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Injection of embryonic stem cell derived macrophages ameliorates fibrosis in a murine model of liver injury.

ABSTRACT: Chronic liver injury can be caused by viral hepatitis, alcohol, obesity, and metabolic disorders resulting in fibrosis, hepatic scarring, and cirrhosis. Novel therapies are urgently required and previous work has demonstrated that treatment with bone marrow derived macrophages can improve liver regeneration and reduce fibrosis in a murine model of hepatic injury and fibrosis. Here, we describe a protocol whereby pure populations of therapeutic macrophages can be produced in vitro from murine embryonic stem cells on a large scale. Embryonic stem cell derived macrophages display comparable morphology and cell surface markers to bone marrow derived macrophages but our novel imaging technique revealed that their phagocytic index was significantly lower. Differences were also observed in their response to classical induction protocols with embryonic stem cell derived macrophages having a reduced response to lipopolysaccharide and interferon gamma and an enhanced response to IL4 compared to bone marrow derived macrophages. When their therapeutic potential was assessed in a murine, carbon tetrachloride-induced injury and fibrosis model, embryonic stem cell derived macrophages significantly reduced the amount of hepatic fibrosis to 50% of controls, down-regulated the number of fibrogenic myofibroblasts and activated liver progenitor cells. To our knowledge, this is the first study that demonstrates a therapeutic effect of macrophages derived in vitro from pluripotent stem cells in a model of liver injury. We also found that embryonic stem cell derived macrophages repopulated the Kupffer cell compartment of clodronate-treated mice more efficiently than bone marrow derived macrophages, and expressed comparatively lower levels of Myb and Ccr2, indicating that their phenotype is more comparable to tissue-resident rather than monocyte-derived macrophages.

SUBMITTER: Haideri SS

PROVIDER: S-EPMC5677947 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Alcoholic liver disease (ALD) is a major cause of chronic liver disease worldwide. Macrophages exhibit different functional states and are classified as classically activated (M1) and alternatively activated (M2) macrophages. However, the mechanisms that govern M1/M2 polarization in chronic ALD remain to be elucidated. Prostacyclin (PGI2) synthase (PTGIS) is an enzyme of the prostaglandin pathway which catalyzes the conversion of Prostaglandin H2 (PGH2) to PGI2. PTGIS has anti-inflammatory properties. However, the function of PTGIS in ALD has not yet been determined. In this study, we demonstrated that PTGIS was downregulated in ALD and forced PTGIS expression in vivo using recombinant adeno-associated viral vector-packed PTGIS overexpression plasmid, which alleviated the inflammatory response and suppressed the macrophage M1 phenotype in mice. Loss- and gain-of function-experiments demonstrated that forced PTGIS expression inhibited the macrophage switch to the M1 phenotype and promoted M2 polarization. Furthermore, we identified the genes regulated by PTGIS through RNA-sequencing (RNA-seq) analysis. Gene ontology and KEGG pathway analyses showed that PTGIS regulates many genes involved in the immune response and is enriched in the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signal transduction pathway, which plays an important role in regulating macrophage polarization. The proteins interacting with JAKs were predicted using the STRING database. The overlap between the RNA-seq and the STRING database was interleukin-6; this indicated that it was involved in macrophage polarization regulated by JAK/STAT signaling. We further explored the microRNAs that could regulate the expression of PTGIS through TargetScan. The results of luciferase assay illustrated that the expression of PTGIS was regulated by miR-140-3p.1. These results imply that PTGIS plays a pivotal role in ALD, partly by influencing macrophage polarization.

Project description:BACKGROUND: Characterization of gene expression signatures for cardiomyocytes derived from embryonic stem cells will help to define their early biologic processes. RESULTS: A transgenic alpha-myosin heavy chain (MHC) embryonic stem cell lineage was generated, exhibiting puromycin resistance and expressing enhanced green fluorescent protein (EGFP) under the control of the alpha-MHC promoter. A puromycin-resistant, EGFP-positive, alpha-MHC-positive cardiomyocyte population was isolated with over 92% purity. RNA was isolated after electrophysiological characterization of the cardiomyocytes. Comprehensive transcriptome analysis of alpha-MHC-positive cardiomyocytes in comparison with undifferentiated alpha-MHC embryonic stem cells and the control population from 15-day-old embryoid bodies led to identification of 884 upregulated probe sets and 951 downregulated probe sets in alpha-MHC-positive cardiomyocytes. A subset of upregulated genes encodes cytoskeletal and voltage-dependent channel proteins, and proteins that participate in aerobic energy metabolism. Interestingly, mitosis, apoptosis, and Wnt signaling-associated genes were downregulated in the cardiomyocytes. In contrast, annotations for genes upregulated in the alpha-MHC-positive cardiomyocytes are enriched for the following Gene Ontology (GO) categories: enzyme-linked receptor protein signaling pathway (GO:0007167), protein kinase activity (GO:0004672), negative regulation of Wnt receptor signaling pathway (GO:0030178), and regulation of cell size (O:0008361). They were also enriched for the Biocarta p38 mitogen-activated protein kinase signaling pathway and Kyoto Encyclopedia of Genes and Genomes (KEGG) calcium signaling pathway. CONCLUSION: The specific pattern of gene expression in the cardiomyocytes derived from embryonic stem cells reflects the biologic, physiologic, and functional processes that take place in mature cardiomyocytes. Identification of cardiomyocyte-specific gene expression patterns and signaling pathways will contribute toward elucidating their roles in intact cardiac function.

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Injection of embryonic stem cell derived macrophages ameliorates fibrosis in a murine model of liver injury.

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