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

• US 9,534,205 B2
• Filed: 03/17/2009
• Issued: 01/03/2017
• 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:
  US12/933,391
• Publication Number:
  US 20110110899A1
• Time in Patent Office: 2,849 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 or ex vivo method of producing induced pluripotent stem cells from mammalian non-pluripotent cells, the method comprising:

(a) contacting the mammalian non-pluripotent cells with a composition comprising a glycogen synthase kinase 3 (GSK3) inhibitor, a MAPK/ERK kinase (MEK) inhibitor, and a transforming growth factor beta (TGFβ

) receptor/activating receptor-like kinase 5 (ALK5) inhibitor prior to the initiation of reprogramming of the non-pluripotent cells; and

(b) introducing one or more expression cassettes comprising a polynucleotide encoding an Klf polypeptide and a polynucleotide encoding a Oct3/4 polypeptide into the mammalian non-pluripotent cells prior to the initiation of reprogramming of the non-pluripotent cells,

thereby producing induced pluripotent stem cells.

6. A method comprising differentiating the induced pluripotent stem cells of claim 1 or claim 2 into a desired cell type.

7. The method of claim 6, wherein the desired cell type is introduced into an animal.

8. The method of claim 7, wherein the animal is a human.

11. The method of claim 1, further comprising introducing one or more expression cassettes comprising a polynucleotide encoding a Sox polypeptide or a polynucleotide encoding a c-Myc polypeptide into the mammalian non-pluripotent cells.

12. The method of claim 1, further comprising introducing one or more expression cassettes comprising a polynucleotide encoding a Sox polypeptide and a polynucleotide encoding a c-Myc polypeptide into the mammalian non-pluripotent cells.

2. An in vitro or ex vivo method of producing induced pluripotent stem cells from mammalian non-pluripotent cells, the method comprising:

(a) introducing one or more expression cassettes comprising a polynucleotide encoding an Oct4 polypeptide and a polynucleotide encoding a Klf4 polypeptide into the mammalian non-pluripotent cells; and

(b) contacting the mammalian non-pluripotent cells with a composition comprising a glycogen synthase kinase 3 (GSK3) inhibitor;

a MAPK/ERK kinase (MEK) inhibitor; and

a transforming growth factor beta (TGFβ

) receptor/activin receptor-like kinase (ALK5) inhibitor, wherein steps (a) and (b) are performed prior to the initiation of reprogramming of the non-pluripotent cells, thereby producing induced pluripotent stem cells.

3. The method of claim 2, wherein the mammalian non-pluripotent cells are somatic cells.

4. The method of claim 2, wherein the mammalian non-pluripotent cells are progenitor cells.

5. The method of claim 2, further comprising, c) selecting cells that exhibit pluripotent stem cell characteristics.

9. The method of claim 4, wherein the progenitor cells are neural progenitor cells, skin progenitor cells or hair follicle progenitor cells.

10. The method of claim 2, wherein the MEK inhibitor is PD0325901.

13. The method of claim 2, further comprising introducing one or more expression cassettes comprising a polynucleotide encoding a Sox2 polypeptide or a polynucleotide encoding a c-Myc polypeptide into the mammalian non-pluripotent cells.

14. The method of claim 2, further comprising introducing one or more expression cassettes comprising a polynucleotide encoding a Sox2 polypeptide and a polynucleotide encoding a c-Myc polypeptide into the mammalian non-pluripotent cells.