Reprogramming cells

• US 10,738,281 B2
• Filed: 01/03/2019
• Issued: 08/11/2020
• 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
• Primary Examiner(s): Ton, Thaian N.
• Application Number:
  US16/239,452
• Publication Number:
  US 20190203182A1
• Time in Patent Office: 586 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. 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

(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 a PDK1 activator (3′

-phosphoinositide-dependent kinase-1), and

(ii) a HDAC (histone deacetylase) inhibitor,

thereby inducing the non-pluripotent mammalian cell into an induced pluripotent stem cell;

wherein contacting the non-pluripotent mammalian 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, wherein the HDAC inhibitor comprises sodium butyrate (NaB) or valproic acid (VPA);

and/or wherein the non-pluripotent cell is a human cell.

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. A mixture comprising:

(a) isolated mammalian cells;

(b) a cell culture medium comprising;

(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 a small molecule PDK1 activator,

wherein the small molecule is present in an amount that enhances reprogramming of a non-pluripotent cell when compared to without the small molecule; and

(ii) a histone deacetylase (HDAC) 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.

6. The mixture of claim 5, wherein the cells are non-pluripotent cells, and/or human cells.

7. The mixture of claim 5, 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-1phenylpropylthio)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”

).

8. The mixture of claim 5, wherein the cell culture medium further comprises a TGFβ

receptor/ALK5 inhibitor.

9. The mixture of claim 7, wherein the PDK1 activator is PS48.

10. The mixture of claim 8, wherein the TGFβ

receptor/ALK5 inhibitor is A-83-01.

11. A kit comprising a mixture of

(A) a cell culture medium,

(B) a HDAC 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:

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

(ii) contacting the non-pluripotent mammalian cell with

(a) 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

(b) the HDAC 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.

12. The kit of claim 11, further comprising one or more of the following:

(a) a non-pluripotent mammalian cell;

(b) 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; and

/or

(c) one or more of a TGFβ

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

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

13. The kit of claim 11, 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 HDAC inhibitor is sodium butyrate (NaB) or valproic acid (VPA).

14. The kit of claim 12, wherein

(a) the TGFβ

receptor/ALK5 inhibitor is A-83-01;

or

(b) the GSK3 inhibitor is CHIR99021.

15. The kit of claim 11, wherein the non-pluripotent mammalian cell is

(a) a human cell;

or

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