Multipotent stem cells from peripheral tissues and uses thereof

• US 20030003574A1
• Filed: 03/15/2002
• Published: 01/02/2003
• Est. Priority Date: 01/24/2000
• Status: Active Grant

• Current Assignee: McGill University
• Original Assignee: McGill University
• Inventors: Fernandes, Karl J. L., Akhavan, Mahnaz, Fortier, Mathieu, Miller, Freda, Toma, Jean
• Granted Patent: US 7,544,509 B2
• Time in Patent Office: Days
• US Class Current: 435/368
• 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: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• 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 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/195 Heregulin, neu differentiation factor
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2506/03 from non-embryonic pluripotent stem cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0619 Neurons
• CPC Class Codes: C12N 5/0622 Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
• CPC Class Codes: C12N 5/0623 Stem cells

Abstract

This invention relates to multipotent stem cells, purified from the peripheral tissue of mammals, and capable of differentiating into neural and non-neural cell types. These stem cells provide an accessible source for autologous transplantation into CNS, PNS, and other damaged tissues.

Claims

1. A cellular composition comprising a purified population of multipotent mammalian cells, which multipotent cells form non-adherent clusters in culture, are self renewing, are positive for nestin and fibronectin protein, and differentiate into both neuronal and non-neuronal cell types.

9. The cellular composition of any of claims 1-8, which multipotent cells differentiate into cells expressing one or more markers selected from the group consisting of Glial Fibrillary Acid Protein (GFAP), neurofilament 160, β

III tubulin, NeuN, neurofilament-M (NFM), neuron-specific enolase, galactocerebroside, GAD, tyrosine hydroxylase (TH), dopamine β

-dehyrdogenase and CNPase.

10. The cellular composition of any of claims 1-8, which multipotent cells can differentiate to produce astrocytes, oligodendrocytes, and neurons.

11. The cellular composition of claim 10, wherein the neuron is a dopaminergic neuron.

12. The cellular composition of any of claims 1-8, which multipotent cells differentiate to form cells selected from the group consisting of epithelial cells, endothelial cells, skeletal muscle cells, cardiac muscle cells, connective tissue cells, lung cells, adipocytes, pancreatic islet cells, hematopoietic cells, chondrocytes, bone, kidney cells, and hepatocytes.

13. The cellular composition of any of claims 1-8, wherein the multipotent cells are present in the composition in non-adherent cell aggregates of 10 cells or more.

14. The cellular composition of any of claims 1-8, including 10⁶ or more of said multipotent cells.

15. The cellular composition of any of claims 1-8, wherein the multipotent cells are engineered to include a heterologous gene.

16. The cellular composition of claim 15, wherein the heterologous gene encodes a therapeutic protein.

17. The cellular composition of claim 15, wherein the heterologous gene encodes a protein which affects the growth or differentiation fate of said multipotent cells or the progeny thereof.

19. The cellular composition of claim 18, wherein said skin is foreskin.

23. The cellular composition of the differentiated cells of any of claims 1-8.

24. A cellular composition comprising differentiated cells prepared by the method comprising plating the non-adherent clusters of any of claims 1-8 on a substratum coated with a substrate that promotes their attachment to the substratum.

27. A kit comprising a cellular composition of any of claims 1-8, and means for introducing the cellular composition into a patient.

28. A kit comprising a composition of differentiated progeny from a multipotent cell of any of claims 1-8, and means for introducing the composition into a patient.

29. The cellular composition comprising any of claims 1-8, formulated in a pharmaceutically acceptable carrier, auxiliary or excipient.

31. The cell line established from the cellular composition of any of claims 1-8.

32. The cell line of claim 31, wherein the cell expresses a heterologous gene.

33. A method of treating a patient with cell damage or disease comprising transplanting the cells of any of claims 1-8.

34. The method of claim 33, wherein the multipotent cells are autologously derived.

35. The method of claim 33, wherein the multipotent cells are derived from a genetically related donor.

36. The method of claim 33, wherein the multipotent cells are derived from a genetically unrelated donor.

37. The method of claim 33, wherein the cell damage or disease is selected from a neurodegenerative disease, diabetes, heart disease, heart attack, or stroke.

38. The method of claim 33, wherein the cell damage or disease is the result of bacterial or viral infection.

39. The method of claim 33, wherein the cell damage or disease is the result of traumatic injury including fractures, lacerations, and burns.

40. The method of claim 33, wherein the multipotent cells are transplanted at the site of cell damage or disease.

41. The method of claim 33, wherein the multipotent cells are delivered to the site of cell damage via the bloodstream.

42. The method of claim 33, wherein the patient is a human patient.

43. A cellular composition comprising a purified preparation of the differentiated cells of claims 23 or 24.

44. A method of treating a patient with cell damage or disease comprising transplanting the cells of claim 43.

45. The method of claim 44, wherein the patient is a human patient.

46. The method of claim 44, wherein the transplant is at the site of cell damage or disease.

47. The method of claim 44, wherein the cell damage or disease is a neurodegenerative disease, diabetes, heart disease, heart attack, or stroke.

48. The method of claim 44, wherein the cell damage or disease is the result of bacterial or viral infection.

49. The method of claim 44, wherein the cell damage or disease is the result of traumatic injury including fractures, lacerations, and burns.

62. A method for conducting a stem cell business, comprising:

(i) identifying one or more agents which affect the proliferation or differentiation of the multipotent cells of any of claims 1-8;

(ii) conducting therapeutic profiling of agents identified in step (i), or analogs thereof, for efficacy and toxicity in animals; and

(iii) formulating a pharmaceutical preparation including one or more agents identified in step (ii) as having an acceptable therapeutic profile.

63. The method of claim 62, wherein step (i) comprises contacting the multipotent stem cells with one or more small molecules and identifying those which affect the proliferation or differentiation of the multipotent stem cells.

64. The method of claim 62, wherein step (i) comprises contacting the multipotent stem cells with one or more extracellular proteins and identifying those which affect the proliferation or differentiation of the multipotent stem cells.

65. The method of claim 62, including an additional step of establishing a distribution system for distributing the pharmaceutical preparation for sale.

66. The method of claim 62 or 65, including establishing a sales group for marketing the pharmaceutical preparation.

67. A method of conducting a drug discovery business, comprising:

(i) identifying one or more agents which affect the proliferation or differentiation of the multipotent cells of any of claims 1-8;

(ii) licensing, to a third party, the rights for further drug development of agents identified in step (i) as able to affect the proliferation or differentiation of the multipotent stem cells.

2. A cellular composition comprising multipotent mammalian cells with fewer than 30 percent lineage committed cells, wherein said multipotent cells form non-adherent clusters in culture, are self renewing, are positive for nestin and fibronectin protein, and differentiate into ectodermal and mesodermal cell types.

3. A cellular composition comprising multipotent cells prepared by the method comprising:

(a) culturing a dissociated sample of epithelial tissue;

(b) isolating, from the culture, non-adherent cells characterized by the following;

are positive for nestin and fibronectin protein, are self renewing, and differentiate into ectodermal and mesodermal cell types.

18. The cellular composition of claim 3, 4 or 5, wherein said epithelial tissue is skin or mucosal tissue.

20. The cellular composition of claim 3, 4 or 5, wherein said epithelial tissue is derived from tongue.

21. The cellular composition of claims 3, 4, 5, 18, 19 or 20, wherein said epithelial tissue is from an adult mammal.

22. The cellular composition of claims 3, 4, 5, 18, 19 or 20, wherein said epithelial tissue is from a juvenile mammal.

25. The cellular composition of claim 24, wherein the method further comprises modulating the plating conditions to influence proliferation, differentiation, and/or survival of the cells.

26. The cellular composition of claim 25, wherein the modulation of plating conditions includes at least one of changing the serum concentration, changing the plating density or the addition of a therapeutic protein.

30. The cellular composition comprising claim 23, formulated in a pharmaceutically acceptable carrier, auxiliary or excipient.

4. A cellular composition comprising multipotent cells prepared by the method comprising:

(a) separating the dermal and epidermal layers of epithelial tissue;

(b) culturing a dissociated sample of the dermal layer of said epithelial tissue;

(c) isolating, from the culture, non-adherent cells characterized by the following;

are self-renewing, and differentiate into ectodermal and mesodermal cell types.

5. A cellular composition comprising a population of multipotent mammalian cells, which multipotent cells form non-adherent clusters in culture, are self-renewing, are positive for nestin and fibronectin protein, differentiate into both neuronal and non-neuronal cell types, and can proliferate in culture in the absence of exogenous EGF.

6. A cellular composition comprising a population of multipotent mammalian cells, which multipotent cells form non-adherent clusters in culture, are self-renewing, differentiate into both neuronal and non-neuronal cell types, and can proliferate in culture in the absence of exogenous LIF.

7. A cellular composition comprising a population of multipotent mammalian cells, which multipotent cells form non-adherent clusters in culture, are self-renewing, are positive for nestin and fibronectin protein, are negative for vimentin and cytokeratin protein, and differentiate into both neuronal and non-neuronal cell types.

8. A cellular composition comprising a population of multipotent mammalian cells, which multipotent cells form non-adherent clusters in culture, are self-renewing, are positive for nestin and fibronectin protein, are negative for vimentin, cytokeratin, and p75 protein, and differentiate into both neuronal and non-neuronal cell types.

50. A method for preparing stem cell preparations, comprising:

(a) obtaining an epithelial tissue sample;

(b) culturing cells dissociated from said tissue sample;

(c) isolating from the culture multipotent cells characterized by the following;

form non-adherent clusters in culture;

are self renewing;

express nestin and fibronectin; and

differentiate into ectodermal and mesodermal cell types.

51. A method for preparing stem cell preparations, comprising:

(a) obtaining an epithelial tissue sample;

(b) separating the dermal and epidermal layers of said tissue sample;

(c) culturing cells dissociated from the dermal layer of said tissue sample;

(d) isolating from the culture multipotent cells characterized by the following;

form non-adherent clusters in culture;

are self renewing; and

differentiate into ectodermal and mesodermal cell types.

52. A method for preparing stem cell preparations, comprising:

(a) obtaining an epithelial tissue sample from a patient;

(b) culturing cells dissociated from said tissue sample;

(c) isolating from the culture multipotent cells characterized by the following;

form non-adherent clusters in culture;

are self renewing;

express nestin and fibronectin; and

differentiate into ectodermal and mesodermal cell types; and

(d) preserving and storing the multipotent cells for later retrieval.

55. The method of any of claims 52, 53 or 54, wherein the preserved cells are formulated in a pharmaceutically acceptable carrier, auxiliary or excipient.

56. The method of any of claims 52, 53 or 54, wherein the step of preserving the multipotent cells or differentiated cells includes cryogenic preservation.

59. The method of claim 57 or 58, wherein said epithelial tissue is skin.

60. The method of claim 59, wherein said skin is selected from foreskin or skin obtained from cosmetic surgery.

61. The method of claim 57 or 58, further including a billing system for billing the client or a medical insurance provider thereof.

53. A method for preparing cell preparations, comprising:

(a) obtaining an epithelial tissue sample from a patient;

(b) culturing cells dissociated from said tissue sample under conditions wherein multipotent cells are expanded, which multipotent cells are characterized by the following;

form non-adherent clusters in culture;

are self renewing;

express nestin and fibronectin; and

differentiate into ectodermal and mesodermal cell types;

(c) differentiating the multipotent cells into one or more lineage committed cell types; and

(d) preserving and storing the differentiated cells for later retrieval.

54. A method for preparing cell preparations, comprising:

(a) obtaining an epithelial tissue sample from a patient;

(b) culturing cells dissociated from said tissue sample under conditions wherein multipotent cells are expanded, which multipotent cells are characterized by the following;

form non-adherent clusters in culture;

are self renewing;

express nestin and fibronectin; and

differentiate into ectodermal and mesodermal cell types;

(c) differentiating the multipotent cells into one or more lineage committed cell types, wherein the conditions for differentiating the multipotent cells include modulating the plating conditions; and

(d) preserving and storing the differentiated cells for later retrieval.

57. A method for conducting a regenerative medicine business, comprising:

(a) a service for accepting and logging in epithelial tissue samples from a client;

(b) a cell culture system for culturing cells dissociated from said tissue sample, which system provides conditions suitable for expanding multipotent cells in said sample, which multipotent cells are characterized by the following;

form non-adherent clusters in culture;

are self renewing; and

differentiate into ectodermal and mesodermal cell types;

(c) a cell preservation system for preserving said multipotent cells for later retrieval on behalf of said client or other third party.

58. A method for conducting a regenerative medicine business, comprising:

(a) a service for accepting and logging in epithelial tissue samples from a client;

(b) a cell culture system for culturing cells dissociated from said tissue sample, which system provides conditions suitable for expanding multipotent cells in said sample, which multipotent cells are characterized by the following;

form non-adherent clusters in culture;

are self renewing; and

differentiate into ectodermal and mesodermal cell types;

(c) a cell differentiation system for differentiating said multipotent cells into one or more lineage committed cell types

(d) a cell preservation system for preserving said lineage committed cells for later retrieval on behalf of said client or other third party.

68. A cellular composition of adult stem cells which (i) will proliferate in an in vitro culture, (ii) maintains the potential to differentiate to derivatives of endoderm, mesoderm, and ectoderm tissues throughout the culture, and (iii) is inhibited from differentiation when cultured under proliferative conditions.

69. A cellular composition of adult stem cells which stem cells which (i) will proliferate in an in vitro culture for over one year, (ii) maintains a karyotype in which the chromosomes are euploid and not altered through prolonged culture, (iii) maintains the potential to differentiate to derivatives of endoderm, mesoderm, and ectoderm tissues throughout the culture, and (iv) is inhibited from differentiation when cultured under proliferative conditions.

70. A method for conducting a regenerative medicine business, comprising:

(a) a service for accepting and logging in epithelial tissue samples from a client;

(b) a cell culture system for culturing cells dissociated from said tissue sample, which system provides conditions suitable for expanding multipotent cells in said sample, which multipotent cells are characterized by the following;

form non-adherent clusters in culture;

are self renewing; and

differentiate into ectodermal, mesodermal, and endodermal cell types;

(c) a cell preservation system for preserving said multipotent cells for later retrieval on behalf of said client or other third party.

72. The method of claim 70 or 71, wherein said epithelial tissue is skin.

73. The method of claim 72, wherein said skin is selected from foreskin or skin obtained from cosmetic surgery.

71. A method for conducting a regenerative medicine business, comprising:

(a) a service for accepting and logging in epithelial tissue samples from a client;

(b) a cell culture system for culturing cells dissociated from said tissue sample, which system provides conditions suitable for expanding multipotent cells in said sample, which multipotent cells are characterized by the following;

form non-adherent clusters in culture;

are self renewing; and

differentiate into ectodermal, mesodermal, and endodermal cell types;

(c) a cell differentiation system for differentiating said multipotent cells into one or more lineage committed cell types

(d) a cell preservation system for preserving said lineage committed cells for later retrieval on behalf of said client or other third party.