Follistatin Enhanced Differentiation of Mouse Embryonic Stem Cells toward Endoderm and Hepatocyte Proteomic Mechanism and Functional Microrna Study

Embryonic stem cells(ESC) are taken from the blastocysts' inner cell mass. ESC has the ability to self-renewal and pluripotent. Embryonic stem cell-derived endoderm or hepatocyte is critical for the development of cellular therapies for the treatment of disease such as diabetes mellitus, liver cirrhosis. Induced ESC to hepatocyte usually results low yield of hepatocyte. Here, in this proposal in order to improve the hepatocyte differentiation ratio to highest level, we use a new method, first of all the mouse ESC are cultured in fibronectin-coated collagen gel surface and adds follistatin induced mouse ESC differentiation to the endoderm, and then use our published differentiation of mouse ESC into hepatocytes method, passing the endoderm cells differentiate into high proportion of mature hepatocytes. In the first year of pilot study, use follistatin can successfully set the mouse embryonic stem cells to the endoderm cell differentiation, and has a unique endoderm FoxA2 and Sox17 genes and proteins. In order to provide a valid therapeutic design method for using embryonic stem cell-derived endoderm or hepatocyte treats liver cirrhosis. In the second year of proposal, we will be from protein levels to study the mechanism of using follistatin induction mouse ESC to the endoderm differentiation and endoderm cells differentiate into hepatocytes respectively. In the second year of preliminary result, we use the SILAC methods to label of mouse ESC and endoderm cells, and nanoLC-ESI-MS/MS to analyze the protein quantitative difference. Total 903 proteins with two-fold change are got in which 719 proteins are up-regulated, 184 proteins are down-regulated. When the biological roles of 903 differentially expressed proteins are assessed via MetaCore analysis, the mouse ESC to differentiate into endoderm cells process may be enhanced G-protein alpha 12 signaling pathway (contains MAPK/ERK, PI3K, and p38MAPK pathway). In order to solve the present use ESC to differentiate into hepatocytes quantity are not enough for clinician to treat liver diseases. Our approach will from microRNA, when the specific microRNA function is promoting the endoderm differentiation or hepatic cells' differentiation, we will be able to modulate mouse ESC specific microRNA expression, have it over or down expression to achieve the higher differentiation ratio of endoderm cells or hepatocytes. In the third year of preliminary result, we use the mouse miRNA chip to analyze mouse ESC and endoderm cells microRNA expression difference (three duplicates) and get 70 microRNAs with 2 times difference (19 microRNAs in mouse ESC are higher expression, 51 microRNAs in mouse ESC are lower expression). In line with the TargetScan software analysis, initially find function of the miR-150 and miR-143 may be promoting the endoderm differentiation. We will deliver the synthesis of Anti-miR inhibitor into mouse ESC to suppress the performance of the miR-150 and miR-143, to confirm their functionality. As a result the future of clinical application, embryonic stem cells may be a good endoderm cells and hepatocytes source. Within the future as cellular therapy for using endoderm cells or hepatocytes to treat acute liver failure patients and using hepatocytes to test a new drug development would make an important contribution.

Project ID:PB9907-12641
External Project ID:NSC99-2221-E182-036