Compositions and methods for differentiating pluripotent stem cells into primitive blood cells and uses thereof
Compositions and methods that employ various combinations of such factors as retinoic acid signaling inhibitors, antioxidants, BMP4, VEGF, prostaglandin E 2 pathway stimulants, TPO, SCF, FLT-3, EPO, TGFβ1, p38 MAPK inhibitors, beta adrenergic receptor agonists, cell cycle inhibitors, RXR agonists, Cripto, and chromatin remodelers to drive differentiation of pluripotent stem cells towards primitive blood cells. Uses of such primitive blood cells are provided.
1. A differentiation medium comprising:
a retinoic acid signaling inhibitor comprising DEAB;
an antioxidant comprising ascorbic acid;
a stimulant of prostaglandin E 2 pathway comprising prostaglandin E 2 (PGE2);
a P38 MAPK inhibitor comprising LY2228820;
BMP4;
vascular endothelial growth factor (VEGF); and
a beta adrenergic receptor agonist comprising norepinephrine.
2. The differentiation medium of claim 1 wherein:
the DEAB is present in an amount of from about 0.3 μM to about 300 μM;
the PGE2 is present in an amount of from about 0.6 μM to about 60 μM;
the LY2228820 is present in an amount of from about 3 nM to about 3 μM;
the BMP4 is present in an amount of from about 0.3 ng/ml to about 300 ng/mL;
the VEGF is present in an amount of from about 0.01 ng/mL to about 30 ng/mL; and
the norepinephrine is present in an amount of from about 10 μM to about 10 mM.
3. The differentiation medium of claim 1 , further comprising thrombopoietin (TPO), stem cell factor (SCF), fms-related tyrosine kinase 3 (FLT3), erythropoietin (EPO), and transforming growth factor beta (TGFβ1).
4. The differentiation medium of claim 3 wherein:
the TPO is present in an amount of from about 0.6 ng/mL to about 600 ng/mL;
the SCF is present in an amount of from about 0.6 ng/mL to about 600 ng/mL;
the FLT3 is present in an amount of from about 0.6 ng/mL to about 600 ng/mL;
the EPO is present in an amount of from about 0.6 ng/mL to about 600 ng/mL; and
the TGFβ1 is present in an amount of from about 0.15 ng/mL to about 150 ng/mL.
5. The differentiation medium of claim 3 wherein:
the DEAB is present in an amount of from about 0.3 μM to about 300 μM;
the PGE2 is present in an amount of from about 0.6 μM to about 60 μM;
the LY2228820 is present in an amount of from about 3 nM to about 3 μM;
the BMP4 is present in an amount of from about 0.3 ng/ml to about 300 ng/mL;
the VEGF is present in an amount of from about 0.01 ng/mL to about 30 ng/mL; and
the norepinephrine is present in an amount of from about 10 μM to about 10 mM
the TPO is present in an amount of from about 0.6 ng/mL to about 600 ng/mL;
the SCF is present in an amount of from about 0.6 ng/mL to about 600 ng/mL;
the FLT3 is present in an amount of from about 0.6 ng/mL to about 600 ng/mL;
the EPO is present in an amount of from about 0.6 ng/mL to about 600 ng/mL; and
the TGFβ1 is present in an amount of from about 0.15 ng/mL to about 150 ng/mL.
6. A method of differentiation comprising contacting a cell with the differentiation medium of claim 1 .
7. The method of claim 6 wherein the cell comprises a pluripotent stem cell and wherein the contacting comprises contacting for a time and under conditions sufficient to generate a hematopoietic stem cell.
8. The method of claim 7 wherein the hematopoietic stem cell is capable of further differentiating into a myeloid cell and is also capable of further differentiating into a lymphoid cell.
9. The method of claim 7 further comprising differentiating the hematopoietic stem cell into a cell selected from the group consisting of a myeloid cell and a lymphoid cell.
10. The method of claim 9 wherein the differentiating the hematopoietic stem cell is performed in vitro.
11. The method of claim 9 wherein the differentiating the hematopoietic stem cell is performed in vivo.
12. The method of claim 6 wherein the cell is a human cell.
13. The method of claim 6 wherein the contacting comprises initially contacting the cell with the differentiation medium by adding a volume of the differentiation medium to an existing volume of non-differentiation medium to generate a mixed medium, contacting the cell with the mixed medium, and subsequently replacing substantially all the mixed medium after about 12-48 hours from the initial contacting with a fresh volume of the differentiation medium.
14. The method of claim 13 wherein after the replacing substantially all the mixed medium the contacting further comprises adding a fresh volume of the differentiation medium only to an existing volume of the differentiation medium, wherein the existing volume comprises medium contacting the cell for a period of at least two hours.
15. The method of claim 6 wherein the cell comprises an intact embryoid body formed for a period of at least 7 days prior to the contacting.
16. The method of claim 15 wherein the contacting comprises contacting the intact embryoid body with the differentiation medium for a period of at least 5 days prior to plating the embryoid body, and then plating the embryoid body.
17. The method of claim 16 wherein the contacting, after the plating, further comprises adding a fresh volume of the differentiation medium only to an existing volume of the differentiation medium without removing any of the existing volume of the differentiation medium, wherein the existing volume comprises medium contacting the cell for a period of at least 15 minutes.
18. The method of claim 6 wherein the contacting comprises contacting the cell with the differentiation medium in the absence of feeder cells.
19. A method of engrafting a blood cell in a human subject comprising administering a hematopoietic stem cell or a cell differentiated therefrom to the subject, wherein the hematopoietic stem cell is generated by contacting a cell with the differentiation medium of claim 1 .