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

Patent metadata

Field	Value
Publication number	US-9540615-B2
Application number	US-201313831289-A
Country	US
Kind code	B2
Filing date	Mar 14, 2013
Priority date	Mar 17, 2008
Publication date	Jan 10, 2017
Grant date	Jan 10, 2017

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.

First claim

What is claimed is:

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.

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.

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.

Assignees

• Scripps Research Inst

Inventors

• Shi Yan
• Desponts Caroline
• Ding Sheng
• Zhou Hongyan
• Lin Tongxiang
• Li Wenlin
• Zhu Saiyong

Classifications

• A61P3/10

  for hyperglycaemia, e.g. antidiabetics · CPC title

• A61P9/10

  for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis · CPC title

• A61P9/00

  Drugs for disorders of the cardiovascular system · CPC title

• A61P25/28

  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 title

• A61P25/00

  Drugs for disorders of the nervous system · CPC title