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

• US 9,926,533 B2
• Filed: 11/30/2016
• Issued: 03/27/2018
• 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:
  US15/365,823
• Publication Number:
  US 20170081640A1
• Time in Patent Office: 482 Days
• 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 for increasing homogeneity of an induced pluripotent stem (iPS) cell population, the method comprising:

(a) obtaining a population of reprogrammed mammalian cells, wherein the population of reprogrammed cells comprises an iPS cell and at least one non-iPS cell; and

(b) culturing the cell population of step (a) in a medium comprising a MAPK/ERK kinase (MEK) inhibitor, wherein the culturing promotes growth of iPS cells;

thereby increasing the homogeneity of the iPS cell population in comparison to the cell population of step (a).

2. The method of claim 1, wherein the method inhibits growth of the non-iPS cells.

3. The method of claim 1, wherein the cell population of step (a) is obtained by introducing one or more expression cassettes comprising a polynucleotide encoding an Oct3/4 polypeptide and a polynucleotide encoding a Klf polypeptide into non-pluripotent mammalian cells, thereby initiating reprogramming of the non-pluripotent cells.

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

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

6. The method of claim 1, further comprising:

(c) selecting cells that exhibit pluripotent stem cell characteristics.

7. The method of claim 1, wherein the MEK inhibitor is PD0325901.

8. The method of claim 3, 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.

9. A composition comprising:

(a) a population of reprogrammed mammalian cells, wherein the population comprises iPS cells comprising a recombinant polynucleotide encoding a transcription factor that induces pluripotency; and

(b) a medium comprising a MAPK/ERK kinase (MEK) inhibitor.

10. The composition of claim 9, wherein the cell population comprises at least one non-pluripotent mammalian cell.

11. The composition of claim 9, wherein the MEK inhibitor is PD0325901.

12. The composition of claim 9, wherein the cell population is obtained by introducing one or more expression cassettes comprising a polynucleotide encoding an Oct3/4 polypeptide and a polynucleotide encoding a Klf polypeptide into non-pluripotent mammalian cells, thereby initiating reprogramming of the non-pluripotent cells.

13. The composition of claim 12, wherein the one or more expression cassettes further comprise a polynucleotide encoding a Sox polypeptide or a polynucleotide encoding a c-Myc polypeptide.

14. The composition of claim 10, wherein the non-pluripotent mammalian cells are somatic cells.

15. The composition of claim 10, wherein the non-pluripotent mammalian cells are progenitor cells.