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, an antioxidant, and BMP4.
2 . The differentiation medium of claim 1 further comprising one, all, or any subcombination of components selected from the group consisting of a stimulant of prostaglandin E 2 pathway, TPO, VEGF, SCF, FLT-3, EPO, and TGFβ1.
3 . The differentiation medium of claim 1 further comprising one, all, or any combination of components selected from the group consisting of a p38 MAPK inhibitor, a beta adrenergic receptor agonist, a cell cycle inhibitor, an RXR agonist, Cripto, and a chromatin remodeler.
4 . The differentiation medium of claim 3 wherein the p38 MAPK inhibitor is LY2228820, the cell cycle inhibitor is rapamycin, the RXR agonist is LG101506, and the chromatin remodeler is valproic acid.
5 . The differentiation medium of claim 3 wherein the beta adrenergic receptor agonist comprises one, all, or any combination of components selected from the group consisting of norepinephrine, epinephrine, salmeterol, and isoproterenol.
6 . The differentiation medium of claim 1 wherein the antioxidant comprises one, all, or any combination of components selected from the group consisting of sodium selenite, butein, glutathione, and N-acetyl cysteine.
7 . The differentiation medium of claim 1 wherein the retinoic acid signaling inhibitor comprises DEAB.
8 . A method of differentiation comprising exposing a cell to a differentiation medium as described in claim 1 .
9 . The method of claim 8 wherein the cell comprises a pluripotent stem cell and wherein the exposing comprises exposing for a time and under conditions sufficient to generate a hematopoietic stem cell.
10 . The method of claim 9 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.
11 . The method of claim 9 further comprising differentiating the hematopoietic stem cell into a cell selected from the group consisting of a myeloid cell and a lymphoid cell.
12 . The method of claim 11 wherein the differentiating the hematopoietic stem cell is performed in vitro.
13 . The method of claim 11 wherein the differentiating the hematopoietic stem cell is performed in vivo.
14 . The method of claim 8 wherein the cell is a human cell.
15 . The method of claim 8 wherein the exposing comprises initially exposing the cell to the differentiation medium by adding a volume of the differentiation medium to an existing volume of non-differentiation medium to generate a mixed medium, exposing the cell to the mixed medium, and subsequently replacing substantially all the mixed medium after about 12-48 hours from the initial exposing with a fresh volume of the differentiation medium.
16 . The method of claim 15 wherein after the replacing substantially all the mixed medium the exposing 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 exposed to the cell for a period of at least two hours.
17 . The method of claim 8 wherein the cell comprises an intact embryoid body formed for a period of at least 7 days prior to the exposing.
18 . The method of claim 17 wherein the exposing comprises exposing the intact embryoid body to the differentiation medium for a period of at least 5 days prior to plating the embryoid body, and then plating the embryoid body.
19 . The method of claim 18 wherein the exposing, 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 exposed to the cell for a period of at least 15 minutes.
20 . The method of claim 8 wherein the exposing comprises exposing the cell to the differentiation medium in the absence of feeder cells.
21 . 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 exposing a cell to a differentiation medium as described in claim 1 .