Methods for epicardial differentiation of human pluripotent stem cells
Methods for generating high-yield, high-purity epicardial cells are described. Wnt/β-catenin signaling is first activated in human cardiac progenitor cells, by, for example, inhibiting Gsk-3 to induce differentiation into epicardial cells. Methods for long-term in vitro maintenance of human cardiac progenitor cell-derived epicardial cells and method comprising chemically defined, xeno-free, and albumin-free culture conditions are also provided.
1. A method for generating a population of epicardial cells from human pluripotent stem cells, the method comprising culturing human cardiac progenitor cells, differentiated from human pluripotent stem cells, in a chemically defined, albumin-free culture medium that comprises an activator of Wnt/β-catenin signaling and that does not comprise Bone Morphogenetic Protein 4 (BMP4), whereby a cell population comprising human epicardial cells is obtained.
2. The method of claim 1 , wherein the human cardiac progenitor cells express one or more of Isl1, Nkx2.5, and Flk-1.
3. The method of claim 1 , wherein at least 95% of cells of the cell population are epicardial cells positive for expression of Wilms' tumor suppressor protein (WT1).
4. The method of claim 3 , wherein the epicardial cells are self-renewing for at least 25 population doublings when cultured in the presence of an inhibitor of TGFβ signaling.
5. The method of claim 1 , wherein the activator of Wnt/β-catenin signaling is a Gsk3 inhibitor.
6. The method of claim 5 , wherein the Gsk3 inhibitor is a small molecule selected from the group consisting of CHIR99021, CHIR98014, BIO-acetoxime, BIO, LiCl, SB216763, SB415286, AR A014418, 1-Azakenpaullone, and Bis-7-indolylmaleimide.
7. The method of claim 5 , wherein the Gsk3 inhibitor is CHIR99021 and is present in a concentration of about 0.2 μM to about 9 μM.
8. The method of claim 1 , wherein the human cardiac progenitor cells are obtained by a method comprising (i) culturing human pluripotent stem cells in a chemically defined, albumin-free culture medium comprising an activator of Wnt/β-catenin signaling to obtain a first cell population comprising mesodermal cells positive for expression of Brachyury/T; and (ii) culturing the first cell population in a chemically defined culture medium that comprises an inhibitor of Wnt/β-catenin signaling, whereby a cell population comprising human cardiac progenitor cells is obtained.
9. The method of claim 8 , wherein the human cardiac progenitor cells express one or more of Isl1, Nkx2.5, and Flk-1.
10. The method of claim 8 , wherein the activator of Wnt/β-catenin signaling is a Gsk3 inhibitor.
11. The method of claim 10 , wherein the Gsk3 inhibitor is a small molecule selected from the group consisting of CHIR99021, CHIR98014, BIO-acetoxime, BIO, LiCl, SB216763, SB415286, AR A014418, 1-Azakenpaullone, and Bis-7-indolylmaleimide.
12. The method of claim 8 , wherein the inhibitor of Wnt/β-catenin signaling is selected from the group consisting of a small molecule that stabilizes axin and stimulates β-catenin degradation, an inhibitor of porcupine, an antibody that blocks activation of a Wnt ligand receptor, an antibody that binds to one or more Wnt ligand family members, and a short hairpin interfering RNA (shRNA) for β-catenin in the first cell population.
13. The method of claim 12 , wherein the small molecule that stimulates β-catenin degradation and stabilizes axin is XAV939.
14. The method of claim 12 , wherein the porcupine inhibitor is selected from the group consisting of IWP2 and IWP4, or a combination thereof.
15. The method of claim 12 , wherein the porcupine inhibitor is present in a concentration of about 1 μM to about 4 μM.
16. The method of claim 1 , wherein no cell separation or selection step is used to obtain the cell population comprising epicardial cells.
17. A method for long-term in vitro maintenance of self-renewing human epicardial cells, the method comprising:
culturing human cardiac progenitor cells differentiated from human pluripotent stem cells in a chemically defined, albumin-free culture medium that comprises an activator of Wnt/β-catenin signaling and that does not comprise Bone Morphogenetic Protein 4 (BMP4), whereby a cell population comprising human epicardial cells is obtained; and
culturing the cell population comprising human epicardial cells in the presence of an inhibitor of TGFβ signaling, whereby the human epicardial cells are maintained in vitro as self-renewing epicardial cells for at least 25 population doublings, are not immortalized, and maintain the ability to undergo epithelial-to-mesenchymal transition (EMT).