Reprogramming cells
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.
1. A method of inducing a non-pluripotent mammalian cell into an induced pluripotent stem cell, the method comprising:
(a) introducing into the non-pluripotent cell a polynucleotide encoding an Oct4 polypeptide; and
(b) contacting the non-pluripotent cell with a small molecule 3′-phosphoinositide-dependent kinase-1 (PDK1) activator, a TGFβ receptor/ALK5 inhibitor, and a MEK inhibitor, thereby inducing the non-pluripotent mammalian cell into an induced pluripotent stem cell.
2. The method of claim 1 , wherein the small molecule PDK1 activator is:
(a) an allosteric PDK1 activator; or
(b) (Z)-5-(4-Chlorophenyl)-3-phenylpent-2-enoic acid (“PS48”), (Z)-5-(4-Bromo-2-fluorophenyl)-3-phenylpent-2-enoic acid (“PS08”), 2-(3-(4-Chlorophenyl)-3-oxo-1-phenylpropylthio)acetic acid, (Z)-5-(Napthalen-2-yl)-3-phenylpent-2-enoic acid (“12Z”), or (Z)-5-(1H-Indol-3-yl)-3-phenylpent-2-enoic acid (“13Z”).
3. The method of claim 1 , further comprising contacting the non-pluripotent cell with a histone deacetylase (HDAC) inhibitor.
4. The method of claim 1 , further comprising one or more of:
(a) introducing into the non-pluripotent cell a polynucleotide encoding a Klf-4 polypeptide;
(b) introducing into the non-pluripotent cell a polynucleotide encoding a Sox-2 polypeptide; or
(c) introducing into the non-pluripotent cell a polynucleotide encoding a c-Myc polypeptide.
5. The method of claim 1 , wherein the non-pluripotent cell is a human cell.
6. The method of claim 1 , wherein contacting comprises:
(a) contacting the non-pluripotent cell with a small molecule PDK1 activator in the absence of a MEK inhibitor, followed by contacting the non-pluripotent cell with a small molecule PDK1 activator and a MEK inhibitor; or
(b) contacting the non-pluripotent cell with a small molecule PDK1 activator, a TGFβ receptor/ALK5 inhibitor, and a histone deacetylase (HDAC) inhibitor in the absence of a MEK inhibitor, followed by contacting the non-pluripotent cell with a small molecule PDK1 activator, a TGFβ receptor/ALK5 inhibitor, a histone deacetylase (HDAC) inhibitor and a MEK inhibitor.
7. A mixture comprising: isolated mammalian cells, a small molecule PDK1 activator, and:
(a) a TGFβ receptor/ALK5 inhibitor;
(b) a MEK inhibitor; and
(c) one or more vectors comprising a polynucleotide encoding one or more exogenous transcription factors selected from the group consisting of an Oct-4 polypeptide, a Klf-4 polypeptide, a c-myc polypeptide, and a Sox-2 polypeptide.
8. The mixture of claim 7 , wherein at least 99% of the cells are non-pluripotent cells.
9. The mixture of claim 7 , wherein the cells are human cells.
10. The mixture of claim 7 , wherein the small molecule PDK1 activator is:
(a) an allosteric PDK1 activator; or
(b) (Z)-5-(4-Chlorophenyl)-3-phenylpent-2-enoic acid (“PS48”), (Z)-5-(4-Bromo-2-fluorophenyl)-3-phenylpent-2-enoic acid (“PS08”), 2-(3-(4-Chlorophenyl)-3-oxo-1-phenylpropylthio)acetic acid, (Z)-5-(Napthalen-2-yl)-3-phenylpent-2-enoic acid (“12Z”), or (Z)-5-(1H-Indol-3-yl)-3-phenylpent-2-enoic acid (“13Z”).
11. The mixture of claim 7 , further comprising a histone deacetylase (HDAC) inhibitor.