The 3Rs guidelines recommend replacing animal testing with alternative models. One of the solutions proposed is organ-on-chip technology in which liver-on-chip is one of the most promising alternatives for drug screening and toxicological assays. The main challenge is to achieve the relevant in vivo-like functionalities of the liver tissue in an optimized cellular microenvironment. Here, we investigated the development of hepatic cells under dynamic conditions inside a 3D hydroscaffold embedded in a microfluidic device. The hydroscaffold is made of hyaluronic acid and composed of liver extracellular matrix components (galactosamine, collagen I/IV) with RGDS (Arg-Gly-Asp-Ser) sites for cell adhesion. The HepG2/C3A cell line was cultured under a flow rate of 10 µL/min for 21 days. After seeding, the cells formed aggregates and proliferated, forming 3D spheroids. The cell viability, functionality, and spheroid integrity were investigated and compared to static cultures. The results showed a 3D aggregate organization of the cells up to large spheroid formations, high viability and albumin production, and an enhancement of HepG2 cell functionalities. Overall, these results highlighted the role of the liver-on-chip model coupled with a hydroscaffold in the enhancement of cell functions and its potential for engineering a relevant liver model for drug screening and disease study.

The liver plays a key role in the metabolism of lipoproteins, controlling both production and catabolism. To accelerate the development of new lipid‐lowering therapies in humans, it is essential to have a relevant in vitro study model available. The current hepatocyte‐like cells (HLCs) models derived from hiPSC can be used to model many genetically driven diseases but require further improvement to better recapitulate the complexity of liver functions. Here, we aimed to improve the maturation of HLCs using a three‐dimensional (3D) approach using Biomimesys®, a hyaluronic acid‐based hydroscaffold in which hiPSCs may directly form aggregates and differentiate toward a functional liver organoid model. After a 28‐day differentiation 3D protocol, we showed that many hepatic genes were upregulated in the 3D model (liver organoids) in comparison with the 2D model (HLCs). Liver organoids, grown on Biomimesys®, exhibited an autonomous cell organization, were composed of different cell types and displayed enhanced cytochromes P450 activities compared to HLCs. Regarding the functional capacities of these organoids, we showed that they were able to accumulate lipids (hepatic steatosis), internalize low‐density lipoprotein and secrete apolipoprotein B. Interestingly, we showed for the first time that this model was also able to produce apolipoprotein (a), the apolipoprotein (a) specific of Lp(a). This innovative hiPSC‐derived liver organoid model may serve as a relevant model for studying human lipopoprotein metabolism, including Lp(a).

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a key regulator of low-density lipoprotein (LDL) cholesterol metabolism and the target of lipid-lowering drugs. PCSK9 is mainly expressed in hepatocytes. Here, we show that PCSK9 is highly expressed in undifferentiated human induced pluripotent stem cells (hiPSCs). PCSK9 inhibition in hiPSCs with the use of short hairpin RNA (shRNA), CRISPR/cas9-mediated knockout, or endogenous PCSK9 loss-of-function mutation R104C/V114A unveiled its new role as a potential cell cycle regulator through the NODAL signaling pathway. In fact, PCSK9 inhibition leads to a decrease of SMAD2 phosphorylation and hiPSCs proliferation. Conversely, PCSK9 overexpression stimulates hiPSCs proliferation. PCSK9 can interfere with the NODAL pathway by regulating the expression of its endogenous inhibitor DACT2, which is involved in transforming growth factor (TGF) β-R1 lysosomal degradation. Using different PCSK9 constructs, we show that PCSK9 interacts with DACT2 through its Cys-His-rich domain (CHRD) domain. Altogether these data highlight a new role of PCSK9 in cellular proliferation and development.

Background Extracellular matrix (ECM) is a three-dimensional (3D) structure found around cells in the tissues of many organisms. It is composed mainly of fibrous proteins, such as collagen and elastin, and adhesive glycoproteins, such as fibronectin and laminin—as well as proteoglycans, such as hyaluronic acid. The ECM performs several essential functions, including structural support of tissues, regulation of cell communication, adhesion, migration, and differentiation by providing biochemical and biomechanical cues to the cells. Pancreatic β-cells have been previously shown to be responsive to the surrounding mechanical stress, impacting their insulin-secreting function. Purpose We aimed to derive a physiologically relevant in vitro model of pancreatic tissue by using an innovative synthesised porous ECM that mimics the native tissue microenvironment and mechanical properties. Methods Here we performed mechanical, physico-chemical and functional characterisation of a synthetic hydrogel ECM, composed of hyaluronic acid cross-linked with collagen types I and VI and modified with fibronectin. The hydrogel was used as a 3D cell culture scaffold for the MIN6 insulinoma cell line. Cell proliferation, viability, gene expression, and insulin secretion in response to glucose stimulus were assessed and contrasted with classic monolayer culture. Results The biomaterial exhibited a shear modulus of 815.37 kPa and a distinctive viscoelastic response. MIN6 cells showed a higher proliferation and viability rates and maintained insulin secretion in response to glucose stimulus and β-cell identity gene expression when cultured in the 3D hydrogel compared to monolayer culture. Conclusion Our study demonstrated the potential of this biomimetic hydrogel scaffold as an innovative matrix enabling better in vitro models to study disease physiopathology.

Les cellules souches pluripotentes induites humaines (hiPSC) offrent une alternative particulièrement attractive pour l ‘étude de nouvelles fonctions liées à des pathologies ainsi que la mise en place de modèles d’études innovants. C’est dans ce contexte que nous étudions un acteur clé de la régulation du métabolisme du cholestérol par les hépatocytes, PCSK9. Lors de la différenciation des hiPSC en hépatocytes, nous avons mis en évidence un rôle inattendu de PCSK9 dans les cellules indifférenciées. Grâce à l’utilisation d’outils de surexpression, d’invalidation de gène par une approche CRISPR/Cas9 ou d’hiPSC d’un patient portant des mutations pertes de fonction de PCSK9, nous avons pu mettre en évidence une action régulatrice de PCSK9 sur la voie TGFß. Cet effet serait médié par une action de PCSK9 sur la protéine DACT2, un régulateur négatif de la sous-unité R1 du récepteur au TGFßR. Les effets de PCSK9 sur DACT2 entrainent une modification de la phosphorylation de SMAD2 et subséquemment la prolifération des hiPSC. En parallèle, afin d’améliorer les fonctions hépatiques des hiPSC différenciées, nous avons mis en place une nouvelle procédure permettant d’obtenir de façon simple des organoïdes hépatiques. Pour cela les hiPSC sont mises en culture dans un hydroscaffold modifié, BIOMIMESYS®, produit par HCS Pharma dans un format 96-puits pour adapter le modèle au criblage moléculaire. Notre procédure récapitulant les étapes clés du développement hépatiques nous a permis d’obtenir des organoïdes hépatiques comprenant en plus des hépatocytes, des cellules biliaires, étoilées et endothéliales. Leur caractérisation fonctionnelle nous a permis de mettre en évidence des activités de cytochrome supérieur au modèle en 2D, ainsi qu’une excellente réponse pharmacologique suite à leur capacité à accumuler les lipides lors d'un traitement à l'amiodarone ou à l’éthanol ainsi qu’à absorber les LDL-bodipy après traitement aux statines. Notre modèle pouvant être personnalisé et automatisable, offre de nouvelles perspectives pour le criblage moléculaire à haut débit.