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

• Main Authors: Catherine Jane Messner, Lmar Babrak, Gaia Titolo, Michaela Caj, Enkelejda Miho, Laura Suter-Dick
• Format: Article
• Language: English
• Published: MDPI AG 2021-04-01
• Series: International Journal of Molecular Sciences
• Subjects: single cell sequencing; in vitro; liver; liver fibrosis; liver microtissues
• Online Access: https://www.mdpi.com/1422-0067/22/9/4372
• ISSN: 1661-6596; 1422-0067

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 (<i>COL15A1</i>), and THP-1 (<i>ALOX5AP</i> and <i>LAPTM5</i>).