GENERATION OF PLURIPOTENT STEM CELLS USING RECOMBINANT PROTEINS

• US 20100233804A1
• Filed: 03/12/2010
• Published: 09/16/2010
• 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
• Granted Patent: US 9,068,170 B2
• Time in Patent Office: Days
• US Class Current: 435/354
• 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 one or more different exogenous polypeptides, the exogenous polypeptides 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 transcription factor is selected from the group consisting of a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide.

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-1 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

(a) a first exogenous polypeptide comprising a first transcription factor selected from a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide;

and

(b) a second exogenous polypeptide comprising a second transcription factor selected from a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide, wherein the first and second transcription factors are different.

5. The method of claim 4, wherein the non-pluripotent cell is further contacted with

(c) a third exogenous polypeptide comprising a third transcription factor selected from a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide, wherein the third transcription factor is different from the first and the second transcription factor.

6. The method of claim 5, wherein the first transcription factor is a Klf polypeptide, the second transcription factor is an Oct polypeptide, and the third transcription factor is a Sox polypeptide.

7. The method of claim 5, wherein the non-pluripotent cell is further contacted with

(d) a fourth exogenous polypeptide comprising a fourth transcription factor selected from a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide, wherein the fourth transcription factor is different from the first and the second and the third transcription factor.

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

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

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

i. contacting the non-pluripotent cell with the one or more exogenous polypeptides followed by culturing the cell in the absence of the exogenous polypeptides.

11. The method of claim 10, further comprising purifying the pluripotent cell to generate a homogenous population of the pluripotent cell.

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

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

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

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

a MEK inhibitor;

a GSK3 inhibitor;

a TGFβ

receptor inhibitor or an ALK5 inhibitor;

an histione deacetylase (HDAC) inhibitor; and

a DNA methyltransferase (DNMT) inhibitor.

16. 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 MEK inhibitor;

a GSK3 inhibitor;

a TGFβ

receptor inhibitor or an ALK5 inhibitor;

an histione deacetylase (HDAC) inhibitor; and

a DNA methyltransferase (DNMT) inhibitor.

17. An isolated polypeptide comprising a transcription factor fused to a heterologous peptide sequence that enhances transport across cell membranes, wherein the transcription factor is selected from the group consisting of:

an Oct polypeptide,

a Klf polypeptide,

a Myc polypeptide, and

a Sox polypeptide.

18. The isolated polypeptide of claim 17, 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.

19. The isolated polypeptide of claim 17, wherein the heterologous peptide is a peptide sequence comprising RRRRRRRRRRR (SEQ ID NO:

1).

20. The isolated polypeptide of claim 17, wherein the transcription factor is a human or mouse polypeptide.

21. An isolated nucleic acid encoding the polypeptide of claim 17.

22. A vector comprising the nucleic acid of claim 21.

23. A cell comprising the vector of claim 22.

24. A mixture comprising:

at least one polypeptide of claim 17;

a non-pluripotent mammalian cell.

25. The mixture of claim 24, comprising:

(a) a first exogenous polypeptide of claim 17 comprising a first transcription factor selected from a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide; and

(b) a second exogenous polypeptide of claim 17 comprising a second transcription factor selected from a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide.

26. The mixture of claim 25, further comprising:

(c) a third exogenous polypeptide of claim 17 comprising a third transcription factor selected from a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide.

27. The mixture of claim 26, wherein the first transcription factor is a Klf polypeptide, the second transcription factor is an Oct polypeptide, and the third transcription factor is a Sox polypeptide.

28. The mixture of claim 26, further comprising:

(d) a fourth exogenous polypeptide of claim 17 comprising a fourth transcription factor selected from a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide.

29. The mixture of claim 24, wherein the mixture does not comprises an Nanog polypeptide.

30. The mixture of claim 24, further comprising one or more of:

a MEK inhibitor;

a GSK3 inhibitor;

a TGFβ

inhibitor;

an histione deacetylase (HDAC) inhibitor; and

a DNA methyltransferase (DNMT) inhibitor.

31. The mixture of claim 24, further comprising 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 MEK inhibitor;

a GSK3 inhibitor;

a TGFβ

receptor inhibitor or an ALK5 inhibitor;

an histione deacetylase (HDAC) inhibitor; and

a DNA methyltransferase (DNMT) inhibitor.

32. A kit comprising at least one polypeptide of claim 17.

33. The kit of claim 32, comprising:

(a) a first exogenous polypeptide of claim 17 comprising a first transcription factor selected from a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide; and

(b) a second exogenous polypeptide of claim 17 comprising a second transcription factor selected from a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide.

34. The kit of claim 33, further comprising:

(c) a third exogenous polypeptide of claim 17 comprising a third transcription factor selected from a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide.

35. The kit of claim 34, further comprising:

(d) a fourth exogenous polypeptide of claim 17 comprising a fourth transcription factor selected from a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide.

36. The kit of claim 32, wherein the kit does not comprise a Nanog polypeptide.

37. The kit of claim 32, further comprising one or more of:

a MEK inhibitor;

a GSK3 inhibitor;

a TGFβ

inhibitor;

an histione deacetylase (HDAC) inhibitor; and

a DNA methyltransferase (DNMT) inhibitor.

38. The kit of claim 32, further comprising 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 MEK inhibitor;

a GSK3 inhibitor;

a TGFβ

receptor inhibitor or an ALK5 inhibitor;

an histione deacetylase (HDAC) inhibitor; and

a DNA methyltransferase (DNMT) inhibitor.