Multi-step method for the differentiation of insulin positive, glucose responsive cells
AssigneeES Cell International PTE Ltd.
InventorsDiana Clarke, Josephine S. D'Alessandro, Kuanghui Lu, Anlai Wang
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Abstract
The present invention provides improved methods of differentiating insulin+, glucose responsive islet-like structures from insulin− cells. The invention further provides methods for using insulin+, glucose responsive islet-like structures, as well as the insulin+, glucose responsive cells which comprise said islet-like clusters.
Claims81
A method for culturing substantially purified, insulin− cells, wherein the insulin− cells differentiate to insulin+ cells, which insulin+ cells are responsive to glucose.
A method for differentiating substantially purified, insulin− cells to insulin+, glucose responsive cells, comprising
- (a) culturing purified cells as non-adherent spheres;
- (b) selecting cells by culturing in the presence of a gp130 agonist;
- (c) dissociating the spheres and culturing in the presence of mitogens, wherein at least one mitogen is an FGF family member;
- (d) culturing the spheres in the presence of at least two growth factors, or growth factor agonists, wherein at least one growth factor is an FGF family member;
- (e) plating the spheres on a coated substratum in high-glucose media;
- and
- (f) culturing the spheres in media containing standard glucose.
A method for differentiating substantially purified, insulin− cells to insulin+, glucose responsive cells, comprising
- (a) culturing purified cells as non-adherent spheres;
- (b) selecting cells by culturing in serum-free media supplemented with cardiotrophin-1;
- (c) dissociating the spheres and culturing in serum-free media supplemented with FGF-18 and a hedgehog polypeptide;
- (d) culturing the spheres in the presence of at least two growth factors, or growth factor agonists, wherein at least one growth factor is FGF-18;
- (e) plating the spheres on a coated substratum in high-glucose media;
- and
- (f) culturing the spheres in media containing standard glucose supplemented with nicotinamide.
A composition comprising an islet-like structure differentiated from substantially purified insulin− cells or progeny thereof.
A composition comprising insulin+, glucose responsive cells differentiated from substantially purified insulin− cells or progeny thereof.
A composition comprising an islet-like structure differentiated from substantially purified insulin− cells or progeny thereof and a pharmaceutically acceptable carrier or excipient.
A composition comprising insulin+, glucose responsive cells differentiated from substantially purified insulin− cells or progeny thereof and a pharmaceutically acceptable carrier or excipient.
A method of increasing the number of Pdx1− cells in a non-adherent sphere of insulin− cells, wherein said Pdx1− can differentiate to Pdx1+ cells comprising
- (a) culturing said insulin− cells to form non-adherent sphere;
- and
- (b) culturing said non-adherent sphere in media comprising an FGF mitogen and a cAMP elevating agent for at least one day, whereby following at least one day in culture in media comprising an FGF mitogen and a cAMP elevating agent the number of Pdx1− cells in said non-adherent sphere which can differentiate to Pdx1+ cells increases.
A method of dissociating a cluster of cells, comprising culturing the cluster of cells in the presence of Protease XXIII.
Use of insulin+, glucose responsive cells in the manufacture of a medicament to treat a condition in a patient, wherein said condition is characterized by an inhibition of glucose responsiveness.
Use of islet-like structures containing insulin+, glucose responsive cells in the manufacture of a medicament to treat a condition in a patient, wherein said condition is characterized by an inhibition of glucose responsiveness.
Dependencies are inferred from the claim text.