Development of Liver-on-Chip Integrating a Hydroscaffold Mimicking the Liver's Extracellular Matrix.

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.

PCSK9 regulates the NODAL signaling pathway and cellular proliferation in hiPSCs.

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) ß-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.