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Directed differentiation and maturation of pluripotent cells into hepatocyte like cells by modulation of Wnt-signalling pathway

  • US 9,394,522 B2
  • Filed: 03/22/2011
  • Issued: 07/19/2016
  • Est. Priority Date: 03/22/2010
  • Status: Active Grant
First Claim
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1. A method for directing the differentiation of human definitive endoderm cells into human hepatocyte-like cells, wherein said method comprises:

  • i) differentiating human definitive endoderm cells into human hepatoblasts using a differentiation medium containing 0.05-5% DMSO; and

    ii) differentiating said human hepatoblasts obtained in step i) into human hepatocyte-like cells using a differentiation medium containing 0.01-5 μ

    M Dexamethasone, 1-50 ng/ml hepatocyte growth factor (HGF), 1-25 ng/ml Oncostatin M (OsM), 0.05-5% DMSO, and 0.1-10 μ

    M ofa GSK-3 inhibitor selected from a group consisting of;

    BIO (2′

    Z,3′

    £

    )-6-Bromoindirubin-3′

    -oxime (GSK3 Inhibitor IX);

    BIO-Acetoxime (2′

    Z,3′

    E)-6-Bromoindirubin-3′

    -acetoxime (GSK3 Inhibitor X);

    (5-Methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine (GSK3-Inhibitor XIII);

    Pyridocarbazole-cyclopenadienylruthenium complex (GSK3 Inhibitor XV);

    TDZD-8 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (GSK3beta Inhibitor I);

    2-Thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3beta Inhibitor II);

    OTDZT 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione (GSK3beta Inhibitor III);

    alpha-4-Dibromoacetophenone (GSK3beta Inhibitor VII);

    AR-AO 14418 N-(4-Methoxybenzyl)-N′

    -(5-nitro-1,3-thiazol-2-yl)urea (GSK-3beta Inhibitor VIII);

    3-(1-(3-Hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione (GSK-3beta Inhibitor XI);

    TWSI 19 pyrrolopyrimidine compound (GSK3beta Inhibitor XII);

    L803 H-KEAPPAPPQSpP-NH2 (SEQ ID NO;

    1) or its Myristoylated form (GSK3beta Inhibitor XIII);

    2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone (GSK3beta Inhibitor VI);

    Aminopyrimidine CHIR99021;

    Kenpaullone (9-Bromo-7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one;

    SB216763 3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; and

    Indirubin-3′

    -monoxime.

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