Methods of specifying mesodermal, endodermal and mesoendodermal cell fates
View Patent ↗We disclose a method for producing a mesodermal or a endodermal cell from a pluripotent stem cell, the method comprising activating a Wnt signalling pathway in the pluripotent stem cell. In some embodiments, the pluripotent stem cell is in a substantially 2 dimensional configuration, such as a monolayer, for at least a portion of the time when the Wnt signalling pathway is activated.
1. A method for producing a mesodermal progenitor cell from an embryonic stem cell, the method comprising activating a Wnt signaling pathway in the embryonic stem cell by culturing the embryonic stem cell in Wnt-3A conditioned medium (CM) or in a medium comprising a chemical inhibitor of glycogen synthase kinase-3β, wherein the Wnt signaling pathway is activated for at least 8 days, thereby producing a mesodermal progenitor cell, wherein the method does not produce an ectodermal cell.
2. The method of claim 1 , in which the embryonic stem cell is in a substantially 2 dimensional configuration for at least a portion of the time when the Wnt signaling pathway is activated.
3. The method of claim 2 , in which the embryonic stem cell is in a substantially 2 dimensional configuration for substantially all of the time when the Wnt signaling pathway is activated.
4. The method of claim 2 , in which the 2 dimensional configuration is a monolayer.
5. The method of claim 1 , further comprising terminally differentiating the mesodeimal cell to a cell selected from the group consisting of an adipocyte, a cardiomyocyte, a chondrocyte, a fibroblast, a hematopoietic cell, a myocyte, an osteoblast, and an endothelial cell.
6. The method of claim 1 , in which the mesodermal cell expresses a mesodermal marker selected from the group consisting of: T-brachyury, Runx 1 and Pitx2.
7. The method of claim 1 , in which the chemical inhibitor comprises iGSK-3β or BIO, or a variant thereof capable of reducing GSK activity.
8. A method of inducing expression of a mesoderm specific marker in a cell comprising culturing an embryonic stem cell in Wnt-3A conditioned medium (CM) or in a medium comprising a chemical inhibitor of glycogen synthase kinase-3β, wherein the culturing is for at least 8 days, thereby inducing expression of a mesoderm specific marker in the cell.
9. The method of claim 8 , in which the mesoderm specific marker is selected from the group consisting of: T-brachyury, Runx1 and Pitx2.
10. The method of claim 1 , wherein the Wnt signaling pathway is activated for at least 10 days.
11. The method of claim 1 , wherein the embryonic stem cell is a human embryonic stem cell.
12. The method of claim 1 , wherein the Wnt signaling pathway is activated for at least 2 weeks.
13. The method of claim 1 , wherein the Wnt signaling pathway is activated for at least 3 weeks.
14. The method of claim 1 , wherein the Wnt signaling pathway is activated for at least 4 weeks.