Single Cell Gene Expression Analysis in a 3D Microtissue Liver Model Reveals Cell Type-Specific Responses to Pro-Fibrotic TGF-β1 Stimulation

• Published in: International journal of molecular sciences 2021-04, Vol.22 (9), p.4372
• Main Authors: Messner, Catherine Jane, Babrak, Lmar, Titolo, Gaia, Caj, Michaela, Miho, Enkelejda, Suter-Dick, Laura
• Format: Article
• Language: English
• Subjects: Albumins; Cell culture; Cell suspensions; Cytokines; Disease; Enzyme-linked immunosorbent assay; Enzymes; Extracellular matrix; Fibrosis; Gene expression; Genotype & phenotype; Hepatocytes; in vitro; Inflammation; LAPTm5 protein; Liver; Liver cancer; liver fibrosis; liver microtissues; Metabolism; Phenotypes; Physiology; single cell sequencing; Spheroids; Telomerase reverse transcriptase; Therapeutic applications; Transforming growth factor-b1
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms22094372

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 ( ), and THP-1 ( and ).