Reprogramming cells
First Claim
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1. A composition comprising an induced mammalian pluripotent stem cell (iPSC) and a cell culture medium, wherein:
- (a) the iPSC comprises an exogenous polynucleotide encoding an Oct4 polypeptide;
(b) the cell culture medium comprises;
(i) a small molecule selected from the group consisting of F2,6P (fructose 2,6-bisphosphate), F6P (fructose 6-phosphate), DNP (2,4-dinitrophenol), NOG (N-oxalylglycine), QC (quercetin), 2-HA (2-hydroxyglutaric acid), NA (nicotinic acid), and PDK1 activator (3′
-phosphoinositide-dependent kinase-1), and(ii) one or both of a TGFβ
receptor/ALK5 inhibitor and a MEK inhibitor; and
(c) the iPSC is reprogrammed from a non-pluripotent mammalian cell;
wherein contacting the non-pluripotent mammalian cell with the small molecule enhances reprogramming when compared to without the small molecule.
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Abstract
The present invention provides for methods, compositions, and kits for producing an induced pluripotent stem cell from a non-pluripotent mammalian cell using a 3′-phosphoinositide-dependent kinase-1 (PDK1) activator or a compound that promotes glycolytic metabolism as well as other small molecules.
43 Citations
20 Claims
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1. A composition comprising an induced mammalian pluripotent stem cell (iPSC) and a cell culture medium, wherein:
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(a) the iPSC comprises an exogenous polynucleotide encoding an Oct4 polypeptide; (b) the cell culture medium comprises; (i) a small molecule selected from the group consisting of F2,6P (fructose 2,6-bisphosphate), F6P (fructose 6-phosphate), DNP (2,4-dinitrophenol), NOG (N-oxalylglycine), QC (quercetin), 2-HA (2-hydroxyglutaric acid), NA (nicotinic acid), and PDK1 activator (3′
-phosphoinositide-dependent kinase-1), and(ii) one or both of a TGFβ
receptor/ALK5 inhibitor and a MEK inhibitor; and(c) the iPSC is reprogrammed from a non-pluripotent mammalian cell; wherein contacting the non-pluripotent mammalian cell with the small molecule enhances reprogramming when compared to without the small molecule. - View Dependent Claims (2, 3, 4, 5)
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6. A composition for use in manufacturing a mammalian pluripotent stem cell for application in regenerative medicine, the composition comprising a cell culture medium comprising a small molecule and one or both of a TGFβ
- receptor/ALK5 inhibitor and a MEK inhibitor, wherein (i) the small molecule is present in an amount that enhances reprogramming of a non-pluripotent mammalian cell comprising an exogenous polynucleotide encoding an Oct4 polypeptide and (ii) the small molecule is selected from the group consisting of F2,6P (fructose 2,6-bisphosphate), F6P (fructose 6-phosphate), DNP (2,4-dinitrophenol), NOG (N-oxalylglycine), QC (quercetin), 2-HA (2-hydroxyglutaric acid), NA (nicotinic acid), or a PDK1 (3′
-phosphoinositide-dependent kinase-1) activator. - View Dependent Claims (7, 8)
- receptor/ALK5 inhibitor and a MEK inhibitor, wherein (i) the small molecule is present in an amount that enhances reprogramming of a non-pluripotent mammalian cell comprising an exogenous polynucleotide encoding an Oct4 polypeptide and (ii) the small molecule is selected from the group consisting of F2,6P (fructose 2,6-bisphosphate), F6P (fructose 6-phosphate), DNP (2,4-dinitrophenol), NOG (N-oxalylglycine), QC (quercetin), 2-HA (2-hydroxyglutaric acid), NA (nicotinic acid), or a PDK1 (3′
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9. A kit comprising a mixture of (A) a cell culture medium, (B) a MEK inhibitor, and (C) a small molecule in an amount that enhances reprogramming of a non-pluripotent mammalian cell when used in a method of obtaining an induced mammalian pluripotent stem cell (iPSC) comprising:
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(a) introducing into a non-pluripotent mammalian cell a polynucleotide encoding an Oct4 polypeptide; (b) contacting the non-pluripotent mammalian cell with (i) the small molecule, wherein the small molecule enhancing reprogramming is selected from the group consisting of F2,6P (fructose 2,6-bisphosphate), F6P (fructose 6-phosphate), DNP (2,4-dinitrophenol), NOG (N-oxalylglycine), QC (quercetin), 2-HA (2-hydroxyglutaric acid), NA (nicotinic acid), and PDK1 activator (3′
-phosphoinositide-dependent kinase-1), and(ii) the MEK inhibitor; and (c) culturing the contacted non-pluripotent mammalian cell of (b) in the culture medium, thereby reprogramming the non-pluripotent mammalian cell to produce an induced mammalian pluripotent stem cell; wherein contacting the non-pluripotent mammalian cell with the small molecule enhances reprogramming when compared to without the small molecule. - View Dependent Claims (10, 11, 12, 13, 14, 15)
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16. A method of making an induced mammalian pluripotent stem cell (iPSC), the method comprising:
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(a) introducing into a non-pluripotent mammalian cell a polynucleotide encoding an Oct4 polypeptide; (b) contacting the non-pluripotent mammalian cell with (i) a small molecule selected from the group consisting of F2,6P (fructose 2,6-bisphosphate), F6P (fructose 6-phosphate), DNP (2,4-dinitrophenol), NOG (N-oxalylglycine), QC (quercetin), 2-HA (2-hydroxyglutaric acid), NA (nicotinic acid), and PDK1 activator (3′
-phosphoinositide-dependent kinase-1), and(ii) one or both of a TGFβ
receptor/ALK5 inhibitor and a MEK inhibitor; and(c) culturing the contacted non-pluripotent mammalian cell of (b), thereby reprogramming the non-pluripotent mammalian cell to produce an induced mammalian pluripotent stem cell; wherein contacting the non-pluripotent mammalian cell with the small molecule enhances reprogramming when compared to without the small molecule; and
further wherein (iii) the MEK inhibitor is PD0325901, or (iv) the method further comprises contacting the non-pluripotent mammalian cell with one or more of a glycogen synthase kinase 3 (GSK3) inhibitor, and a histone deacetylase (HDAC) inhibitor. - View Dependent Claims (17, 18, 19, 20)
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Specification