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

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 co...

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Autores principales: Messner, Catherine Jane, Babrak, Lmar, Titolo, Gaia, Caj, Michaela, Miho, Enkelejda, Suter-Dick, Laura
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8122664/
https://www.ncbi.nlm.nih.gov/pubmed/33922101
http://dx.doi.org/10.3390/ijms22094372
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author Messner, Catherine Jane
Babrak, Lmar
Titolo, Gaia
Caj, Michaela
Miho, Enkelejda
Suter-Dick, Laura
author_facet Messner, Catherine Jane
Babrak, Lmar
Titolo, Gaia
Caj, Michaela
Miho, Enkelejda
Suter-Dick, Laura
author_sort Messner, Catherine Jane
collection PubMed
description 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).
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spelling pubmed-81226642021-05-16 Single Cell Gene Expression Analysis in a 3D Microtissue Liver Model Reveals Cell Type-Specific Responses to Pro-Fibrotic TGF-β1 Stimulation Messner, Catherine Jane Babrak, Lmar Titolo, Gaia Caj, Michaela Miho, Enkelejda Suter-Dick, Laura Int J Mol Sci Article 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). MDPI 2021-04-22 /pmc/articles/PMC8122664/ /pubmed/33922101 http://dx.doi.org/10.3390/ijms22094372 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Messner, Catherine Jane
Babrak, Lmar
Titolo, Gaia
Caj, Michaela
Miho, Enkelejda
Suter-Dick, Laura
Single Cell Gene Expression Analysis in a 3D Microtissue Liver Model Reveals Cell Type-Specific Responses to Pro-Fibrotic TGF-β1 Stimulation
title Single Cell Gene Expression Analysis in a 3D Microtissue Liver Model Reveals Cell Type-Specific Responses to Pro-Fibrotic TGF-β1 Stimulation
title_full Single Cell Gene Expression Analysis in a 3D Microtissue Liver Model Reveals Cell Type-Specific Responses to Pro-Fibrotic TGF-β1 Stimulation
title_fullStr Single Cell Gene Expression Analysis in a 3D Microtissue Liver Model Reveals Cell Type-Specific Responses to Pro-Fibrotic TGF-β1 Stimulation
title_full_unstemmed Single Cell Gene Expression Analysis in a 3D Microtissue Liver Model Reveals Cell Type-Specific Responses to Pro-Fibrotic TGF-β1 Stimulation
title_short Single Cell Gene Expression Analysis in a 3D Microtissue Liver Model Reveals Cell Type-Specific Responses to Pro-Fibrotic TGF-β1 Stimulation
title_sort single cell gene expression analysis in a 3d microtissue liver model reveals cell type-specific responses to pro-fibrotic tgf-β1 stimulation
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8122664/
https://www.ncbi.nlm.nih.gov/pubmed/33922101
http://dx.doi.org/10.3390/ijms22094372
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