Reprogramming cells

• US 9,315,779 B2
• Filed: 05/16/2013
• Issued: 04/19/2016
• 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
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US13/896,259
• Publication Number:
  US 20130323833A1
• Time in Patent Office: 1,069 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:

(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.