Generation of functional and durable endothelial cells from human amniotic fluid-derived cells
This disclosure is directed to methods for reproducibly generating substantial amounts of endothelial cells from amniotic cells. The endothelial cells generated in accordance with the present methodology, as well as therapeutic methods utilizing these cells, are also disclosed.
1. A method of generating endothelial cells, comprising
obtaining amniotic cells; and
culturing the amniotic cells for at least 21 days under conditions where transcription factors ETV2, FLI1 and ERG are expressed in the amniotic cells in the presence of a TGFβ signaling inhibitor, wherein said amniotic cells express ETV2 for the first 13-15 days, said TGFβ signaling inhibitor is present for the first 20-21 days, and FLI1 and ERG are constitutively expressed, thereby obtaining endothelial cells.
2. The method of claim 1 , wherein the amniotic cells are transduced with vectors comprising nucleic acids encoding transcription factors ETV2, FLI1 and ERG to achieve expression of the transcription factors.
3. The method of claim 1 , wherein mRNAs encoding the transcription factors are delivered into amniotic cells to achieve expression of the transcription factors.
4. The method of claim 1 , wherein the transcription factors are delivered to amniotic cells in polypeptide forms.
5. The method of claim 1 , wherein the TGFβ signaling inhibitor is an inhibitor specific for the type I TGFβ receptors.
6. The method of claim 5 , wherein said inhibitor is a polypeptide comprising a soluble form of a type I TGFβ receptor, an antibody directed to a type I TGFβ receptor or ligand, or a small molecule compound.
7. The method of claim 6 , wherein said inhibitor is a small molecule compound selected from SB-431542, A 83-01, D 4476, LY 364947, SB 525334, SD 208, and SJN 2511.
8. The method of claim 7 , wherein said inhibitor is SB-431542.
9. The method of claim 1 , wherein the cells are cultured for a total duration of at least 28 days.
10. The method of claim 1 , wherein the cells are cultured for a total duration of at least 42 days.
11. The method of claim 1 , wherein ERG is ERG1.
12. The method of claim 1 , wherein cells characterized by expression of surface markers, VE-cadherin, CD31 and VEGFR2, are isolated.