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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Authors
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Publication date
22.04.2021
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01A - Journal article
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Parent work
International Journal of Molecular Sciences
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Volume
22
Issue / Number
9
Pages / Duration
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Publisher / Publishing institution
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
1422-0067
Language
English
Created during FHNW affiliation
Yes
Strategic action fields FHNW
Publication status
Published
Review
Peer review of the complete publication
Open access category
Gold
License
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