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International audience; The liver is a complex organ composed of several cell types organized hierarchically. Among these, liver sinusoidal endothelial cells (LSECs) are specialized vascular cells known to interact with hepatocytes and hepatic stellate cells (HSCs), and to be involved in the regulation of important hepatic processes in healthy and pathological situations. Protocols for the differentiation of LSECs from human induced pluripotent stem cells, hiPSCs, have been proposed and in-depth analysis by transcriptomic profiling of those cells has been performed. In the present work, an extended analysis of those cells in terms of proteome and metabolome has been implemented. The proteomic analysis confirmed the expression of important endothelial markers and pathways. Among them, the expression of patterns typical of LSECs such as PECAM1, VWF, LYVE1, STAB1 (endothelial markers), CDH13, CDH5, CLDN5, ICAM1, MCAM-CD146, ICAM2, ESAM (endothelial cytoskeleton), NOSTRIN, NOS3 (Nitric Oxide endothelial ROS), ESM1, ENG, MMRN2, THBS1, ANGPT2 (angiogenesis), CD93, MRC1 (mannose receptor), CLEC14A (C-type lectin), CD40 (antigen), and ERG (transcription factor) was highlighted. Besides, the pathway analysis revealed the enrichment of the endocytosis, Toll-like receptor, Nod-like receptor, Wnt, Apelin, VEGF, cGMP-PCK, and PPAR related signaling pathways. Other important pathways such as vasopressin regulated water reabsorption, fluid shear stress, relaxin signaling, and renin secretion were also highlighted. At confluence, the metabolome profile appeared consistent with quiescent endothelial cell patterns. The integration of both proteome and metabolome datasets revealed a switch from fatty acid synthesis in undifferentiated hiPSCs to a fatty oxidation in LSECs and activation of the pentose phosphate pathway and polyamine metabolism in hiPSCs-derived LSECs. In conclusion, the comparison between the signature of LSECs differentiated following the protocol described in this work, and data found in the literature confirmed the particular relevance of these cells for future in vitro applications.

As a novel method of ivermectin (IVM) administration for the treatment of scabies, we devised a whole-body bathing (WBB), in which patients are immersed in a fluid that contains IVM. A multi-institutional trial for elderly patients with scabies was conducted to investigate the efficacy and safety of IVM-WBB. Seven elderly patients with scabies were enrolled and received IVM-WBB up to four times at 1-week interval. The cure for scabies was defined as the absence of mites in two consecutive microscopic or dermoscopic examinations at weekly intervals and the absence of new skin lesions indicative of scabies. Consequently, the cure rate on day 22, the primary end-point, was 71.4%, and all patients had been cured until day 29. Additionally, neither significant adverse events nor clinically problematic abnormal blood test values were obtained. Furthermore, no IVM was detected in the optional plasma (five cases) collected for IVM measurement after bathing. These results suggest that IVM-WBB was effective to treat scabies, causing no serious adverse events and with a very low internal exposure of IVM.

Objectives The combination therapy of phenothrin (PHT) and ivermectin (PI regimen) was effective for pyrethroid‐resistant head lice in Okinawa; however, further investigations have been required to improve the regimen. This study aimed to investigate the efficacy of the modified PI (mPI) regimen, which increased the maximum number of PHT treatments in the PI regimen by one, in Tsukuba, which has a low mutation rate compared to Okinawa. Methods An open‐label, uncontrolled, exploratory study was conducted at the Department of Dermatology, University of Tsukuba Hospital to investigate the treatment effect of mPI regimen. Genotypes were investigated for the knockdown (kdr) mutations at voltage‐sensitive sodium channel (VSSC) and glutamate‐gated chloride receptor‐encoding loci to identify their correlation with drug resistance. Results All the 8 patients treated with the mPI regimen resulted in lice free on day 29. Compared to baseline, the number of bodies, eggs, and itching grade significantly decreased on day 29. Mild erythema appeared after the second application of PHT on day 8 in one patient, persisted for 2 days, and recovered on day 15. No other adverse events were reported. Mutation rates were more than double in head lice collected from one patient for whom PHT was ineffective compared to those in lice from patients who responded to PHT. Furthermore, we identified four new haplotypes of VSSC loci in head lice. Conclusions The mPI regimen is a potential effective treatment for economically combating both PHT‐sensitive and resistant head lice.

Oncostatin M (OSM), a member of the IL-6 family of cytokines, has important roles in renal diseases. The relationship between OSM and kidney stone disease, however, remains unclear. To investigate the roles of OSM in the development of kidney stone disease, we generated a mouse model of renal crystal formation using OSM receptor β (OSMRβ)-deficient mice (OSMRβ−/− mice). There were fewer renal crystal deposits in OSMRβ−/− mice than in wild-type (WT) mice. Crystal-binding molecules (osteopontin, annexin A1, and annexin A2), inflammatory cytokines (TNF-α and IL-1β), and fibrosis markers (TGF-β, collagen 1a2, and α-smooth muscle actin) were also decreased in the kidneys of OSMRβ−/− mice compared with those in WT mice. Immunofluorescence staining showed that OSMRβ was expressed in renal tubular epithelial cells (RTECs) and renal fibroblasts in the model of renal crystal formation. In the cultured RTECs and renal fibroblasts, OSM directly induced the expression of crystal-binding molecules and fibrosis markers. Expressions of inflammatory cytokines were increased by stimulation with OSM in cultured renal fibroblasts. OSM may promote the formation of renal crystal deposits by directly acting on RTECs and renal fibroblasts to produce crystal-binding molecules and inflammatory cytokines.

Laminin is a family of basement membrane proteins, whose selective and spatiotemporal expression profiles are linked to their various functions in development, maintenance, and functional regulation of different tissues. In the liver, α1-and α5-containing laminin isoforms have been documented to be critically involved in the developmental process of the epithelial tissue of the bile duct. However, possible roles of other laminin isoforms in bile duct formation and function remain elusive. Here, we evaluated public single-cell RNA sequencing databases on human liver cells to reveal expression landscape of laminin genes, and found that genes for laminin-332 subunits were conjointly expressed in the EPCAM+ biliary epithelial cell population. Expression of the β3 and γ2 subunit genes was restricted to biliary epithelial cells in the liver and, remarkably, showed apparent heterogeneity among them. We confirmed the heterogeneous nature of the laminin-β3 expression in murine livers, which was firmly related to morphological substructures in the biliary epithelium. Finally, we generated the liver epithelial tissue-specific laminin- β3 knockout mice and found that this laminin subunit was dispensable under physiological conditions. Together, our present findings have identified the β3 subunit and the related laminin-332 isoform as useful markers and potentially important regulatory molecules for future understanding of pathophysiology in the hepatobiliary system.

Liver Sinusoidal Endothelial Cells (LSECs) are an important component of the liver as they compose the microvasculature which allows the supply of oxygen, blood, and nutrients. However, maintenance of these cells in vitro remains challenging as they tend to rapidly lose some of their characteristics such as fenestration or as their immortalized counterparts present poor characteristics. In this work, human induced pluripotent stem cells (hiPSCs) have been differentiated toward an LSEC phenotype. After differentiation, the RNA quantification allowed demonstration of high expression of specific vascular markers (CD31, CD144, and STAB2). Immunostaining performed on the cells was found to be positive for both Stabilin-1 and Stabilin-2. Whole transcriptome analysis performed with the nanoCAGE method further confirmed the overall vascular commitment of the cells. The gene expression profile revealed the upregulation of the APLN, LYVE1, VWF, ESAM and ANGPT2 genes while VEGFA appeared to be downregulated. Analysis of promoter motif activities highlighted several transcription factors (TFs) of interest in LSECs (IRF2, ERG, MEIS2, SPI1, IRF7, WRNIP1, HIC2, NFIX_NFIB, BATF, and PATZ1). Based on this investigation, we compiled the regulatory network involving the relevant TFs, their target genes as well as their related signaling pathways. The proposed hiPSC-derived LSEC model and its regulatory network were then confirmed by comparing the experimental data to primary human LSEC reference datasets. Thus, the presented model appears as a promising tool to generate more complex in vitro liver multi-cellular tissues.

/ Introductory paragraphTubular epithelial tissues in the body play fundamental roles as infrastructure constituting conduits to transport various types of biological fluids, for which contiguous and integrated epithelial tissue structures should be maintained continuously and even under stressed conditions. Compared to tissue morphological processes that take place during ontogeny, the mechanisms whereby tubular epithelial tissues maintain their structural integrity in adulthood remains largely unclear. Here, we show that the transcription factor Klf5 is crucial for maintaining the biliary epithelial integrity in tissue remodeling processes induced under cholestatic injury conditions in the adult liver. Loss of Klf5 in the biliary epithelia led to tissue collapse in vivo in injured mouse livers, as well as in vitro in bile ductular organoids in a tissue-autonomous manner and independent of cell proliferation. Klf5 regulated cell junction organization and cell adhesion, along with extracellular matrix remodeling around the expanding biliary epithelia through deposition of Lamb3-containing laminin complexes. Targeting the Lamb3 expression in biliary epithelia in mice recapitulated the tissue collapse phenotype. Together, our results highlight a novel mechanism whereby the epithelial tissue maintains its integrity while undergoing unstable structural transformation.

Number of liver organoids have been reported, though it is not clearly shown whether the functional connection between hepatocytes and cholangiocytes is recapitulated in those organoids. Here, we report generation of a hepatobiliary tubular organoid (HBTO) using mouse hepatocyte progenitors called small hepatocytes (SHs) and cholangiocytes. SHs differentiate and form the bile canalicular network in HBTOs and secret metabolites into the canaliculi, which are then transported into the biliary structure. Hepatocytes in the organoid acquire and maintain metabolic functions including albumin secretion and cytochrome P450 activities, over the long term. We provide the step-by-step protocol for induction of HBTO including isolation of cholangiocytes and SHs and co-culture of these two types of cell to generate functional connections between hepatocytes and cholangiocytes.

Notch signaling is one of the most common drivers of carcinogenesis in many types of cancers, including hepatocellular carcinoma (HCC); however, it occasionally suppresses tumor progression. Moreover, it is virtually unknown how different sets of Notch ligands and receptors regulate the HCC development. In this study, we demonstrate that the expression of the Notch ligands, Delta-like 4 (Dll4) and Jagged-1 (Jag1), is upregulated during diethylnitrosamine-induced hepatocarcinogenesis. Dll4 is detected in the preneoplastic hepatocytes and HCC cells, but not in the normal hepatocytes, while Jag1 is expressed in the desmin-positive mesenchymal cells. Hepatocyte-specific Dll4 knockout abolishes the Notch1 signaling and suppresses the tumor progression. In contrast, Jag1 deletion induces the ectopic expression of Dll4 in hepatocytes along with the loss of Notch2 signaling, leading to the tumor progression. These results indicate that the two distinct Notch signals, Dll4/Notch1 and Jag1/Notch2, are antagonistic to each other, exerting opposite effects on HCC progression., Dll4/Notch1 signal promotes the progression of HCC, while Jag1/Notch2 signal antagonistically suppresses it in murine chemical hepatocarcinogenesis. Nakano et al. report that two distinct Notch signals regulate the progression of hepatocellular carcinoma (HCC) using tissue specific loss of function mouse mutants. They find Dll4/Notch1 signal promotes HCC progression, while the Jag1/Notch2 signal antagonistically suppresses it.

Mesenchymal stromal cells (MSCs) are used for ameliorating liver fibrosis and aiding liver regeneration after cirrhosis; Here, we analyzed the therapeutic potential of small extracellular vesicles (sEVs) derived from interferon-γ (IFN-γ) pre-conditioned MSCs (γ-sEVs). γ-sEVs effectively induced anti-inflammatory macrophages with high motility and phagocytic abilities in vitro, while not preventing hepatic stellate cell (HSC; the major source of collagen fiber) activation in vitro. The proteome analysis of MSC-derived sEVs revealed anti-inflammatory macrophage inducible proteins (e.g., annexin-A1, lactotransferrin, and aminopeptidase N) upon IFN-γ stimulation. Furthermore, by enabling CX3CR1+ macrophage accumulation in the damaged area, γ-sEVs ameliorated inflammation and fibrosis in the cirrhosis mouse model more effectively than sEVs. Single cell RNA-Seq analysis revealed diverse effects, such as induction of anti-inflammatory macrophages and regulatory T cells, in the cirrhotic liver after γ-sEV administration. Overall, IFN-γ pre-conditioning altered sEVs resulted in efficient tissue repair indicating a new therapeutic strategy.

International audience; Maturation of human-induced pluripotent stem cells (hiPSCs)-derived hepatocytes-like cells (HLCs) toward a complete hepatocyte phenotype remains a challenge as primitiveness patterns are still commonly observed. In this study, we propose a modified differentiation protocol for those cells which includes a prematuration in Petri dishes and a maturation in microfluidic biochip. For the first time, a large range of biomolecular families has been extracted from the same sample to combine transcriptomic, proteomic, and metabolomic analysis. After integration, these datasets revealed specific molecular patterns and highlighted the hepatic regeneration profile in biochips. Overall, biochips exhibited processes of cell proliferation and inflammation (via TGFB1) coupled with anti-fibrotic signaling (via angiotensin 1-7, ATR-2, and MASR). Moreover, cultures in this condition displayed physiological lipid-carbohydrate homeostasis (notably via PPAR, cholesterol metabolism, and bile synthesis) coupled with cell respiration through advanced oxidative phosphorylation (through the overexpression of proteins from the third and fourth complex). The results presented provide an original overview of the complex mechanisms involved in liver regeneration using an advanced in vitro organ-on-chip technology.

It is important to predict drug-drug interactions via inhibition of intestinal cytochrome P450 3A (CYP3A) which is a determinant of bioavailability of orally administered CYP3A substrates. However, inhibitory effects of macrolide antibiotics on CYP3A-mediated metabolism are not entirely identical between humans and rodents.We investigated the effects of macrolide antibiotics, clarithromycin and erythromycin, on in vitro and in vivo metabolism of triazolam, a CYP3A substrate, in CYP3A-humanised mice generated by using a mouse artificial chromosome vector carrying a human CYP3A gene.Metabolic activities of triazolam were inhibited by macrolide antibiotics in liver and intestine microsomes of CYP3A-humanised mice.The area under the plasma concentration-time curve ratios of 4-hydroxytriazolam to triazolam after oral dosing of triazolam were significantly decreased by multiple administration of macrolide antibiotics. The plasma concentrations ratios of α-hydroxytriazolam and 4-hydroxytriazolam to triazolam in portal blood were significantly decreased by multiple administration of clarithromycin in CYP3A-humanised mice.These results suggest that intestinal CYP3A activity was inhibited by macrolide antibiotics in CYP3A-humanised mice in vitro and in vivo. The plasma concentrations of triazolam and its metabolites in the portal blood of CYP3A-humanised mice would be useful for direct evaluation of intestinal CYP3A-mediated drug-drug interactions. It is important to predict drug-drug interactions via inhibition of intestinal cytochrome P450 3A (CYP3A) which is a determinant of bioavailability of orally administered CYP3A substrates. However, inhibitory effects of macrolide antibiotics on CYP3A-mediated metabolism are not entirely identical between humans and rodents. We investigated the effects of macrolide antibiotics, clarithromycin and erythromycin, on in vitro and in vivo metabolism of triazolam, a CYP3A substrate, in CYP3A-humanised mice generated by using a mouse artificial chromosome vector carrying a human CYP3A gene. Metabolic activities of triazolam were inhibited by macrolide antibiotics in liver and intestine microsomes of CYP3A-humanised mice. The area under the plasma concentration-time curve ratios of 4-hydroxytriazolam to triazolam after oral dosing of triazolam were significantly decreased by multiple administration of macrolide antibiotics. The plasma concentrations ratios of α-hydroxytriazolam and 4-hydroxytriazolam to triazolam in portal blood were significantly decreased by multiple administration of clarithromycin in CYP3A-humanised mice. These results suggest that intestinal CYP3A activity was inhibited by macrolide antibiotics in CYP3A-humanised mice in vitro and in vivo. The plasma concentrations of triazolam and its metabolites in the portal blood of CYP3A-humanised mice would be useful for direct evaluation of intestinal CYP3A-mediated drug-drug interactions.

In the liver, the bile canaliculi of hepatocytes are connected to intrahepatic bile ducts lined with cholangiocytes, which remove cytotoxic bile from the liver tissue. We have developed a hepatobiliary organoid using mouse hepatocyte progenitors and cholangiocytes. Hepatocyte metabolites were secreted into the bile canaliculi, and then transported into the biliary structure. Hepatocytes in the organoid acquired and maintained metabolic functions including albumin secretion and cytochrome P450 activities, over the long term. In this study, we established functional liver tissue incorporating a bile drainage system ex vivo. This hepatobiliary organoid enabled us to reproduce the transport of hepatocyte metabolites in liver tissue, and to investigate the way in which the two types of epithelial cells establish functional connections.

International audience; Liver modeling in disease via advanced in vitro physiological tissues remains a challenging issue, yet crucial for the future development of relevant tools for drug screening. In that regard, advanced technics such as human induced Pluripotent Stem Cells (hiPSCs) and organ-on-chip are promising technologies for the relevant reproduction of the in vivo micro physiology in in vitro culture conditions. In the present work, the maturation of carboxypeptidase M positive (CPM+) Hepatocyte-Like-Cells (HLCs) in microfluidic culture conditions was investigated. hiPSCs were differentiated in culture dishes until sorting and further amplified until seeding in biochips. After 2 weeks of maturation in biochips, evaluation of the cell metabolism was performed, and samples were collected for characterization via RNA sequencing, proteomics and metabolomics. The omics profile of biochips loaded with CPM+ HLCs was then compared with the one of previously cultured biochips loaded with unsorted HLCs. CPM+ HLCs presented advanced liver characteristics in terms of a higher activity of important Transcriptions Factors (TFs) such as HNF1, HNF4A, and CEBP/A, of the upregulation of steroids/corticoids-related nuclear receptors (FXR, NR3C2, NR4A1 genes), of phase I, and phase II metabolism genes (CYP1A1, CYP1B1, CYP2C18, CYP27A1, several UGT, SULT and GST genes). Further differences in the cellular reorganization, in the lipids and steroids metabolisms, in the OXPHOS respiration and in the expression of TGFβ signaling were also observed. The present study introduces a novel protocol using advances hiPSCs differentiation and sorting methods as well as the organ-on-chip technology to highlight important in vitro liver regeneration and hepatic maturation processes.

Hepatocytes derived from human iPSCs are useful to study hepatitis B virus (HBV) infection, however infection efficiency is rather poor. In order to improve the efficiency of HBV infection to iPSC-derived hepatocytes, we set a co-culture of hepatocytes with liver non-parenchymal cells and found that liver sinusoidal endothelial cells (LSECs) enhanced HBV infection by secreting epidermal growth factor (EGF). While EGF receptor (EGFR) is known as a co-receptor for HBV, we found that EGF enhanced HBV infection at a low dose of EGF, whereas EGF at a high dose suppressed HBV infection. EGFR is internalized by clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) pathways depending on the dose of EGF. At a high dose of EGF, the endocytosed EGFR via CIE is degraded in the lysosome. This study is the first to provide evidence that HBV is endocytosed via CME and CIE pathways at a low and high dose of EGF, respectively. In conclusion, we developed an in vitro system of HBV infection using iPSC-derived liver cells, and show that EGF secreted from LSECs modulates HBV infection in a dose dependent manner.

Cell senescence plays a key role in age-associated organ dysfunction, but the in vivo pathogenesis is largely unclear. Here, we generated a p16-Cre

The capability to produce and maintain functional human adult hepatocytes remains one of the major challenges for the use of in-vitro models toward liver cell therapy and industrial drug-screening applications. Among the suggested strategies to solve this issue, the use of human-induced pluripotent stem cells (hiPSCs), differentiated toward hepatocyte-like cells (HLCs) is promising. In this work, we propose a 31-day long protocol, that includes a final 14-day long phase of oncostatin treatment, as opposed to a 7-day treatment which led to the formation of a hepatic tissue functional for CYP1A2, CYP2B6, CYP2C8, CYP2D6, and CYP3A4. The production of albumin, as well as bile acid metabolism and transport, were also detected. Transcriptome profile comparisons and liver transcription factors (TFs) motif dynamics revealed increased expression of typical hepatic markers such as HNF1A and of important metabolic markers like PPARA. The performed analysis has allowed for the extraction of potential targets and pathways which would allow enhanced hepatic maturation in-vitro. From this investigation, NRF1 and SP3 appeared as transcription factors of importance. Complex epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) patterns were also observed during the differentiation process. Moreover, whole transcriptome analysis highlighted a response typical of the one observed in liver regeneration and hepatocyte proliferation. While a complete maturation of hepatocytes was yet to be obtained, the results presented in this work provide new insights into the process of liver development and highlight potential targets aimed to improve in-vitro liver regeneration.

Upon severe and/or chronic liver injury, ectopic emergence and expansion of atypical biliary epithelial-like cells in the liver parenchyma, known as the ductular reaction, is typically induced and implicated in organ regeneration. Although this phenomenon has long been postulated to represent activation of facultative liver stem/progenitor cells that give rise to new hepatocytes, recent lineage-tracing analyses have challenged this notion, thereby leaving the pro-regenerative role of the ductular reaction enigmatic. Here, we show that the expanded and remodelled intrahepatic biliary epithelia in the ductular reaction constituted functional and complementary bile-excreting conduit systems in injured parenchyma where hepatocyte bile canalicular networks were lost. The canalicular collapse was an incipient defect commonly associated with hepatocyte injury irrespective of cholestatic statuses, and could sufficiently provoke the ductular reaction when artificially induced. We propose a unifying model for the induction of the ductular reaction, where compensatory biliary epithelial tissue remodeling ensures bile-excreting network homeostasis., Kenji Kamimoto et al. use multidimensional imaging technologies to study changes in the mouse biliary system following liver injury. They find an unexpected role of the ductular reaction – the process of ectopic expansion of biliary-like cells following liver injury – in restoring functional biliary structures in injured livers.

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.

Human induced pluripotent stem cells (hiPSCs) are potentially an invaluable source of cells for regenerative medicine, disease modeling and drug discovery. However, the differentiation of hiPSCs into fully functional hepatocytes remains a major challenge. Despite the importance of the information carried by metabolomes, the exploitation of metabolomics for characterizing and understanding hiPSC differentiation remains largely unexplored. Here, to increase knowledge of hiPSC maturation into mature hepatocytes, we investigated their metabolomics profiles during sequential step-by-step differentiation: definitive endoderm (DE), specification into hepatocytes (HB-pro (hepatoblast progenitors)), progenitor hepatocytes (Pro-HEP) and mature hepatocyte-like cells (HLCs). Metabolomics analysis illustrated a switch from glycolysis-based respiration in DE step to oxidative phosphorylation in HLCs step. DE was characterized by fatty acid beta oxidation, sorbitol metabolism and pentose phosphate pathway, and glutamine and glucose metabolisms as various potential energy sources. The complex lipid metabolism switch was monitored via the reduction of lipid production from DE to HLCs step, whereas high glycerol production occurred mainly in HLCs. The nitrogen cycle, via urea production, was also a typical mechanism revealed in HLCs step. Our analysis may contribute to better understanding of differentiation and suggest new targets for improving iPSC maturation into functional hepatocytes.

International audience; The differentiation of human induced pluripotent stem cells (hiPSCs) into functional hepatocytes has the potential to solve the shortage of human primary liver cells and would be of use in drug screening. In this frame, we developed a hiPSCs maturation strategy in microfluidic biochips, using a liver-on-chip approach, a promising technology mimicking in vivo physiology. Hepato-like tissues differentiated in biochips presented advanced liver features, including ALB and CYP3A4 expressing cells. The metabolomics and transcriptomics profiles of hepato-likes cells differentiated either in biochips or Petri dishes were integrated to compare their functionalities. The multi-omics analysis revealed 41 metabolites and 302 genes differentially expressed. Overall, biochip environment lead to higher degree of hepatic differentiation demonstrated by an increase in the metabolic production of lipids, fatty acids and biliary acids, which was confirmed at the transcriptome level by the modulation of expression for genes involved in related signaling pathways. This observation was correlated with higher production of fructose in biochips, together with down-regulation of genes engaged in glycolysis. In parallel, increased activity of the Krebs cycle, pentose phosphate shuffle, and fatty acid beta oxidation was observed in tissues cultured in Petri. Besides, the modulation of nitrogen metabolism was observed in transcriptomic data and confirmed by an intense production of glutamine, putrescine and creatinine and by the higher consumption of spermidine measured in Petri.