Reprogramming cells

• US 9,657,274 B2
• Filed: 03/14/2016
• Issued: 05/23/2017
• Est. Priority Date: 03/31/2010
• Status: Active Grant

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Ding, Sheng, Zhu, Saiyong
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US15/069,730
• Publication Number:
  US 20160257938A1
• Time in Patent Office: 435 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. An in vitro method of reprogramming a non-pluripotent mammalian cell to produce an induced phiripotent stem cell, the method comprising:

a) introducing into a non-plurripotent cell, a polynucleotide encoding an Oct4 polpeptide;

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-dependentb 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.

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 polypeptide; and

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

5. The method of claim 1, wherein the contacting in step b) comprises:

(a) contacting the non-pluripotent cell with 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 (2hydroxyglutaric acid), NA (nicotinic acid), and PDK1 activator (3′

-phosphoinositide-dependent kinase-1) in the absence of a MEK inhibitor, followed by contacting the non-pluripotent cell with 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-oxaloylglycine), OC (quercetin), 2-HA (2-hydroxyglutaric acid), NA (nicotinic acid), and PDK1 activator O′

-phosphoinositide-dependent kinase-1) and a MEK inhibitor;

or

(b) contacting the non-pluripotent cell with a small molecule selected from the group consisting of F2,6P (fructose 2,6 bisphosphate), F6P (fructose 6-phosphate), DNP (2,4dinitrophenol), NOG (N-oxalylglycine), QC (quercetin), 2-HA (2 droxyglutaric acid), NA (nicotinic acid), and PDK1 activator (3′

-phosphoinositide-dependent kinase1), 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 selected from the group consisting of F2,6P (fructose 2,6-bisphosphate), F6P (fructose 6-hosphate), DNP (2,4-dinitrophenol), NOG (N-oxaloylglycine), OC (quercetin), 2-HA (2-hydroxyglutaric acid), NA (nicotinic acid), and PDK1 activator O′

-phosphoinositide-dependent kinase-1), a TGFβ

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

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

7. The method of claim 6, wherein the non-pluripotent cell is a somatic cell, a progenitor cell, or a fully differentiated cell.

8. A mixture comprising:

(a) isolated mammalian cells;

(b) 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 kinase1);

(c) one or more of;

a TGFβ

receptor/ALK5 inhibitor and a MEK inhibitor; and

(d) one or more vectors comprising a polynucleotide encoding one or more exogenous transcription factors selected from the group consisting of Oct4, Klf and Sox2.

9. The mixture of claim 8, 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).

10. The mixture of claim 8, wherein the mammalian cells are human cells.

11. The mixture of claim 8, wherein the mammalian cells are somatic cells, progenitor cells, fully differentiated cells, non-pluripotent cells, or pluripotent cells.