Reprogramming of cells to a new fate

• US 9,376,664 B2
• Filed: 06/14/2011
• Issued: 06/28/2016
• Est. Priority Date: 06/14/2010
• Status: Active Grant

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Kim, Janghwan, Efe, Jem A., Zhu, Saiyong, Hilcove, Simon, Ding, Sheng
• Primary Examiner(s): Weidner, Adam M
• Application Number:
  US13/704,214
• Publication Number:
  US 20130280809A1
• Time in Patent Office: 1,841 Days
• US Class Current: 1/1
• CPC Class Codes: A61K 35/30 Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
• CPC Class Codes: A61K 35/33 Fibroblasts
• CPC Class Codes: A61K 35/34 Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes vascular smooth muscle A61K35/44
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• 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 2501/606 c-Myc
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/08 from cells of the nervous system
• CPC Class Codes: C12N 2506/1307 from adult fibroblasts
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0602 Vertebrate cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• CPC Class Codes: C12N 5/0607 : Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0618 : Cells of the nervous system
• CPC Class Codes: C12N 5/0619 : Neurons
• CPC Class Codes: C12N 5/0623 : Stem cells
• CPC Class Codes: C12N 5/0657 : Cardiomyocytes; Heart cells
• CPC Class Codes: C12N 5/0676 : Pancreatic cells
• CPC Class Codes: C12N 5/0678 : Stem cells; Progenitor cells; Precursor cells

Abstract

Methods and compositions for transdifferentiation of an animal cell from (i) a first pluripotent cell fate to a second nonpluripotent cell fate or (ii) from a non-pluripotent mesodermal, endodermal, or ectodermal cell fate to a different non-pluripotent mesodermal, endodermal, or ectodermal cell fate.

Claims

1. A method of converting an animal cell from a first non-pluripotent cell fate to a second non-pluripotent cell fate, the method comprising:

(a) introducing a polynucleotide encoding an Oct4 polypeptide, and optionally one or more reprogramming factors comprising polynucleotides encoding a Klf4 polypeptide, a Sox2 polypeptide, or a c-Myc polypeptide, into a first non-pluripotent cell, or

contacting a first non-pluripotent cell with an Oct4 polypeptide, and optionally one or more reprogramming factors comprising a Klf4 polypeptide, a Sox2 polyeptide, or a c-Myc polypeptide,

(b) limiting the expression of endogenous Nanog in the cells from step (a) to a level substantially lower than the level of expression of endogenous Nanog in an induced pluripotent cell (iPSC), thereby generating a non-pluripotent intermediate cell and

(c) inducing differentiation of the cell under conditions to generate a cell having a second non-pluripotent cell fate.

2. The method of claim 1, wherein step (b) further comprises contacting the non-pluripotent intermediate cell with a JAK inhibitor.

3. The method of claim 1, wherein step (b) comprises eliminating exogenous LIF.

4. The method of claim 1, wherein the first non-pluripotent cell is a fibroblast or a neural precursor cell.

5. The method of claim 1, wherein the method is conducted in vitro.

6. The method of claim 1, wherein the non-pluripotent intermediate cell is not capable of forming a teratoma.

7. The method of claim 1, wherein polynucleotides encoding an Oct4 polypeptide, a Klf4 polypeptide, and a Sox2 polypeptide are introduced into the first non-pluripotent cell or wherein the first non-pluripotent cell is contacted with an Oct4 polypeptide, a Klf4 polypeptide, and a Sox2 polypeptide.

8. The method of claim 1, wherein polynucleotides encoding an Oct4 polypeptide, a Klf4 polypeptide, a Sox2 polypeptide, and a c-Myc polypeptide are introduced into the first non-pluripotent cell or wherein the first non-pluripotent cell is contacted with an Oct4 polypeptide, a Klf4 polypeptide, a Sox2 polypeptide, and a c-Myc polypeptide.

9. The method of claim 1, wherein the cell having the second non-pluripotent cell fate is a cell of mesodermal lineage, a cell of ectodermal lineage, or a cell of endodermal lineage.

10. The method of claim 9, wherein inducing differentiation of the non-pluripotent intermediate cell to the cell having the second non-pluripotent cell fate comprises contacting the non-pluripotent intermediate cell with BMP4, a calcium channel agonist, a Gα

s activating agent, a cAMP analog, and/or a GSK-3 inhibitor.

11. The method of claim 9, wherein the cell of the mesodermal lineage is a cardiomyocyte, the cell of the ectodermal lineage is a neuronal cell, or the cell of the endodermal lineage is a pancreatic cell.

12. A method of differentiating an animal cell into a cardiomyocyte, the method comprising:

(a) generating a non-pluripotent intermediate cell by (i) introducing a first non-pluripotent animal cell having a first non-pluripotent cell fate a polynucleotide encoding Oct4, and optionally, one more reprogramming factors comprising polynucleotides encoding a Klf4 polypeptide, a Sox2 polyeptide, or a c-Myc polypeptide;

or (ii) contacting a first non-pluripotent animal cell having a first non-pluripotent cell fate with Oct4, and optionally, one or more reprogramming factors comprising a Klf4 polypeptide, a Sox2 polypeptide, or a c-Myc polypeptide;

(b) limiting the expression of endogenous Nanog in the cells from step (a) to a level substantially lower than the level of expression of endogenous Nanog in an induced pluripotent cell (iPSC), thereby generating a non-pluripotent intermediate cell; and

(c) contacting the non-pluripotent intermediate cell with a GSK-3 inhibitor and/or a BMP protein under conditions sufficient to generate a cardiomyocyte.

13. The method of claim 12, wherein the animal cell is a human cell.

14. The method of claim 12, wherein the method is conducted in vitro.

15. A method of transdifferentiating an animal cell into a cardiomyocyte, comprising:

(a) introducing into a first non-pluripotent animal cell having a first non-pluripotent cell fate a polynucleotide encoding Oct4, and optionally, one or more reprogramming factors comprising polynucleotides encoding a Klf4 polypeptide, a Sox2 polypeptide, or a c-Myc polypeptide;

or contacting a non-pluripotent animal cell having a first non-pluripotent cell fate with an Oct4 polypeptide, and optionally, one or more reprogramming factors comprising a Klf4 polypeptide, a Sox2 polypeptide, or a c-Myc polypeptide;

(b) limiting the expression of endogenous Nanog in the cells from step (a) to a level substantially lower than the level of expression of endogenous Nanog in an induced pluripotent cell (iPSC), thereby generating a non-pluripotent intermediate cell; and

(c) contacting the non-pluripotent intermediate cell with a GSK-3 inhibitor, a calcium channel agonist, a Gα

s activating agent, a cAMP analog, and/or a BMP protein under conditions to generate the cardiomyocyte;

thereby differentiating the non-pluripotent intermediate cell into the cardiomyocyte.

16. The method of claim 15, wherein the method further comprises contacting the first non-pluripotent animal cell with a JAK inhibitor.

17. The method of claim 16, wherein the first non-pluripotent animal cell is contacted with the JAK inhibitor prior to contacting the first non-pluripotent animal cell with the GSK-3 inhibitor, the calcium channel agonist, the Gα

s activating agent, the cAMP analog, and/or the BMP protein.

18. A method of transdifferentiating an animal cell into a neural progenitor cell, the method comprising:

(a) introducing into a non-pluripotent animal cell having a first non-pluripotent cell fate a polynucleotide encoding an Oct4 polypeptide, and optionally, one or more polynucleotides encoding one or more polypeptides selected from the group consisting of a Klf4 polypeptide, a Sox2 polypeptide and a c-Myc polypeptide;

or contacting a non-pluripotent animal cell having a first non-pluripotent cell fate with an Oct4 polypeptide, and optionally, one or more polypeptides selected from the group consisting of a Klf4 polypeptide, a Sox2 polypeptide and a c-Myc polypeptide;

(b) limiting the expression of endogenous Nanog in the cells from step (a) to a level substantially lower than the level of expression of endogenous Nanog in an induced pluripotent cell (iPSC), thereby generating a non-pluripotent intermediate cell; and

(c) contacting the non-pluripotent intermediate cell with FGF2, FGF4, and EGF under conditions to generate the neural progenitor cell;

thereby differentiating the non-pluripotent animal cell into the neural progenitor cell.

19. The method of claim 18, wherein the method further comprises contacting the non-pluripotent animal cell with a Wnt inhibitor, a JAK inhibitor, or both a Wnt inhibitor and a JAK inhibitor.

20. The method of claim 19, wherein the non-pluripotent animal cell is contacted with the Wnt inhibitor, the JAK inhibitor, or both the Wnt inhibitor and the JAK inhibitor prior to contacting the cell with FGF2, FGF4, and EGF.

21. A method of transdifferentiating an animal cell into an induced definitive endoderm (iDE) cell, the method comprising:

(a) introducing into a non-pluripotent animal cell having a first non-pluripotent cell fate a polynucleotide encoding an Oct4 polypeptide, and optionally, one or more polynucleotides encoding one or more polypeptides selected from the group consisting of a Klf4 polypeptide, a Sox2 polypeptide and a c-Myc polypeptide;

or contacting a non-pluripotent animal cell having a first non-pluripotent cell fate with an Oct4 polypeptide, and optionally, one or more polypeptides selected from the group consisting of a Klf4 polypeptide, a Sox2 polypeptide and a c-Myc polypeptide;

(b) limiting the expression of endogenous Nanog in the cells from step (a) to a level substantially lower than the level of expression of endogenous Nanog in an induced pluripotent cell (iPSC), thereby generating a non-pluripotent intermediate cell; and

(c) contacting the non-pluripotent intermediate cell with a GSK-3 inhibitor, an HDAC inhibitor, and a TGFβ

/Activin/Nodal family member under conditions to generate the iDE cell;

thereby differentiating the non-pluripotent animal cell into the iDE cell.

22. A method of transdifferentiating an animal cell into a pancreatic beta cell, the method comprising:

(a) introducing into a non-pluripotent animal cell having a first non-pluripotent cell fate a polynucleotide encoding an Oct4 polypeptide, and optionally, one or more polynucleotides encoding one or more polypeptides selected from the group consisting of a Klf4 polypeptide, a Sox2 polypeptide and a c-Myc polypeptide;

or contacting a non-pluripotent animal cell having a first non-pluripotent cell fate with an Oct4 polypeptide, and optionally, one or more polypeptides selected from the group consisting of a Klf4 polypeptide, a Sox2 polypeptide and a c-Myc polypeptide;

(b) limiting the expression of endogenous Nanog in the cells from step (a) to a level substantially lower than the level of expression of endogenous Nanog in an induced pluripotent cell (iPSC), thereby generating a non-pluripotent intermediate cell;

(c) contacting the non-pluripotent intermediate cell with a GSK-3 inhibitor, an HDAC inhibitor, and a TGFβ

/Activin/Nodal family member under conditions to generate an iDE cell; and

(d) under conditions to generate the pancreatic beta cell,

(1) contacting the iDE cell with FGF7, RA, a Hedgehog pathway inhibitor, a BMP inhibitor, and a TGFβ

/ALK5 receptor inhibitor;

(2) contacting the cell of step (d)(1) with EGF and a Notch inhibitor; and

(3) contacting the cell of step (d)(2) with bFGF and nicotinamide;

thereby differentiating the non-pluripotent animal cell into the pancreatic beta cell.

23. The method of claim 22, wherein step (d) further comprises contacting with extendin 4.