Characterization of liver zonation‐like transcriptomic patterns in HLCs derived from hiPSCs in a microfluidic biochip environment

Abstract

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.

Engineering Hydrogels for the Development of Three-Dimensional In Vitro Models

“…The results showed hepatocyte polarity and the allowance of hepatobiliary function. In a later follow-up study, an oxygen measurement system was incorporated for toxicological screening [ 353 ]. In another study conducted by Lee et al, porcine liver dECM was used as bio-ink to print a 3D liver-on-a-chip platform.…”

Multi‐omics analysis of hiPSCs‐derived HLCs matured on‐chip revealed patterns typical of liver regeneration

“…In those optimized conditions, the tissue was still found to be positive to AFP and to CK19, and the mRNA levels of mature markers such as ALB and CYP3A4 were not found to reach the levels observed in Liver Total RNA samples. While the transcriptomics analysis of the comparison with Total Liver RNA samples as a positive control highlighted remaining patterns of primitiveness in the hiPSCs‐derived tissue (Figure S3), the current protocol has allowed detecting a CYP3A4 activity as well as an improved activity of several cytochromes over Petri dishes and over previously published protocols using biochips (Danoy et al, 2019, 2020). The multi‐omics analysis performed in the biochips revealed a signature, typical of a liver regenerative process, illustrated in Figures 5 and S4.…”

“…In this study, we present an extended maturation of hiPSCs‐derived HLCs in microfluidic biochip for 14 days under perfusion and stimulation with OSM. In addition, as we observed that the 5‐cm‐long microfluidic biochip that was used in our previous experiment would lead to a largely hypoxic culture condition (Danoy et al, 2020), we decided to shorten the microfluidic biochip to 1 cm. This design has shown its suitability for primary liver human hepatic cultures (Jellali et al, 2016) and allows the reduction of the required number of inoculated cells, which make it more suitable for larger‐scale experiments.…”

Investigation of the hepatic development in the coculture of hiPSCs-derived LSECs and HLCs in a fluidic microenvironment

“…Indeed, as cells are injected in biochips at a progenitor stage, they still retain part of their pluripotency which allows them to differentiate toward a different lineage in the biochip. Especially, the presence of endothelial-like cells and hypoxia-related phenotypes was confirmed in longer biochips after 7 days of maturation ( Danoy et al , 2019 , Danoy et al , 2020b ). In the current setup, the length of the biochip was reduced by 5 folds and hypoxia-related phenotype was not highlighted.…”