Method for culturing stem cell
AssigneesRiken, Sumitomo Chemical Company, Limited
InventorsYoshiki Sasai, Hidetaka Suga
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Abstract
The present invention provides a method of obtaining aggregates containing a rostral hypothalamus tissue and a rostral head ectodermal tissue, a hypophysis precursor tissue and a hypophysis hormone producing cell, by using a serum-free medium (preferably substantially free of growth factor and insulins), forming homogeneous aggregates of stem cells from pluripotent stem cells such as ES cell and the like, which are plated at a high cell concentration, and subjecting the formed aggregates to floating-culture.
Claims24
A method of producing an aggregate comprising both a central nervous tissue and a non-neural head ectodermal tissue, comprising
- a step of forming an aggregate of stem cells in a serum-free medium, and
- a step of floating-culturing the formed aggregate in a serum-free medium,
- wherein a Shh signal promoter and a BMP signal promoter are added to the serum-free medium used for the floating-culturing,
- wherein the Shh signal promoter is SAG and is added within 192 hours from the start of the floating-culture at a concentration of 50-1000 nM, and
- wherein the BMP signal promoter is BMP2 and/or BMP4 and is added within 96 hours from the start of the floating-culture at a concentration of 0.5-5 nM,
- thereby producing an aggregate comprising both a central nervous tissue and a non-neural head ectodermal tissue.
A method of producing an aggregate comprising both a hypothalamus tissue and a non-neural head ectoderm, comprising
- a step of forming an aggregate of stem cells in a serum-free medium, and
- a step of floating-culturing the formed aggregate in a serum-free medium,
- wherein a Shh signal promoter and a BMP signal promoter are added to the serum-free medium used for the floating-culturing,
- wherein the Shh signal promoter is SAG and is added within 192 hours from the start of the floating-culture at a concentration of 50-1000 nM, and
- wherein the BMP signal promoter is BMP2 and/or BMP4 and is added within 96 hours from the start of the floating-culture at a concentration of 0.5-5 nM,
- thereby producing an aggregate comprising both a hypothalamus tissue and a non-neural head ectoderm.
A method of producing an aggregate comprising both a hypothalamus tissue and a non-neural head ectoderm, comprising
- (1) forming an aggregate of stem cells in a serum-free medium,
- (2) floating-culturing the formed aggregate in a serum-free medium,
- wherein a Shh signal promoter and a BMP signal promoter are added to the serum-free medium used for the floating-culturing,
- wherein the Shh signal promoter is SAG and is added within 192 hours from the start of the floating-culture at a concentration of 50-1000 nM, and
- wherein the BMP signal promoter is BMP2 and/or BMP4 and is added within 96 hours from the start of the floating-culture at a concentration of 0.5-5 nM,
- thereby obtaining an aggregate that comprises Rx-positive neural precursor cells and E-cad and cytokeratin-positive non-neural ectoderm cells, the neural precursor cells being present in the aggregate, and the non-neural ectoderm cells being present on the surface of the aggregate and forming an epithelial structure, and
- (3) floating-culturing the aggregate obtained in (2) in a serum-free medium containing SAG, thereby obtaining an aggregate comprising both a hypothalamus tissue and a non-neural head ectoderm.
Dependencies are inferred from the claim text.