REPROGRAMMING CELLS

• US 20170283776A1
• Filed: 04/17/2017
• Published: 10/05/2017
• 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 10,214,729 B2
• Time in Patent Office: Days
• 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

1-19. -19. (canceled)

20. A composition comprising an induced pluripotent stem cell (iPSC), wherein the iPSC is obtained by a method comprising:

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

(b) contacting the non-pluripotent 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 cell of (b), thereby reprogramming the non-pluripotent mammalian cell to produce an induced pluripotent stem cell;

wherein contacting the non-pluripotent cell with the small molecule enhances reprogramming when compared to without the small molecule.

21. The composition of claim 20, 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).

23. The composition of claim 20, wherein the method further comprises one or more of:

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

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

25. The composition of claim 20, wherein the non-pluripotent cell is (a) a human cell;

or (b) a somatic cell, a progenitor cell, or a fully differentiated cell.

22. (canceled)

24. (canceled)

26. (canceled)

27. A therapeutic composition comprising an induced pluripotent stem cell (iPSC), wherein the iPSC is obtained by a method comprising:

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

(b) contacting the non-pluripotent 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 cell of (b), thereby reprogramming the non-pluripotent mammalian cell to produce an induced pluripotent stem cell;

wherein contacting the non-pluripotent cell with the small molecule enhances reprogramming when compared to without the small molecule.

28. A composition for use in manufacturing a pluripotent stem cell for application in regenerative medicine, wherein the composition comprises a small molecule, wherein the small molecule enhances reprogramming of a mammalian cell, and wherein 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.

29. The composition of claim 28, 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).

30. The composition of claim 28, wherein the mammalian cell is (a) a human cell;

or (b) a somatic cell, a progenitor cell, a fully differentiated cell, a non-pluripotent cell, or a pluripotent cell.

31. (canceled)

32. A kit comprising a small molecule that enhances reprogramming when used for a method of obtaining an induced pluripotent stem cell (iPSC) comprising:

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

(b) contacting the non-pluripotent 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) a MEK inhibitor; and

(c) culturing the contacted non-pluripotent cell of (b), thereby reprogramming the non-pluripotent mammalian cell to produce an induced pluripotent stem cell;

wherein contacting the non-pluripotent cell with the small molecule enhances reprogramming when compared to without the small molecule.

33. The kit of claim 32, further comprising a non-pluripotent mammalian cell.

34. The kit of claim 32, further comprising at least one exogenous transcription factor comprising Oct4, and optionally one or both of Sox2 and Klf;

wherein the at least one exogenous transcription factor is a polypeptide, or a polynucleotide encoding the polypeptide.

35. The kit of claim 32, wherein

(a) the PDK1 activator is an allosteric PDK1 activator;

or

(b) the PDK1 activator is (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);

or

(c) the MEK inhibitor is PD0325901.

37. The kit of claim 32, further comprising one or more of a TGFβ

receptor/ALK5 inhibitor, a glycogen synthase kinase 3 (GSK3) inhibitor, and a histone deacetylase (HDAC) inhibitor and wherein said method further comprises contacting the non-pluripotent cell with said one or more of a TGFβ

receptor/ALK5 inhibitor, a glycogen synthase kinase 3 (GSK3) inhibitor, and a histone deacetylase (HDAC) inhibitor.

38. The kit of claim 37, wherein

(a) the TGFβ

receptor/ALK5 inhibitor is A-83-01;

or

(b) the HDAC inhibitor is sodium butyrate (NaB) or valproic acid (VPA).

39. The kit of claim 32, wherein the non-pluripotent cell is (a) a human cell;

or (b) a somatic cell, a progenitor cell, or a fully differentiated cell.

36. (canceled)

40. (canceled)

41. A method of making a therapeutic composition comprising an induced pluripotent stem cell (iPSC) for tissue regeneration in a subject in need thereof, wherein said iPSC is obtained by a method comprising:

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

(b) contacting the non-pluripotent 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 cell of (b), thereby reprogramming the non-pluripotent mammalian cell to produce an induced pluripotent stem cell;

wherein contacting the non-pluripotent cell with the small molecule enhances reprogramming when compared to without the small molecule.

43. The method of claim 41, wherein:

(a) the PDK1 activator is an allosteric PDK1 activator;

or

(b) the PDK1 activator is (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);

or

(c) the MEK inhibitor is PD0325901.

45. The method of claim 41, wherein said method further comprises contacting the non-pluripotent cell with one or more of a TGFβ

receptor/ALK5 inhibitor, a glycogen synthase kinase 3 (GSK3) inhibitor, and a histone deacetylase (HDAC) inhibitor.

46. The method of claim 45, wherein

(a) the TGFβ

receptor/ALK5 inhibitor is A-83-01;

or

(b) the HDAC inhibitor is sodium butyrate (NaB) or valproic acid (VPA).

47. The method of claim 41, wherein the non-pluripotent cell is (a) a human cell;

or (b) a somatic cell, a progenitor cell, or a fully differentiated cell.

42. (canceled)

44. (canceled)

48. (canceled)