hepatic organization. We observed the overexpression since 24 hr of perfusion of the APC and PCK1 proteins upstream in the oxygen-rich area of the device. The overexpression of GS, GCK, CYP1A, and HIFα proteins were observed downstream in the oxygen-poor area. Then, CYP3A2 and β-catenin spatial reorganization was achieved after 48 hr of culture. The results presented a partial zonation-like pattern that was superimposed with an oxygen gradient profile.

In the present study, we evaluated the performance of different protocols for the hepatic differentiation of human-induced pluripotent stem cells (hiPSCs) in microfluidic biochips. Strategies for complete and partial on-chip differentiation were tested. Unlike full on-chip differentiation, the transfer of iPSCs from Petri dishes to biochips during the differentiation process produced a heterogeneous tissue with enhanced hepatic features compared with control cultures in Petri dishes. The tissue in biochips was constituted of cells expressing either stabilin-1 or albumin, while no stabilin-1 was detected in controls. Functional analysis also revealed double the production rate for albumin in biochips (about 2,000 ng per day per 106 cells). Besides this, tissues obtained in biochips and controls exhibited the metabolism of a specific bile acid. Whole transcriptome analysis with nanoCAGE exhibited a differential expression of 302 genes between control and biochip cultures and a higher degree of hepatic differentiation in biochips, together with increased promoter motif activity for typical liver transcription factors such as estrogen related receptor alpha ( ESRRA), hepatic nuclear factor 1 ( HNF1A), hepatic nuclear factor 4 ( HNF4A), transcription factor 4 ( TCF4), and CCAAT enhancer binding protein alpha ( CEBPA). Gene set enrichment analysis identified several pathways related to the extracellular matrix, tissue reorganization, hypoxia-inducible transcription factor, and glycolysis that were differentially modulated in biochip cultures. However, the presence of CK19/ALB-positive cells and the ɑ-fetoprotein levels measured in the cultures still reflect primitive differentiation patterns. Overall, we identified key parameters for improved hepatic differentiation on-chip, including the maturation stage of hepatic progenitors, inoculation density, adhesion time, and perfusion flow rate. Optimization of these parameters further led to establish a protocol for reproducible differentiation of hiPSCs into hepatocyte-like cells in microfluidic biochips with significant improvements over Petri dish cultures.

Microfluidics is the emerging technologies that could bring favorable features to tissue engineering applications. Fundamental techniques to fabricated microfluidic cell-culture devices and experimental attempts towards in vitro liver tissue reconstitution are presented for further discussion on the possible developments in the field of lab-on-a-chip for cellomics.