Single Cell Gene Expression analysis in a 3D microtissue liver model reveals cell type-specific responses to pro-fibrotic TGF-β1 stimulation

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Publication date
22.04.2021
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01A - Journal article
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International Journal of Molecular Sciences
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22
Issue / Number
9
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MDPI
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Abstract
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).
Keywords
Single cell sequencing, In vitro, Liver, Liver fibrosis, Liver microtissues
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1661-6596
1422-0067
Language
English
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Yes
Strategic action fields FHNW
Publication status
Published
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Peer review of the complete publication
Open access category
Gold
License
'http://creativecommons.org/licenses/by/3.0/us/'
Citation
MESSNER, Catherine, Lmar BABRAK, Gaia TITOLO, Michaela CAJ, Enkelejda MIHO und Laura SUTER-DICK, 2021. Single Cell Gene Expression analysis in a 3D microtissue liver model reveals cell type-specific responses to pro-fibrotic TGF-β1 stimulation. International Journal of Molecular Sciences. 22 April 2021. Bd. 22, Nr. 9. DOI 10.3390/ijms22094372. Verfügbar unter: https://doi.org/10.26041/fhnw-4152