Endothelial cell production by programming
The invention generally regards methods for providing endothelial cells and precursors of endothelial cells from a variety of cell sources, such as pluripotent stem cells. Also provided are therapeutic compositions including the provided endothelial cells, and methods of using them for the treatment of subjects.
1. A method of providing endothelial cells by forward programming of pluripotent stem cells, comprising contacting the pluripotent stem cells with one or more endothelial programming factor genes, or products of such genes, in an amount sufficient to cause forward programming of the pluripotent stem cells into endothelial cells, said one or more endothelial programming factors or gene products of such genes comprises an ETV2 and/or ERG gene or gene product or an isoform thereof.
2. The method of claim 1 , wherein the pluripotent stem cells are induced pluripotent stem cells.
3. The method of claim 1 , wherein said contacting said pluripotent stem cells comprises contacting said pluripotent stem cells with an expression cassette encoding one or more endothelial reprogramming factor genes, wherein said expression cassette is introduced into said pluripotent stem cells and expressed by said pluripotent stem cells.
4. The method of claim 1 , wherein said contacting said pluripotent stem cells comprises contacting said pluripotent stem cells with said products of such genes, wherein said products of such genes are polypeptide products of said one or more endothelial programming factor genes.
5. The method of claim 1 , wherein said contacting said pluripotent stem cells comprises contacting said pluripotent stem cells with said products of such genes, wherein said products of such genes are RNA transcript products of said one or more endothelial programming factor genes.
6. The method of claim 1 , wherein the one or more endothelial programming factor genes or products of such genes comprises an ERG gene or gene product or isoform thereof.
7. The method of claim 6 , wherein the ERG gene or gene product or isoform thereof is ERG isoform 3.
8. The method of claim 1 , wherein the one or more endothelial programming factor genes or products of such genes comprises an ETV2 gene or gene product or isoform thereof.
9. The method of claim 1 , wherein the endothelial cells comprise one or more characteristics of endothelial cells comprising:
(i) expression of one or more endothelial cell markers;
(ii) the ability to take up acetylated low density lipoprotein (ac-LDL), form tube-like structures in a three dimensional matrix, upregulate expression of one or more cell adhesion molecules in response to one or more pro-inflammatory stimuli, or a combination of two or more of these abilities; or
(iii) endothelial cell morphological features.
10. The method of claim 9 , wherein the one or more endothelial cell markers are selected from the group consisting of: CD144, CD31, CD34, ESAM, CD102, CD143, CD93, CD184, CD105, CD146, von Willebrand factor, ZO-1, CD304, and DLL4.
11. The method of claim 9 , wherein the one or more pro-inflammatory stimuli are selected from the group consisting of TNF and IL-1, and the one or more cell adhesion molecules are selected from the group consisting of CD54, CD62E, and CD106.
12. The method of claim 1 , wherein the provided endothelial cells are mature endothelial cells.
13. The method of claim 1 , wherein the pluripotent stem cells or progeny cells thereof are cultured in a medium comprising one or more growth factors.
14. The method of claim 13 , wherein the one or more growth factors include basic fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), or both.
15. The method of claim 1 , further defined as a method of generating hematopoietic cells, said method comprising culturing the pluripotent stem cells contacted with the one or more endothelial programming factor genes, or products of such genes, in a hematopoietic culture medium, wherein said one or more endothelial programming factors genes, or products of such genes comprises an EVT2 gene or gene product or isoform thereof, thereby producing endothelial cells comprising hematopoietic cells that are CD31+, CD43+, CD235a/CD41a-, and CD45+.
16. The method of claim 1 , further defined as a method of generating mesenchymal cells, said method comprising culturing the pluripotent stem cells contacted with the one or more endothelial programming factor genes, or products of such genes, in a mesenchymal medium comprising FGF and a TGFβ inhibitor, thereby producing endothelial cells comprising mesenchymal cells that that are CD31−, CD73+, and CD105+.