Methods for programming differentiated cells into hematopoietic stem cells
The disclosure relates to the development of methods for making hematopoietic stem cells from differentiated cells by introducing and expressing transcription factors. More particularly, the disclosure provides methods for redirecting differentiated cells to a hematopoietic stem cell state or to a hemogenic endothelial cell state by direct programming with specific combinations of transcription factors.
1. A method for programming a differentiated cell into a hematopoietic stem cell, the method comprising introducing a combination of transcription factors in the differentiated cell, wherein the combination comprises GATA binding protein 2 (GATA2), growth factor independent 1B (GFI1B), and c-Fos.
2. The method of claim 1 , wherein the combination of transcription factors further comprises ETS translocation variant 6 (ETV6).
3. The method of claim 1 , wherein the combination of transcription factors further comprises a transcription factor selected from the group consisting of stem cell leukemia (SCL/TAL1), runt-related transcription factor 1 (RUNX1), and B lymphoma Mo-MLV insertion region 1 homolog (BMI1).
4. The method of claim 1 further comprising the step of screening the hematopoietic stem cell for:
(a) expression of a hemogenic endothelial cell marker or a hematopoietic stem cell marker; or
(b) uptake of acetylated low density lipoprotein (acLDL).
5. The method of claim 4 wherein the hemogenic endothelial cell marker or the hematopoietic stem cell marker is a marker selected from the group consisting of: CD31, CD34, CD38 lo/− , CD41, CD43, CD45, CD49f, Thy1/CD90, CD105, CD117/c-kit, CD133, CD150, Sca-1, Tie2, VE-Cadherin, KDR/FLK1, Flk-2/Flt3, and CXCR4.
6. The method of claim 5 , wherein the hematopoietic stem cell marker is selected from the group consisting of: CD31, CD34, CD41, CD117/c-kit, CD133, Sca-1, Tie2, VE-Cadherin, and CD150.
7. The method of claim 2 further comprising the step of screening the hematopoietic stem cell for a lack of expression of a differentiated hematopoietic lineage (lin) marker.
8. The method of claim 7 , wherein the lin − marker is selected from the group consisting of CD4, CD5, CD8, CD45RA/B220, Gr-1/Ly-6G/C, and Ter119.
9. The method of claim 4 further comprising the step of isolating the cell expressing the hematopoietic stem cell marker.
10. The method of claim 9 further comprising the step of co-culturing the hematopoietic stem cell with a stromal cell.
11. The method of claim 10 , wherein the stromal cell is an AFT024 stromal cell.
12. The method of claim 2 , wherein the combination of transcription factors further comprises a transcription factor selected from the group consisting of stem cell leukemia (SCL/TAL1), runt-related transcription factor 1 (RUNX1), and B lymphoma Mo-MLV insertion region 1 homolog (BMI1).
13. The method of claim 2 further comprising the step of screening the hematopoietic stem cell for:
(a) expression of a hemogenic endothelial cell marker or a hematopoietic stem cell marker; or
(b) uptake of acetylated low density lipoprotein (acLDL).
14. The method of claim 13 , wherein the hemogenic endothelial cell marker or the hematopoietic stem cell marker is a marker selected from the group consisting of: CD31, CD34, CD38 lo/− , CD41, CD43, CD45, CD49f, Thy1/CD90, CD105, CD117/c-kit, CD133, CD150, Sca-1, Tie2, VE-Cadherin, KDR/FLK1, Flk-2/Flt3, and CXCR4.
15. The method of claim 14 , wherein the hematopoietic stem cell marker is selected from the group consisting of: CD31, CD34, CD41, CD117/c-kit, CD133, Sca-1, Tie2, VE-Cadherin, and CD150.
16. The method of claim 3 further comprising the step of screening the hematopoietic stem cell for:
(a) expression of a hemogenic endothelial cell marker or a hematopoietic stem cell marker; or
(b) uptake of acetylated low density lipoprotein (acLDL).
17. The method of claim 12 further comprising the step of screening the hematopoietic stem cell for:
(a) expression of a hemogenic endothelial cell marker or a hematopoietic stem cell marker; or
(b) uptake of acetylated low density lipoprotein (acLDL).
18. The method of claim 1 further comprising the step of screening the hematopoietic stem cell for a lack of expression of a differentiated hematopoietic lineage (lin) marker.
19. The method of claim 18 , wherein the lin − marker is selected from the group consisting of CD4, CD5, CD8, CD45RA/B220, Gr-1/Ly-6G/C, and Ter119.
20. The method of claim 13 further comprising the step of isolating the cell expressing the hematopoietic stem cell marker.
21. The method of claim 20 further comprising the step of co-culturing the hematopoietic stem cell with a stromal cell.
22. The method of claim 21 , wherein the stromal cell is an AFT024 stromal cell.