Back to results
US 12,485,147 B2Granted
Method for producing cell mass including pituitary tissue, and cell mass thereof
AssigneeSumitomo Chemical Company, Limited
InventorTokushige Nakano
Life of this patent
Estimating the term…
Abstract
The present invention aims to provide a method for efficiently producing a cell mass containing pituitary tissue from pluripotent stem cells. A method for producing a cell mass containing pituitary tissue, including the following steps (1) and (2): (1) a first step of suspension-culturing pluripotent stem cells to form a cell aggregate in the presence of a Wnt signal transduction pathway inhibiting substance, (2) a second step of suspension-culturing the aggregate obtained in the first step in the presence of a BMP signal transduction pathway activating substance and a Sonic hedgehog signal transduction pathway activating substance, thereby obtaining a cell mass comprising pituitary tissue.
Claims31
A method for producing a cell mass comprising pituitary tissue, the method comprising:
- (1) culturing pluripotent stem cells in the absence of feeder cells and in a medium comprising 1) a TGFβ family signal transduction pathway inhibiting substance and/or a Sonic hedgehog signal transduction pathway activating substance, and 2) a factor for maintaining an undifferentiated state for 6 to 48 hours,
- (2) suspension-culturing the pluripotent stem cells obtained in (1), to form a cell aggregate in the absence of a factor for maintaining an undifferentiated state and in the presence of a Wnt signal transduction pathway inhibiting substance for 72 hours or less, and
- (3) suspension-culturing the cell aggregate obtained in (2) in the presence of a BMP signal transduction pathway activating substance and a Sonic hedgehog signal transduction pathway activating substance, to obtain a cell mass comprising pituitary tissue,
- wherein the TGFβ family signal transduction pathway inhibiting substance is selected from a group consisting of a TGFβ signal transduction pathway inhibitor, Nodal/Activin signal transduction inhibitor and BMP signal transduction inhibitor,
- wherein the BMP signal transduction pathway activating substance in (3) is added during a period when not less than 10% of the cells of the surface layer of the aggregate formed in (2) form a tight junction, and
- wherein the factor for maintaining an undifferentiated state comprises a FGF signal transduction pathway activating substance.
3 more elements
A method for producing a cell mass comprising pituitary tissue, the method comprising:
- (1) suspension-culturing pluripotent stem cells to form a cell aggregate in the absence of a factor for maintaining an undifferentiated state and in the presence of a Wnt signal transduction pathway inhibiting substance,
- (2) suspension-culturing the cell aggregate obtained in (1) in the presence of a BMP signal transduction pathway activating substance and a Sonic hedgehog signal transduction pathway activating substance, and
- (3) suspension-culturing the cell aggregate obtained in (2) in the presence of a BMP signal transduction pathway inhibiting substance, to obtain a cell mass comprising pituitary tissue,
- wherein the BMP signal transduction pathway activating substance in (2) is added during a period when not less than 10% of the cells of the surface layer of the aggregate formed in (1) form a tight junction.
1 more element
A method for producing a cell mass comprising pituitary tissue, the method comprising:
- (1) culturing pluripotent stem cells in the absence of feeder cells and in a medium comprising 1) a TGFβ family signal transduction pathway inhibiting substance and/or a Sonic hedgehog signal transduction pathway activating substance, and 2) a factor for maintaining an undifferentiated state for 6 to 48 hours,
- (2) suspension-culturing the pluripotent stem cells obtained in (1), to form a cell aggregate in the absence of a factor for maintaining an undifferentiated state and in the presence of a Wnt signal transduction pathway inhibiting substance,
- (3) suspension-culturing the cell aggregate obtained in (2) in the presence of a BMP signal transduction pathway activating substance and a Sonic hedgehog signal transduction pathway activating substance, and
- (4) suspension-culturing the cell aggregate obtained in (3) in the presence of a BMP signal transduction pathway inhibiting substance, to obtain a cell mass comprising pituitary tissue,
- wherein the TGFβ family signal transduction pathway inhibiting substance is selected from a group consisting of a TGFβ signal transduction pathway inhibitor, Nodal/Activin signal transduction inhibitor and BMP signal transduction inhibitor,
- wherein the BMP signal transduction pathway activating substance in (3) is added during a period when not less than 10% of the cells of the surface layer of the aggregate formed in (2) form a tight junction, and
- wherein the factor for maintaining an undifferentiated state comprises a FGF signal transduction pathway activating substance.
4 more elements
A method for producing a pituitary tissue, the method comprising:
- (1) culturing pluripotent stem cells in the absence of feeder cells and in a medium comprising 1) a TGFβ family signal transduction pathway inhibiting substance and/or a Sonic hedgehog signal, transduction pathway activating substance, and 2) a factor for maintaining an undifferentiated state for 6 to 48 hours,
- (2) suspension-culturing the pluripotent stem cells obtained in (1), to form a cell aggregate in the absence of a factor for maintaining an undifferentiated state and in the presence of a Wnt signal transduction pathway inhibiting substance for 72 hours or less,
- (3) suspension-culturing the cell aggregate obtained in (2) in the presence of a BMP signal transduction pathway activating substance and a Sonic hedgehog signal transduction pathway activating substance, to obtain a cell mass comprising pituitary tissue, and
- (4) collecting pituitary tissue from the cell mass obtained in (3),
- wherein the TGFβ family signal transduction pathway inhibiting substance is selected from the group consisting of a TGFβ signal transduction pathway inhibitor, Nodal/Activin signal transduction inhibitor and BMP signal transduction inhibitor,
- wherein the BMP signal transduction pathway activating substance in the (3) is added during a period when not less than 10% of the cells of the surface layer of the aggregate formed in the (2) form a tight junction, and
- wherein the factor for maintaining an undifferentiated state comprises a FGF signal transduction pathway activating substance.
4 more elements
Dependencies are inferred from the claim text.