Compositions, methods and kits relating to reprogramming adult differentiated cells and production of embryonic stem cell-like cells

• US 20060222636A1
• Filed: 03/10/2006
• Published: 10/05/2006
• Est. Priority Date: 09/19/2003
• Status: Abandoned Application

• Current Assignee: Rockefeller University
• Original Assignee: Rockefeller University
• Inventors: Rambukkana, Anura
• Application Number:
  US11/372,612
• Publication Number:
  US 20060222636A1
• Time in Patent Office: Days
• US Class Current: 424/93.210
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: C12N 2500/72 from bacteria
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/13 Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
• CPC Class Codes: C12N 2501/135 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C12N 2501/405 Cell cycle regulated proteins, e.g. cyclins, cyclin-dependant kinases
• CPC Class Codes: C12N 2506/08 from cells of the nervous system
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2533/32 Polylysine, polyornithine
• CPC Class Codes: C12N 5/0619 Neurons
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS
• CPC Class Codes: G01N 2333/35 from Mycobacteriaceae (F)
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity
• CPC Class Codes: G01N 33/5017 for testing neoplastic activity
• CPC Class Codes: G01N 33/5041 involving analysis of members of signalling pathways
• CPC Class Codes: G01N 33/5058 Neurological cells
• CPC Class Codes: G01N 33/5073 Stem cells

Abstract

The present invention includes compositions, methods and kits for non-nuclear transfer reprogramming an adult differentiated cell obtained from an adult tissue into an ES-like cell. The reprogrammed cell can be converted into an ES-like cell which can be re- or trans-differentiated into various differentiated cell types. The present invention further relates to identification of a novel signaling pathway, and components thereof, which effect reprogramming of a cell. The present invention further comprises compositions, methods and kits for regulating the mammalian cell cycle and cellular proliferation, as well as for treating diseases and for identifying components that affect the cell cycle, and reprogram cells, among other things.

Claims

1. A method of producing a reprogrammed embryonic stem-cell like cell (ES-like cell), said method comprising contacting a differentiated adult cell with a Mycobacterium leprae bacterium, or a component thereof, thereby producing said reprogrammed ES-like cell.

2. The method of claim 1, wherein said bacterium is viable or non-viable.

3. The method of claim 1, wherein said adult differentiated cell is a eukaryotic cell.

4. The method of claim 3, wherein said eukaryotic cell is a mammalian cell.

5. The method of claim 4, wherein said mammalian cell is selected from the group consisting of a Schwann cell, a keratinocyte, a beta-islet cell, a hepatocyte, and a heart muscle cell.

6. The method of claim 5, wherein said cell is a Schwann cell.

7. The method of claim 1, wherein said differentiated cell is incubated with said bacterium, or said component thereof, for at least 15 to about 30 days.

8. The method of claim 1, wherein said component is at least one component selected from the group consisting of a PGL-1, a whole cell wall fraction, a cell wall protein, a cell wall lipid, a cell wall carbohydrate, a protein released from a viable bacterium, and a protein secreted from a viable bacterium.

9. A cell produced by the method of claim 1.

10. The method of claim 1, said method further comprising converting said reprogrammed cell to a stem cell-like cell, wherein said conversion comprises contacting said reprogrammed cell with a progenitor medium, thereby converting said reprogrammed cell to a stem cell-like cell.

11. A cell produced by the method of claim 10.

41. A method of treating a neurological disease in an animal in need thereof, said method comprising administering to said animal an effective amount of an ES-like cell produced by the method of claim 1, thereby treating a neurological disease in said animal.

42. The method of claim 41, wherein said neurological disease is selected from the group consisting of Alzheimer'"'"'s disease, Parkinson'"'"'s disease, central nervous system injury, and multiple sclerosis.

43. The method of claim 42, wherein the central nervous system injury is selected from the group consisting of spinal cord injury, stroke, ischemia, and brain injury.

12. An isolated ES-like cell, wherein said cell is produced by contacting an adult differentiated cell with a Mycobacterium leprae bacterium, or a component thereof.

13. The isolated ES-like cell of claim 12, wherein said bacterium is selected from the group consisting of a viable bacterium and an irradiated bacterium.

14. A method of producing a re-differentiated cell, said method comprising

(a) contacting a differentiated adult cell with a Mycobacterium leprae bacterium, or a component thereof, thereby producing a reprogrammed ES-like cell;

(b) incubating said reprogrammed ES-like cell in a progenitor cell medium, thereby producing a stem cell-like cell and;

(c) re-differentiating said stem cell-like cell into a differentiated cell of the same cell type as said differentiated adult cell, thereby producing a re-differentiated cell.

15. The method of claim 14, wherein said differentiated adult cell is an adult human Schwann cell, and wherein said re-differentiated cell is a neural-like cell.

16. The method of claim 15, further wherein said neural-like cell is selected from the group consisting of a neuron-like cell and an oligodendrocyte-like cell.

17. A re-differentiated cell produced using the method of claim 14.

18. The method of claim 14, wherein said stem cell-like cell of step (b) is grown to produce at least two stem cell-like cells prior to the re-differentiation of (c).

19. A method of producing a trans-differentiated cell, said method comprising

(a) contacting a differentiated adult cell with a Mycobacterium leprae bacterium, or a component thereof, thereby producing a reprogrammed ES-like cell;

(b) incubating said reprogrammed ES-like cell in a progenitor cell medium, thereby producing a stem cell-like cell and;

(c) trans-differentiating said stem cell-like cell into a differentiated cell of a different cell lineage than said differentiated adult cell,

thereby producing a trans-differentiated cell.

20. A trans-differentiated cell produced using the method of claim 19.

21. The method of claim 19, wherein said stem cell-like cell of step (b) is grown to produce at least two stem cell-like cells prior to the trans-differentiation of (c).

22. A method of producing a neurosphere, said method comprising contacting a differentiated adult Schwann cell with a Mycobacterium leprae bacterium, or a component thereof, thereby reprogramming said Schwann cell to produce an ES-like cell, and incubating said reprogrammed ES-like cell in a progenitor medium, thereby producing a neurosphere.

23. An isolated neurosphere produced by the method of claim 22.

24. A method of producing a neuron-like cell, said method comprising contacting a differentiated adult Schwann cell with a Mycobacterium leprae bacterium, or a component thereof, thereby reprogramming said Schwann cell into an ES-like cell, incubating said reprogrammed ES-like cell in a progenitor medium to produce a neurosphere, and incubating said neurosphere in a neuronal differentiation medium, thereby producing a neuron-like cell.

25. A neuron-like cell produced using the method of claim 24.

44. A method of treating a neurological disease in an animal in need thereof, said method comprising administering to said animal an effective amount of a neuron-like cell produced by the method of claim 24, thereby treating a neurological disease in said animal.

26. A method of producing an oligodendrocyte-like cell, said method comprising contacting a differentiated adult Schwann cell with a Mycobacterium leprae bacterium, or a component thereof, thereby reprogramming said Schwann cell into an ES-like cell, incubating said reprogrammed ES-like cell in a progenitor medium to produce a neurosphere, and incubating said neurosphere in an oligodendrocyte differentiation medium, thereby producing an oligodendrocyte-like cell.

27. An oligodendrocyte-like cell produced using the method of claim 26.

45. A method of treating a neurological disease in an animal in need thereof, said method comprising administering to said animal an effective amount of a oligodendrocyte-like cell produced by the method of claim 26, thereby treating a neurological disease in said animal.

28. A method of affecting the passage of a cell through the cell cycle, said method comprising contacting a cell with a Mycobacterium leprae bacterium, or a component thereof, thereby affecting the passage of said cell through the cell cycle.

29. A method of increasing the level of cyclin D1 expression in a cell, said method comprising contacting a cell with a Mycobacterium leprae bacterium, or component thereof, thereby increasing the level of cyclin D1 expression in said cell.

30. A method of inducing MEK/Pi3K-independent phosphorylation of Erk1/2 in a cell, said method comprising contacting a cell with a Mycobacterium leprae bacterium, or component thereof, thereby inducing MEK/Pi3K-independent phosphorylation of Erk1/2 in said cell.

31. The method of claim 30, said method comprising phosphorylating the serine 158 amino acid residue of Lck.

32. A method of inducing MEK/Pi3K-independent phosphorylation of GSK3β

in a cell, said method comprising contacting a cell with a Mycobacterium leprae bacterium, or component thereof, thereby inducing MEK/Pi3K-independent phosphorylation of GSK3β

in said cell.

33. The method of claim 32, said method further comprising phosphorylating the serine 158 amino acid residue of Lck.

34. A method of identifying a component a Mycobacterium leprae bacterium that reprograms a differentiated cell, said method comprising contacting a differentiated adult cell with a component of a Mycobacterium leprae bacterium, and comparing the level of a marker of an undifferentiated state in said cell contacted with said component with the level of said marker in an otherwise identical cell not contacted with said component, wherein a higher level said marker in said cell contacted with said component compared with said level of said marker in said cell not contacted with said component is an indication that said component reprogrammed said cell, thereby identifying a component a Mycobacterium leprae bacterium that reprograms a differentiated cell.

35. The method of claim 34, wherein said marker is selected from the group consisting of the level of cyclin D1 expression in a cell, the level of p21 expression in a cell, the level of phosphorylation of Lck at serine residue number 158, the level of proliferation without transformation, the level of MEK/Pi3K-independent Erk1/2 phosphorylation, the level of MEK/Pi3K-independent GSK3β

phosphorylation, expression of an embryonic-stage marker protein, and expression of an embryonic-stage gene.

36. A component identified by the method of claim 34.

37. A method of identifying a component of a Mycobacterium leprae bacterium that affects progression of a cell through the cell cycle, said method comprising contacting a cell with a component of a Mycobacterium leprae bacterium, and comparing the progression of said cell through the cell cycle with the progression through the cell cycle by an otherwise identical cell not contacted with said component, wherein a faster progression through said cell cycle by said cell contacted with said component compared with said progression through said cell cycle by said cell not contacted with said component is an indication that said component affects progression of a cell through the cell cycle, thereby identifying a component of a Mycobacterium leprae bacterium that affects progression of a cell through the cell cycle.

38. A component identified by the method of claim 37.

39. A method of identifying a component of a Mycobacterium leprae bacterium that increases cell proliferation, said method comprising contacting a cell with a component of a Mycobacterium leprae bacterium, and comparing the level of proliferation of said cell with the level of proliferation of an otherwise identical cell not contacted with said component, wherein a greater level of proliferation of said cell contacted with said component compared with said level of proliferation of said otherwise identical cell not contacted with said component is an indication that said component increases cell proliferation, thereby identifying a component of a Mycobacterium leprae bacterium that increases cell proliferation.

40. A component identified by the method of claim 39.

46. A kit for reprogramming an adult differentiated cell, said kit comprising an irradiated Mycobacterium leprae bacterium, or a component thereof, and said kit further comprising an applicator and an instructional material for use thereof.

47. The kit of claim 46, said kit further comprising a progenitor medium.

48. The kit of claim 46, said kit further comprising a differentiation medium.

49. The kit of claim 46, wherein said component is a cell wall obtained from a Mycobacterium leprae bacterium.