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:Adipose tissue dysfunction results in hepatic lipid overload, driving the progression of liver steatosis and non-alcoholic steatohepatitis. The involvement of adipose tissue-derived small extracellular vesicles (sEVs) in initiating and propagating nonalcoholic fatty liver disease (NAFLD) remains unexplored. Herein, we unveiled that transplanting adipose tissue from high-fat diet (HFD)-fed mice exacerbated insulin resistance and liver steatosis in lean mice, and Sirt3 expression was markedly reduced in adipose tissue from both NAFLD patients and murine NAFLD models. Interestingly, fat-specific Sirt3 overexpression (Sirt3AKI) mice enhanced insulin sensitivity and improved liver steatosis under HFD feeding. Furthermore, adipose tissue-derived sEVs were found to regulate hepatic lipid metabolism in Sirt3AKI mice. In vitro studies demonstrated that Sirt3 overexpressing adipocytes (Sirt3OE) released sEVs to ameliorate lipid accumulation in palmitic acid/oleic acid (P/O)-stimulated hepatocytes. Conversely, sEVs derived from NAFLD patients exacerbated hepatic lipid accumulation in HFD-fed Sirt3AKI mice. MicroRNA sequencing further identified a significant decrease in miR-30a-3p levels in sEVs derived from Sirt3OE adipocytes. Fat-specific miR-30a-3p overexpression worsened hepatic steatosis in HFD-fed Sirt3AKI mice. In contrast, engineered sEVs loaded with miR-30a-3p antagonist or miR-30a-3p inhibition robustly enhanced insulin sensitivity and mitigated hepatic accumulation in HFD-fed mice. Mechanistically, CHIP-qPCR and CHIP-sequencing analyses revealed that Sirt3 suppressed miR-30a-3p transcription through H3K56 acetylation. Dual-luciferase reporter assays validated Beclin1 as a bona fide target mRNA of miR-30a-3p. Taken together, these findings highlight the critical role of exosomal microRNAs in mediating adipocyte-hepatocyte crosstalk, provide novel insights into the mechanisms underlying lipotoxicity in hepatocytes, and suggest miR-30a-3p inhibitor could be a therapeutic agent for NAFLD.

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Mus musculus

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