Methods for producing hepatocytes
First Claim
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1. A method for producing hepatocytes, comprising:
- plating pluripotent human stem cells on a cell culture substrate comprising (i) a first laminin which is laminin-521 and (ii) a second laminin selected from the group consisting of laminin-111 and laminin-221, wherein the laminin-521 and the second laminin are each an intact protein; and
culturing the pluripotent human stem cells to obtain the hepatocytes;
wherein the culturing of the pluripotent human stem cells is performed by;
culturing the cells on the cell culture substrate in an endoderm differentiation medium containing activin A and Wnt3a;
culturing the cells on the cell culture substrate in a hepatoblast differentiation medium;
culturing the cells on the cell culture substrate in a hepatocyte maturation medium containing hydrocortisone (HC), hepatocyte growth factor (HGF), and endostatin m (OSM); and
wherein the resulting hepatocytes exhibit CYP1A2 activity of at least 600,000 RLU/mg/mL or CYP3A activity of at least 800,000 RLU/mg/mL.
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Abstract
Methods for producing hepatocytes from pluripotent human stem cells are disclosed herein. The stem cells are plated on a cell culture substrate comprising two laminins. The stem cells are then exposed to different cell culture mediums to induce differentiation. The resulting hepatocytes have higher metabolic capacity compared to hepatocytes cultured on different substrates.
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Citations
30 Claims
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1. A method for producing hepatocytes, comprising:
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plating pluripotent human stem cells on a cell culture substrate comprising (i) a first laminin which is laminin-521 and (ii) a second laminin selected from the group consisting of laminin-111 and laminin-221, wherein the laminin-521 and the second laminin are each an intact protein; and culturing the pluripotent human stem cells to obtain the hepatocytes; wherein the culturing of the pluripotent human stem cells is performed by; culturing the cells on the cell culture substrate in an endoderm differentiation medium containing activin A and Wnt3a; culturing the cells on the cell culture substrate in a hepatoblast differentiation medium; culturing the cells on the cell culture substrate in a hepatocyte maturation medium containing hydrocortisone (HC), hepatocyte growth factor (HGF), and endostatin m (OSM); and wherein the resulting hepatocytes exhibit CYP1A2 activity of at least 600,000 RLU/mg/mL or CYP3A activity of at least 800,000 RLU/mg/mL. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method for producing hepatocytes, comprising:
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plating pluripotent human stem cells on a cell culture substrate comprising (i) a first laminin which is laminin-521 and (ii) a second laminin which is laminin-221, wherein the laminin-521 and the second laminin are each an intact protein; and culturing the pluripotent human stem cells on the cell culture substrate to obtain the hepatocytes; wherein the culturing of the pluripotent human stem cells is performed by; culturing the cells in an endoderm differentiation medium containing activin A and Wnt3a; culturing the cells in a hepatoblast differentiation medium; and culturing the cells in a hepatocyte maturation medium containing hydrocortisone (HC), hepatocyte growth factor (HGF), and oncostatin m (OSM). - View Dependent Claims (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
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Specification