Simplified methods for generating endothelial cells from human pluripotent stem cells under defined conditions
Methods for generating human endothelial cells from human pluripotent stem cells under defined conditions in the absence of VEGF are described. Wnt/β-catenin signaling is activated in human pluripotent stem cells for a defined period, e.g., by inhibition of Gsk3, and then cultured without further exogenous activation of Wnt/β-catenin signaling to obtain a cell population containing human endothelial cells.
1. A method for generating a cell population comprising endothelial cells from human pluripotent stem cells, comprising the steps of:
(i) contacting cultured human pluripotent stem cells with an activator of Wnt/β-catenin signaling for a period of about two days in a cell culture medium suitable for maintenance of human endothelial cells, but substantially free of exogenous growth factors; and
(ii) obtaining a cell population comprising endothelial cells by culturing the contacted cells in the absence of the activator, for at least about three days to about ten days, in a cell culture medium suitable for maintenance of human endothelial cells but substantially free of exogenous growth factors.
2. The method of claim 1 , wherein the cultured human pluripotent stem cells are cultured at a density of about 250,000 cells/cm 2 to about 400,000 cells/cm 2 at the beginning of the contacting step.
3. The method of claim 1 , wherein the cell culture medium is selected from the group consisting of Advanced™ DMEM, VcG-Advanced™ DMEM, LaSR medium, Advanced™ DMEM-F12; VcG-Advanced™ DMEM-F12, StemPro® 34 medium, Advanced™ RPMI, and VcG-Advanced™ RPMI.
4. The method of claim 1 , wherein the cell culture medium substantially free of exogenous growth factors is LaSR medium.
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 selected from the group consisting of CHIR 99021, CHIR 98014, BIO-acetoxime, BIO, LiCl, SB 216763, SB 415286, AR A014418, 1-Azakenpaullone, and Bis-7-indolylmaleimide.
7. The method of claim 1 , wherein the Gsk3 inhibitor is CHIR 99021, CHIR 98014, or BIO-acetoxime.
8. The method of claim 7 , wherein the Gsk3 inhibitor is CHIR 99021 or CHIR 98014 at a concentration of at least about 4 μM to about 10 μM in the medium.
9. The method of claim 1 , wherein the obtained cell population comprises at least 50% endothelial cells.
10. The method of claim 1 , wherein in step (ii) the contacted cells are cultured in the absence of the activator for about ten days.
11. A kit for differentiating human pluripotent stem cells into endothelial cells, the kit comprising:
(i) a Gsk3 inhibitor;
(ii) a culture medium substantially free of exogenous growth factors and suitable for differentiation of human pluripotent stem cells into endothelial cells; and
(iii) instructions describing a method for differentiating human pluripotent stem cells into endothelial cells, the method employing the Gsk3 inhibitor and the culture medium.
12. The kit of claim 11 , wherein the culture medium is selected from the group consisting of Advanced™ DMEM, VcG-Advanced™ DMEM, LaSR medium, Advanced™ DMEM-F12; VcG-Advanced™ DMEM-F12, StemPro® 34 medium, Advanced™ RPMI, and VcG-Advanced™ RPMI.
13. A method for generating a cell population comprising CD31 + cells from human pluripotent stem cells, comprising the steps of:
(i) contacting cultured human pluripotent stem cells with an activator of Wnt/β-catenin signaling for a period of about two days in a cell culture medium suitable for maintenance of human endothelial cells, but substantially free of exogenous growth factors; and
(ii) obtaining a cell population comprising CD31 − cells by culturing the contacted cells in the absence of the activator, for about three days to about ten days, in a cell culture medium suitable for maintenance of human endothelial cells but substantially free of exogenous growth factors.
14. The method of claim 13 , wherein the cell population from step (ii) is cultured for about ten days in the cell culture medium suitable for maintenance of human endothelial cells.
15. The method of claim 13 , further comprising subjecting the cell population of step (ii) to selection for CD34 + cells to obtain a cell population enriched for angioblasts.