Generation of pluripotent stem cells using recombinant proteins

• US 9,068,170 B2
• Filed: 03/12/2010
• Issued: 06/30/2015
• Est. Priority Date: 03/17/2008
• Status: Active Grant

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Zhou, Hongyan, Ding, Sheng
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US12/723,063
• Publication Number:
  US 20100233804A1
• Time in Patent Office: 1,936 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 methods, compositions, and kits of producing an induced pluripotent stem cell from a mammalian non-pluripotent cell using exogenous transcription factors.

Claims

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

contacting the non-pluripotent cell with a plurality of exogenous transcription factor polypeptides, each exogenous transcription factor polypeptide comprising a transcription factor fused to a heterologous peptide sequence that enhances transport across cell membranes, under conditions to produce the induced pluripotent stem cell from the mammalian non-pluripotent cell;

wherein the exogenous transcription factor polypeptides are selected from the group consisting of;

(i) Oct4, Klf4, and Sox2; and

(ii) Oct4, Klf4, Sox2, and c-Myc; and

contacting the non-pluripotent cell with a MEK inhibitor, a GSK3 inhibitor, and a TGFβ

receptor inhibitor/ALK5 inhibitor.

2. The method of claim 1, wherein the heterologous peptide is selected from the group consisting of Drosophila homeoprotein antennapedia transcription protein (AntHD), herpes simplex virus structural protein VP22, the HIV-1 transcriptional activator TAT protein, Kaposi FGF signal sequence (kFGF), protein transduction domain-4 (PTD4), Penetratin, M918, Transportan-10, a nuclear localization sequence, a PEP-I peptide;

an amphipathic peptide;

a delivery enhancing transporter, and a peptide sequence comprising at least 5 or more contiguous arginines.

3. The method of claim 1, wherein the heterologous peptide is a peptide sequence comprising RRRRRRRRRRR (SEQ ID NO:

1).

4. The method of claim 1, wherein the non-pluripotent cell is contacted with an exogenous Klf4 polypeptide, an exogenous Oct4 polypeptide, and an exogenous Sox2 polypeptide.

5. The method of claim 1, wherein the non-pluripotent cell is contacted with an exogenous Klf4 polypeptide, an exogenous Oct4 polypeptide, an exogenous c-Myc polypeptide, and an exogenous Sox2 polypeptide.

6. The method of claim 1, wherein the non-pluripotent cell is not contacted with a Nanog polypeptide.

7. The method of claim 1, wherein the non-pluripotent cell is not contacted with a protein delivery agent.

8. The method of claim 1, wherein the contacting step comprises at least two cycles of:

(i) contacting the non-pluripotent cell with the exogenous polypeptides followed by culturing the cell in the absence of the exogenous polypeptides.

9. The method of claim 8, further comprising purifying the pluripotent cell to generate a homogenous population of pluripotent cells.

10. The method of claim 1, wherein the cell is a mouse cell.

11. The method of claim 1, wherein the cell is a human cell.

12. The method of claim 1, wherein the cell is a non-human cell.

13. The method of claim 1, further comprising contacting the cell with one or more of:

a histone deacetylase (HDAC) inhibitor; and

a DNA methyltransferase (DNMT) inhibitor.

14. The method of claim 1, further comprising contacting the cell with one or more of:

an agent that inhibits H3K9 methylation;

an L-type Ca channel agonist;

an activator of the cAMP pathway;

a nuclear receptor ligand;

an erk inhibitor

a histone deacetylase (HDAC) inhibitor; and

a DNA methyltransferase (DNMT) inhibitor.