2021-04-22, Messner, Catherine, Babrak, Lmar, Titolo, Gaia, Caj, Michaela, Miho, Enkelejda, Suter-Dick, Laura

3D cell culture systems are widely used to study disease mechanisms and therapeutic interventions. Multicellular liver microtissues (MTs) comprising HepaRG, hTERT-HSC and THP-1 maintain multicellular interactions and physiological properties required to mimic liver fibrosis. However, the inherent complexity of multicellular 3D-systems often hinders the discrimination of cell type specific responses. Here, we aimed at applying single cell sequencing (scRNA-seq) to discern the molecular responses of cells involved in the development of fibrosis elicited by TGF-β1. To obtain single cell suspensions from the MTs, an enzymatic dissociation method was optimized. Isolated cells showed good viability, could be re-plated and cultured in 2D, and expressed specific markers determined by scRNA-seq, qRT-PCR, ELISA and immunostaining. The three cell populations were successfully clustered using supervised and unsupervised methods based on scRNA-seq data. TGF-β1 led to a fibrotic phenotype in the MTs, detected as decreased albumin and increased αSMA expression. Cell-type specific responses to the treatment were identified for each of the three cell types. They included HepaRG damage characterized by a decrease in cellular metabolism, prototypical inflammatory responses in THP-1s and extracellular matrix remodeling in hTERT-HSCs. Furthermore, we identified novel cell-specific putative fibrosis markers in hTERT-HSC (COL15A1), and THP-1 (ALOX5AP and LAPTM5).

2020-12-30, Lu, Yan, Messner, Catherine, Suter-Dick, Laura

Exposure to environmental chemicals, particularly those with persistent and bioaccumulative properties have been linked to liver diseases. Induction of fibrotic pathways is considered as a pre-requirement of chemical induced liver fibrosis. Here, we applied 3D in vitro human liver microtissues (MTs) composed of HepaRG, THP-1 and hTERT-HSC that express relevant hepatic pathways (bile acid, sterol, and xenobiotic metabolism) and can recapitulate key events of liver fibrosis (e.g. extracellular matrix-deposition). The liver MTs were exposed to a known profibrotic chemical, thioacetamide (TAA) and three representative environmental chemicals (TCDD, benzo [a] pyrene (BaP) and PCB126). Both TAA and BaP triggered fibrotic pathway related events such as he-patocellular damage (cytotoxicity and decreased albumin release), hepatic stellate cell activation (transcriptional upregulation of α-SMA and Col1α1) and extracellular matrix remodelling. TCDD or PCB126 at measured con-centrations did not elicit these responses in the 3D liver MTs system, though they caused cytotoxicity in HepaRG monoculture at high concentrations. Reduced human transcriptome (RHT) analysis captured molecular re-sponses involved in liver fibrosis when MTs were treated with TAA and BaP. The results suggest that 3D, multicellular, human liver microtissues represent an alternative, human-relevant, in vitro liver model for assessing fibrotic pathways induced by environmental chemicals.