Chemical approaches for generation of induced pluripotent stem cells

• US 9,394,524 B2
• Filed: 04/02/2010
• Issued: 07/19/2016
• Est. Priority Date: 03/17/2008
• Status: Active Grant

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Lin, Tongxiang, Li, Wenlin, Ding, Sheng
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US12/753,386
• Publication Number:
  US 20100267141A1
• Time in Patent Office: 2,300 Days
• US Class Current: 1/1
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 11/08 Bronchodilators
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2500/14 Calcium; Ca chelators; Calcitonin
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• 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 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/604 : Klf-4
• CPC Class Codes: C12N 2501/605 : Nanog
• CPC Class Codes: C12N 2501/608 : Lin28
• CPC Class Codes: C12N 2501/727 : Kinases (EC 2.7.)
• CPC Class Codes: C12N 2506/08 : from cells of the nervous system
• CPC Class Codes: C12N 2506/094 : from keratinocytes
• CPC Class Codes: C12N 2506/1307 : from adult fibroblasts
• CPC Class Codes: C12N 2506/28 : from vascular endothelial cells
• CPC Class Codes: C12N 2510/00 : Genetically modified cells
• CPC Class Codes: C12N 5/0696 : Artificially induced pluripotent stem cells, e.g. iPS

Abstract

The present invention provides for identification and use of small molecules to induce pluripotency in mammalian cells as well as other methods of inducing pluripotency.

Claims

1. A method of producing an induced pluripotent stem cell from a mammalian non-pluripotent cell, the method comprising

(a) introducing into the mammalian non-pluripotent cell, one or more expression cassettes comprising an Oct4 polynucleotide, a Sox2 polynucleotide, and a Klf4 polynucleotide, and

(b) contacting the cell of step (a) with a TGFβ

receptor/ALK5 inhibitor and a GSK-3 inhibitor,

thereby producing an induced pluripotent stem cell from the mammalian non-pluripotent cell.

2. The method of claim 1, further comprising contacting the cell with a second agent selected from the group consisting of a MEK inhibitor and an Erk inhibitor.

3. The method of claim 2, wherein the second agent is a MEK inhibitor.

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

5. The method of claim 1, wherein the TGFβ

receptor/ALK5 inhibitor is selected from the group consisting of;

A-83-01 and SB431542.

6. The method of claim 2 or claim 3, wherein the MEK inhibitor is PD0325901.

7. The method of claim 1, wherein the GSK-3 inhibitor is selected from the group consisting of:

CHIR99021, CHIR98014, and 6-bromoindirubin-3′

-oxime (BIO).

8. The method of claim 1, wherein the one or more expression cassettes further comprises a c-Myc polynucleotide.