Cargando…
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...
Autores principales: | , , , , , |
---|---|
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 |
_version_ | 1783692679328038912 |
---|---|
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). |
format | Online Article Text |
id | pubmed-8122664 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT messnercatherinejane singlecellgeneexpressionanalysisina3dmicrotissuelivermodelrevealscelltypespecificresponsestoprofibrotictgfb1stimulation AT babraklmar singlecellgeneexpressionanalysisina3dmicrotissuelivermodelrevealscelltypespecificresponsestoprofibrotictgfb1stimulation AT titologaia singlecellgeneexpressionanalysisina3dmicrotissuelivermodelrevealscelltypespecificresponsestoprofibrotictgfb1stimulation AT cajmichaela singlecellgeneexpressionanalysisina3dmicrotissuelivermodelrevealscelltypespecificresponsestoprofibrotictgfb1stimulation AT mihoenkelejda singlecellgeneexpressionanalysisina3dmicrotissuelivermodelrevealscelltypespecificresponsestoprofibrotictgfb1stimulation AT suterdicklaura singlecellgeneexpressionanalysisina3dmicrotissuelivermodelrevealscelltypespecificresponsestoprofibrotictgfb1stimulation |