Combined chemical and genetic approaches for generation of induced pluripotent stem cells

• US 9,540,615 B2
• Filed: 03/14/2013
• Issued: 01/10/2017
• Est. Priority Date: 03/17/2008
• Status: Active Grant

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Shi, Yan, Desponts, Caroline, Ding, Sheng, Zhou, Hongyan, Lin, Tongxiang, Li, Wenlin, Zhu, Saiyong
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US13/831,289
• Publication Number:
  US 20130273536A1
• Time in Patent Office: 1,398 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. An in vitro method of producing an induced pluripotent stem cell from a mammalian non-pluripotent cell, the method comprising:

(a) contacting the mammalian non-pluripotent cell with a MEK inhibitor; and

(b) introducing into the mammalian non-pluripotent cell, one or more expression cassettes comprising nucleic acids encoding an Oct4 polypeptide and a Klf4 polypeptide, thereby producing an induced pluripotent stem cell;

wherein the introducing step does not comprise introducing a Myc polypeptide or introducing an expression cassette comprising a nucleic acid encoding a Myc polypeptide.

2. The method of claim 1, wherein the contacting step further comprises contacting the mammalian non-pluripotent cell with a TGFβ

receptor/ALK5 inhibitor.

3. The method of claim 1, wherein the contacting step further comprises contacting the mammalian non-pluripotent cell with a TGFβ

receptor/ALK5 inhibitor and a GSK3 inhibitor.

4. The method of claim 2 or 3, wherein the TGFβ

receptor/ALK5 inhibitor is selected from the group consisting of SB431542 and A-83-01.

5. The method of claim 3, wherein the TGFβ

receptor/ALK5 inhibitor is selected from the group consisting of SB431542 and A-83-01; and

the GSK3 inhibitor comprises CHIR99021.

8. The method of claim 1, further comprising contacting the mammalian non-pluripotent cell with at least one of:

an agent that inhibits H3K9 methylation or promotes H3K9 dernethylation;

an L-type Ca channel agonist;

an activator of the cAMP pathway;

DNA methyltransferase (DNMT) inhibitor;

a nuclear receptor ligand;

a GSK3 inhibitor;

a TGFβ

receptor/ALK5 inhibitor;

a HDAC inhibitor; and

an Erk inhibitor.

9. The method of claim 1, wherein the MEK inhibitor comprises PD0325901.

6. A composition comprising a mammalian non-pluripotent cell;

one or more expression cassettes comprising nucleic acids encoding a Klf4 polypeptide, an Oct4 polypeptide, and/or a Sox2 polypeptide; and

at least one of a TGFβ

receptor/ALK5 inhibitor, a GSK3 inhibitor, and a MEK inhibitor, wherein the one or more expression cassettes does not encode a Myc polypeptide.

7. The composition of claim 6, comprising an expression cassette comprising a nucleic acid encoding the Oct4 polypeptide.