Single Cell RNA Sequencing Identifies Subsets of Hepatic Stellate Cells and Myofibroblasts in Liver Fibrosis
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ABSTRACT:
PROVIDER: PRJNA548680 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9290860_1.fastq.gz | Fastqsanger.gz | |||
| SRR9290860_2.fastq.gz | Fastqsanger.gz | |||
| SRR9290861_1.fastq.gz | Fastqsanger.gz | |||
| SRR9290861_2.fastq.gz | Fastqsanger.gz | |||
| SRR9290862_1.fastq.gz | Fastqsanger.gz |
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Project description:Hepatic stellate cells and activated myofibroblasts display a high heterogeneity in healthy and fibrotic liver characterized by differential expression of collagens and chemokines.
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Project description:Activated hepatic stellate cells (HSC) that transdifferentiate to myofibroblasts in the injured liver are responsible for scar formation that leads to fibrosis and eventually cirrhosis. To investigate the gene expression profile during different stages of this process, we performed serial analysis of gene expression (SAGE), representing a quantitative and qualitative description of all expressed genes. Stellate cells were isolated from human livers and cultured. SAGE was performed on RNA isolated from quiescent, activated and transdifferentiated HSC. Comparison of the three resulting transcriptomes showed that less than 5% of all genes changed significantly in expression. Established markers of liver fibrosis showed enhanced expression in accordance with the transdifferentiation process. In addition, induction was seen for several genes not yet recognized to be involved in liver fibrosis, such as insulin-like growth factor binding proteins (IGFBP) and antagonists of bone morphogenic proteins: follistatin and gremlin. The induction of these genes was validated in vivo in mice developing liver fibrosis. The expression of IGFBPs and gremlin was measurable in the livers of these mice while it was low or undetectable in control mice without liver fibrosis. Since gremlin modulates the activity of bone morphogenic growth factors, it may represent a novel pathway and a target for therapeutic intervention and together with IGFBPs as a specific marker of liver fibrosis. In conclusion, the comparison of the three transcriptomes of (activated) stellate cells reveals novel genes involved in fibrogenesis and provides an appreciation of the sequence and timing of the fibrotic process in liver. Keywords: cell type comparison Human hepatic stellate cells (HSC) were isolated from wedge sections of normal human liver unsuitable for transplantation or from tumor-free human liver after partial hepatectomy as previously reported. Briefly, after a combined digestion with collagenase/pronase, HSC were separated from other liver nonparenchymal cells by ultracentrifugation over gradients of Larcoll (Sigma). The percentage of HSC in these isolates was > 90% as assessed by transmission microscopy, autofluorescence of vitamin A and immunofluorescence of vimentin. Immediately after isolation RNA was extracted for the construction of the SAGE library of quiescent HSC. To obtain activated stellate cells, these cells were cultured for 15 days on plastic culture dishes in modified Dulbecco's medium supplemented with 0.6 U/ml insulin, 2.0 mmol/L glutamine, 0.1 mmol/L nonessential amino acids, 1.0 mmol/L sodium pyruvate, antibiotic antifungal solution (Gibco) and 20% fetal bovine serum. To obtain fully transdifferentiated hepatic stellate cells, or myofibroblasts, the cells were cultured until they had reached passage 6 to 7. Myofibroblast phenotype was confirmed by detection of vimentin and ?-smooth muscle actin using immunofluorescence with monoclonal antibodies from Dako and Sigma, respectively. Medium was refreshed twice a week and at the indicated period RNA was extracted using Trizol (Quiagen)
2006-07-01 | E-GEOD-5022 | biostudies-arrayexpress
Project description:Hepatic stellate cells are the primary cell type responsible for development of fibrosis in chronic liver disease. We used directional RNA sequencing (RNA-seq) and chromatin immunoprecipitation and sequencing (ChIP-seq) to identify the lncRNAs expressed in human HSCs. We also identified the lncRNAs that change in expression with differentiation of nonfibrotic quiescent HSCs into fibrotic HSC myofibroblasts and those that are regulated by TGF-beta signaling. ChIP-seq was also performed to identify DNA regions occupied by H3K27ac to define super-enhancers in HSC myofibroblasts. This study identified lncRNAs expressed HSCs that may regulate fibrosis. Analysis of genome-wide lncRNA expression using RNA-seq and ChiP-seq in human HSCs under four different conditions
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2017-03-02 | GSE78853 | GEO
Project description:Direct induction of induced hepatocytes (iHeps) from fibroblasts holds potential as a strategy for regenerative medicine, but until now has only been shown in culture settings. Here, we describe in vivo iHep formation using transcription factor induction and genetic fate tracing in mouse models of chronic liver disease. We show that ectopic expression of the transcription factors FOXA3, GATA4, HNF1A and HNF4A from a polycistronic lentiviral vector converts mouse myofibroblasts into cells with a hepatocyte phenotype. In vivo expression of the same set of transcription factors from a p75 neurotrophin receptor peptide (p75NTRp)-tagged adenovirus enabled the generation of hepatocyte-like cells from myofibroblasts in fibrotic mouse livers and reduced liver fibrosis. We have therefore been able to convert profibrogenic myofibroblasts in the liver into hepatocyte-like cells with positive functional benefits. This direct in vivo reprogramming approach may open new avenues for the treatment of chronic liver disease. Whole Mouse Genome Oligo Microarray v2 (4x44K) (Agilent Technologies) was used to characterize global gene expression profiles of iHeps compared to myofibroblasts and primary mouse hepatocytes. All microarrays were performed at the Research Core Unit Transcriptomics of the Hanover Medical School. Briefly, total RNA was used to prepare the aminoallyl-UTPmodified (aaUTP) cRNAs (Amino Allyl MessageAmp⢠II Kit; #AM1753; Life Technologies) as directed by the company. The aaUTP-cRNAs were labelled with Alexa Fluor 555 Reactive Dye (#A32756; LifeTechnologies). Prior to the reverse transcription reaction, 1μl of a 1:5000 dilution of Agilentâs One-Color spike-in Kit stock solution (#5188-5282, Agilent Technologies) was added to 100ng of total RNA of each analyzed sample. The cRNA fragmentation, hybridization, and washing steps were carried out according to Agilentâs One-Color Microarray-Based Gene Expression Analysis Protocol V5.7 except that 500ng of each labelled cRNA sample were used for hybridization. Slides were scanned on the Agilent Micro Array Scanner G2565 CA (pixel resolution 5 μm, bit depth 20). Data extraction was performed with the Feature Extraction Software V10.7.3.1. 12 samples were analyzed: Pr-mHSC: Primary myofibroblasts derived from hepatic stellate cells (HSCs), 2 replicates; In-vivo iHep: In-vivo myofibroblasts-derived from induced hepatocytes (iHep), 3 replicates; In-vivo eHep: In-vivo endogenous hepatocytes (eHep), 3 replicates; In-vitro iHep: In-vitro myofibroblasts-derived from induced hepatocytes (iHep), 3 replicates; PH24h: Primary hepatocytes (PH) cultured for 24 hours, 1 replicate.
2016-02-22 | E-GEOD-76843 | biostudies-arrayexpress
Project description:Activated hepatic stellate cells (HSC) that transdifferentiate to myofibroblasts in the injured liver are responsible for scar formation that leads to fibrosis and eventually cirrhosis. To investigate the gene expression profile during different stages of this process, we performed serial analysis of gene expression (SAGE), representing a quantitative and qualitative description of all expressed genes. Stellate cells were isolated from human livers and cultured. SAGE was performed on RNA isolated from quiescent, activated and transdifferentiated HSC. Comparison of the three resulting transcriptomes showed that less than 5% of all genes changed significantly in expression. Established markers of liver fibrosis showed enhanced expression in accordance with the transdifferentiation process. In addition, induction was seen for several genes not yet recognized to be involved in liver fibrosis, such as insulin-like growth factor binding proteins (IGFBP) and antagonists of bone morphogenic proteins: follistatin and gremlin. The induction of these genes was validated in vivo in mice developing liver fibrosis. The expression of IGFBPs and gremlin was measurable in the livers of these mice while it was low or undetectable in control mice without liver fibrosis. Since gremlin modulates the activity of bone morphogenic growth factors, it may represent a novel pathway and a target for therapeutic intervention and together with IGFBPs as a specific marker of liver fibrosis. In conclusion, the comparison of the three transcriptomes of (activated) stellate cells reveals novel genes involved in fibrogenesis and provides an appreciation of the sequence and timing of the fibrotic process in liver. Keywords: cell type comparison
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2015-02-19 | E-GEOD-52253 | biostudies-arrayexpress
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