Reprogramming Cells

• US 20160257938A1
• Filed: 03/14/2016
• Published: 09/08/2016
• Est. Priority Date: 03/31/2010
• Status: Active Grant

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Zhu, Saiyong, Ding, Sheng
• Granted Patent: US 9,657,274 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 2501/065 Modulators of histone acetylation
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/604 Klf-4
• CPC Class Codes: C12N 2501/606 c-Myc
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/73 Hydrolases (EC 3.)
• CPC Class Codes: C12N 2506/03 from non-embryonic pluripotent stem cells
• CPC Class Codes: C12N 2506/094 from keratinocytes
• CPC Class Codes: C12N 2506/28 from vascular endothelial cells
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

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.

Claims

1. A method of inducing a non-pluripotent mammalian cell into an induced pluripotent stem cell, the method comprising:

contacting the non-pluripotent cell with a compound that promotes glycolytic metabolism, a TGFβ

receptor/ALK5 inhibitor, and a MEK inhibitor, under conditions sufficient to induce the cell to become a pluripotent stem cell, thereby inducing the non-pluripotent mammalian cell into an induced pluripotent stem cell.

2. The method of claim 1, wherein the compound that promotes glycolytic metabolism enhances reprogramming when compared to without the compound.

3. The method of claim 1, wherein the compound that promotes glycolytic metabolism is a glycolysis activator, a substrate for glycolysis, a glycolic intermediate or its metabolic precursors, a glucose uptake transporter activator, a mitochondrial respiration modulator, an oxidative phosphorylation inhibitor, or a hypoxia-inducible factor activator.

4. The method of claim 1, wherein the compound that promotes glycolytic metabolism up-regulates key glycolytic genes comprising GLUT1, HK2, PFK1, and/or LDHA.

5. The method of claim 2, wherein the compound that promotes glycolytic metabolism comprises F2,6P (fructose 2,6-bisphosphate), F6P (fructose 6-phosphate), DNP (2,4-dinitrophenol), NOG (N-oxaloylglycine), QC (quercetin), 2-HA (2-hydroxyglutaric acid), NA (nicotinic acid), or a PDK1 (3′

-phosphoinositide-dependent kinase-1) activator.

6. The method of claim 5, wherein the 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”

).

7. The method of claim 1, further comprising contacting the non-pluripotent cell with a histone deacetylase (HDAC) inhibitor.

8. The method of claim 1, further comprising one or more of:

(a) introducing into the non-pluripotent cell a polynucleotide encoding an Oct4 polypeptide;

(b) introducing into the non-pluripotent cell a polynucleotide encoding a Klf-4 polypeptide; and

(c) introducing into the non-pluripotent cell a polynucleotide encoding a Sox-2 polypeptide.

9. The method of claim 1, wherein the contacting comprises:

(a) contacting the non-pluripotent cell with a compound that promotes glycolytic metabolism in the absence of a MEK inhibitor, followed by contacting the non-pluripotent cell with a compound that promotes glycolytic metabolism and a MEK inhibitor;

or

(b) contacting the non-pluripotent cell with a compound that promotes glycolytic metabolism, 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 compound that promotes glycolytic metabolism, a TGFβ

receptor/ALK5 inhibitor, a histone deacetylase (HDAC) inhibitor and a MEK inhibitor.

10. The method of claim 1, wherein the non-pluripotent cell is a human cell.

11. The method of claim 10, wherein the non-pluripotent cell is a somatic cell, a progenitor cell, or a fully differentiated cell.

12. A mixture comprising:

mammalian cells, a compound that promotes glycolytic metabolism, and one or more of;

a TGFβ

receptor/ALK5 inhibitor,

a MEK inhibitor, or

one or more exogenous transcription factors comprising one or more of Oct4, Klf4, and Sox2.

13. The mixture of claim 12, wherein the compound that promotes glycolytic metabolism is a glycolysis activator, a substrate for glycolysis, a glycolic intermediate or its metabolic precursors, a glucose uptake transporter activator, a mitochondrial respiration modulator, an oxidative phosphorylation inhibitor, or a hypoxia-inducible factor activator.

14. The mixture of claim 12, wherein the compound that promotes glycolytic metabolism up-regulates key glycolytic genes comprising GLUT1, HK2, PFK1, and/or LDHA.

15. The mixture of claim 12, wherein the compound that promotes glycolytic metabolism comprises F2,6P (fructose 2,6-bisphosphate), F6P (fructose 6-phosphate), DNP (2,4-dinitrophenol), NOG (N-oxaloylglycine), QC (quercetin), 2-HA (2-hydroxyglutaric acid), NA (nicotinic acid), or a PDK1 (3′

-phosphoinositide-dependent kinase-1) activator.

16. The mixture of claim 15, wherein the 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”

).

17. The mixture of claim 12, wherein the mammalian cells are human cells.

18. The mixture of claim 12, wherein the mammalian cells are somatic cells, progenitor cells, fully differentiated cells, non-pluripotent cells, or pluripotent cells.

19. The mixture of claim 12, wherein the mammalian cells are isolated mammalian cells.