Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Prospective isolation of a bipotential clonogenic liver progenitor cell in adult mice

ABSTRACT: The molecular identification of adult hepatic stem/progenitor cells has been hampered by the lack of truly specific markers. To isolate putative adult liver progenitor cells, we used cell surface-marking antibodies, including MIC1-1C3, to isolate subpopulations of liver cells from normal adult mice or those undergoing an oval cell response and tested their capacity to form bilineage colonies in vitro. Robust clonogenic activity was found to be restricted to a subset of biliary duct cells antigenically defined as CD45–/CD11b–/CD31–/MIC1-1C3+/CD133+/CD26–, at a frequency of one of 34 or one of 25 in normal or oval cell injury livers, respectively. Gene expression analyses revealed that Sox9 was expressed exclusively in this subpopulation of normal liver cells and was highly enriched relative to other cell fractions in injured livers. In vivo lineage tracing using Sox9creERT2-R26RYFP mice revealed that the cells that proliferate during progenitor-driven liver regeneration are progeny of Sox9-expressing precursors. A comprehensive array-based comparison of gene expression in progenitor-enriched and progenitor-depleted cells from both normal and DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine or diethyl1,4-dihydro-2,4,6-trimethyl- 3,5-pyridinedicarboxylate)-treated livers revealed new potential regulators of liver progenitors. Treated and untreated cells were sorted by cell surface markers then compared. M+/133+ untreated: 4 biological and 1 technical replicate - total of 5 M-/133- untreated: 2 biological and 1 technical replicate - total of 3 M+/133+ DDC-treated: 4 biological replicates - total of 4 M-/133- DDC-treated: 4 biological replicates - total of 4

ORGANISM(S): Mus musculus

SUBMITTER: Jonathan Schug

PROVIDER: E-GEOD-29121 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The ability to purify to homogeneity a population of hepatic progenitor cells from adult liver is critical for their characterization prior to any therapeutic application. As a step in this direction, we have utilized gene profiling of a bipotential liver cell line from dpc 14 mouse embryonic liver to catalog genes expressed by liver progenitor cells. These cells, known as Bipotential Mouse Embryonic Liver (BMEL) cells, proliferate in an undifferentiated state and are capable of differentiating into hepatocyte-like and cholangiocyte-like cells in vitro. Upon transplantation, BMEL cells are capable of differentiating into hepatocytes and cholangiocytes in vivo. Microarray analysis of gene expression in the 9A1 and 14B3 BMEL cell lines grown under proliferating and differentiating conditions was used to identify cell surface markers preferentially expressed in the bipotential undifferentiated state. This analysis revealed that proliferating BMEL cells express many genes involved in cell cycle regulation whereas differentiation of BMEL cells by cell aggregation causes a switch in gene expression to functions characteristic of mature hepatocytes. In addition, microarray data and protein analysis indicated that the Notch signaling pathway could be involved in maintaining BMEL cells in an undifferentiated stem cell state. Using GO annotation, a list of cell surface markers preferentially expressed on undifferentiated BMEL cells was generated. One marker, Cd24a, is specifically expressed on progenitor oval cells in livers of DDC treated animals. We therefore consider Cd24a expression a candidate molecule for purification of hepatic progenitor cells. Experiment Overall Design: RNA was extracted from two independently isolated BMEL cell lines (9A1 and 14B3) after culture under three conditions (basal, aggregate 1 day, and aggregate 5 days). Duplicate biological replicates were collected for each cell line:culture condition combination for a total of 12 samples. Samples were biotin-labeled, hybridized to mouse 430 2.0 chips, and scanned according to established Affymetrix protocols.

Project description:The ability to purify to homogeneity a population of hepatic progenitor cells from adult liver is critical for their characterization prior to any therapeutic application. As a step in this direction, we have utilized gene profiling of a bipotential liver cell line from dpc 14 mouse embryonic liver to catalog genes expressed by liver progenitor cells. These cells, known as Bipotential Mouse Embryonic Liver (BMEL) cells, proliferate in an undifferentiated state and are capable of differentiating into hepatocyte-like and cholangiocyte-like cells in vitro. Upon transplantation, BMEL cells are capable of differentiating into hepatocytes and cholangiocytes in vivo. Microarray analysis of gene expression in the 9A1 and 14B3 BMEL cell lines grown under proliferating and differentiating conditions was used to identify cell surface markers preferentially expressed in the bipotential undifferentiated state. This analysis revealed that proliferating BMEL cells express many genes involved in cell cycle regulation whereas differentiation of BMEL cells by cell aggregation causes a switch in gene expression to functions characteristic of mature hepatocytes. In addition, microarray data and protein analysis indicated that the Notch signaling pathway could be involved in maintaining BMEL cells in an undifferentiated stem cell state. Using GO annotation, a list of cell surface markers preferentially expressed on undifferentiated BMEL cells was generated. One marker, Cd24a, is specifically expressed on progenitor oval cells in livers of DDC treated animals. We therefore consider Cd24a expression a candidate molecule for purification of hepatic progenitor cells. Experiment Overall Design: RNA was extracted from two independently isolated BMEL cell lines (9A1 and 14B3) after culture under three conditions (basal, aggregate 5 days, and Matrigel). Duplicate biological replicates were collected for each cell line:culture condition combination for a total of 12 samples. Samples were biotin-labeled, hybridized to mouse 430a chips, and scanned according to established Affymetrix protocols.

Project description:Background and aims: Liver is a major target organ for alcohol-induced disease and the spectrum of pathological states elicited by alcohol in liver comprises steatosis, alcoholic steatohepatitis, progressive fibrosis and cirrhosis, conditions that may progress to hepatocellular carcinoma. Many experimental animal models of alcoholic steatohepatitis exist that vary in duration, mode of alcohol administration and the degree and types of liver injury produced. While most of these models, regardless whether alcohol is administered through liquid diet or intragastrically, produce steatohepatitis and mild fibrosis, it is widely acknowledged that these models fail to fully recapitulate key characteristics of severe forms of alcoholic liver disease, such as alcoholic hepatitis. Recent studies attempted to combine alcohol and fibrosis and achieved promising results in mouse models that achieve some of the key features of alcoholic liver disease accompanied by exacerbated fibrosis and acute renal injury. This study combined a chronic cholestatic liver fibrosis model induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) with a mouse model of intragastric alcohol feeding. Methods: Adult male C57BL6/J mice were treated with 3,5-diethoxycarbonyl-1,4-dihydrocolidine (DDC) containing diet (0.05% w/w) to induce chronic liver fibrosis. Following DDC-induced fibrogenesis, ethyl alcohol (EtOH) (up to 27 g/ kg/day, up to 28 days) was administered continuously to mice via a gastric feeding tube (Tsukamoto-Frenchmodel of alcoholic liver disease). Results: Exposure to DDC or EtOH alone resulted in liver fibrosis or steatosis, respectively. Combined treatment with DDC and EtOH lead to an additive effect on liver injury, as evident by the development of hepatic inflammation, steatosis, and pericellular fibrosis, and by increased serum transaminase levels, compared to mice treated with either agent alone. Liver transcriptomic changes specific to combined treatment group included pathways involved in the cell cycle and DNA damage. Analyses of feces from these mice revealed alcohol-associated changes to the bile acid profile and gut microbiome. Conclusions: Mice treated with DDC and EtOH displayed several key characteristics of human alcoholic hepatitis, including pericellular fibrosis, increased hepatic bacterial load with dysbiosis, reduced capacity of the microbiome to synthesize secondary bile acids.

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Prospective isolation of a bipotential clonogenic liver progenitor cell in adult mice

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