Single Nucleus RNA Sequencing of Pre-Malignant Liver Reveals Disease-Associated Hepatocyte State with HCC Prognostic Potential

Carlessi, Rodrigo; Denisenko, Elena; Boslem, Ebru; Koehn, Julia; Main, Nathan; Bakar, N. Dianah B. Abu; Shirolkar, Gayatri D.; Jones, Matthew; Poppe, Daniel; Dwyer, Benjamin J.; Jackaman, Connie; Tjiam, M. Christian; Lister, Ryan; Karin, Michael; Fallowfield, Jonathan; Kendall, Timothy J.; Forbes, Stuart J.; Olynyk, John K.; Yeoh, George; Forrest, Alistair R.R.; Ramm, Grant A.; Febbraio, Mark A.; Tirnitz–Parker, Janina E.E.

doi:10.1101/2022.03.25.485695

Cited by 5 publications

(16 citation statements)

References 81 publications

(132 reference statements)

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“…To study and characterize the transcriptomic profile of human hepatocytes before and after engraftment in the FRG xenograft model, we employed a droplet-based (10x Chromium) single-nucleus transcriptomics (sn-RNA-seq) approach, as described recently. 22 We isolated and profiled human hepatic nuclei from a 15-month-old female donor before (n = 2) and 6 months after liver engraftment in the FRG model, from flash-frozen ∼25 mg of FRG liver chunks (n = 2). We obtained a total of 7,309 single human nucleus liver cell transcriptomes.…”

Section: Resultsmentioning

confidence: 99%

“…We next wanted to analyze the performance of each capsid with sn-RNA-seq. To do so, as discussed previously, we extracted and sorted nuclei from an unperfused snap-frozen fragment of the left lobe of the same highly engrafted hFRG animals 22 ( Figure 5 B).…”

Section: Resultsmentioning

confidence: 99%

“… 44 , 45 , 46 Given the inherent complexity of the xenograft hFRG model, where both human and murine cell types co-exist in the murine liver scaffold, we opted for sn-RNA-seq instead of sc-RNA-seq. 22 By snap-freezing the liver tissue and extracting and sorting the nuclei a posteriori , it is possible to minimize the biases introduced by collagenase-based cell dissociation, both at the level of cell-type representation and the level of de novo transcriptional stress responses. 28 , 29 One caveat when using sn-RNA-seq could be related to the relatively lower amount of AAV transgene transcripts present in nuclei than in the cytoplasm.…”

Section: Discussionmentioning

confidence: 99%

“…In the present study, we first investigated the validity of the xenograft FRG mouse model as a biologically predictive pre-clinical model of the human liver, using single-nucleus RNA sequencing (sn-RNA-seq) 22 to study the transcriptomic profile of primary human hepatocytes. Immunofluorescence analyses confirmed that in highly engrafted FRG animals the human hepatocytes organize and present appropriate patterns of zone-dependent enzyme expression.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Characterization of the humanized FRG mouse model and development of an AAV-LK03 variant with improved liver lobular biodistribution

Cabanes‐Creus¹,

Navarro²,

Liao³

et al. 2023

Molecular Therapy - Methods & Clinical Development

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Characterization of the humanized FRG mouse model and development of an AAV-LK03 variant with improved liver lobular biodistribution

Cabanes‐Creus¹,

Navarro²,

Liao³

et al. 2023

Molecular Therapy - Methods & Clinical Development

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“…Recently, single-cell transcriptomic (scRNAseq) analyses in both healthy and diseased human and mouse livers have identified heterogeneity within the LSEC population and several biomarkers to assess disease development [ 142 , 143 ].…”

Section: Endothelial Cells In the Pathogenesis Of Nafldmentioning

confidence: 99%

Endothelial Cell Dysfunction and Nonalcoholic Fatty Liver Disease (NAFLD): A Concise Review

Nasiri-Ansari

Ανδρουτσάκος

Flessa

et al. 2022

Cells

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Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide. It is strongly associated with obesity, type 2 diabetes (T2DM), and other metabolic syndrome features. Reflecting the underlying pathogenesis and the cardiometabolic disorders associated with NAFLD, the term metabolic (dysfunction)-associated fatty liver disease (MAFLD) has recently been proposed. Indeed, over the past few years, growing evidence supports a strong correlation between NAFLD and increased cardiovascular disease (CVD) risk, independent of the presence of diabetes, hypertension, and obesity. This implies that NAFLD may also be directly involved in the pathogenesis of CVD. Notably, liver sinusoidal endothelial cell (LSEC) dysfunction appears to be implicated in the progression of NAFLD via numerous mechanisms, including the regulation of the inflammatory process, hepatic stellate activation, augmented vascular resistance, and the distortion of microcirculation, resulting in the progression of NAFLD. Vice versa, the liver secretes inflammatory molecules that are considered pro-atherogenic and may contribute to vascular endothelial dysfunction, resulting in atherosclerosis and CVD. In this review, we provide current evidence supporting the role of endothelial cell dysfunction in the pathogenesis of NAFLD and NAFLD-associated atherosclerosis. Endothelial cells could thus represent a “golden target” for the development of new treatment strategies for NAFLD and its comorbid CVD.

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Epigenetic Regulation of Hepatic Lipid Metabolism by DNA Methylation

et al. 2023

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While extensive investigations have been devoted to the study of genetic pathways related to fatty liver diseases, much less is known about epigenetic mechanisms underlying these disorders. DNA methylation is an epigenetic link between environmental factors (e.g., diets) and complex diseases (e.g., non-alcoholic fatty liver disease). Here, it is aimed to study the role of DNA methylation in the regulation of hepatic lipid metabolism. A dynamic change in the DNA methylome in the liver of high-fat diet (HFD)-fed mice is discovered, including a marked increase in DNA methylation at the promoter of Beta-klotho (Klb), a co-receptor for the biological functions of fibroblast growth factor (FGF)15/19 and FGF21. DNA methyltransferases (DNMT) 1 and 3A mediate HFD-induced methylation at the Klb promoter. Notably, HFD enhances DNMT1 protein stability via a ubiquitination-mediated mechanism. Liver-specific deletion of Dnmt1 or 3a increases Klb expression and ameliorates HFD-induced hepatic steatosis. Single-nucleus RNA sequencing analysis reveals pathways involved in fatty acid oxidation in Dnmt1-deficient hepatocytes. Targeted demethylation at the Klb promoter increases Klb expression and fatty acid oxidation, resulting in decreased hepatic lipid accumulation. Up-regulation of methyltransferases by HFD may induce hypermethylation of the Klb promoter and subsequent down-regulation of Klb expression, resulting in the development of hepatic steatosis.

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Single Nucleus RNA Sequencing of Pre-Malignant Liver Reveals Disease-Associated Hepatocyte State with HCC Prognostic Potential

Cited by 5 publications

References 81 publications

Characterization of the humanized FRG mouse model and development of an AAV-LK03 variant with improved liver lobular biodistribution

Characterization of the humanized FRG mouse model and development of an AAV-LK03 variant with improved liver lobular biodistribution

Endothelial Cell Dysfunction and Nonalcoholic Fatty Liver Disease (NAFLD): A Concise Review

Epigenetic Regulation of Hepatic Lipid Metabolism by DNA Methylation

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