Method for identification of cell growth or differentiation factors

• US 20020187548A1
• Filed: 01/22/2001
• Published: 12/12/2002
• Est. Priority Date: 11/21/1994
• Status: Active Grant

• Current Assignee: National Jewish Medical and Research Center
• Original Assignee: National Jewish Medical and Research Center
• Inventors: Keller, Gordon M., Choi, Kyunghee, Hawley, Robert G.
• Granted Patent: US 6,555,318 B2
• Time in Patent Office: Days
• US Class Current: 435/325
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: C12N 2500/24 Iron; Fe chelators; Transferrin
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/14 Erythropoietin [EPO]
• CPC Class Codes: C12N 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2501/22 Colony stimulating factors (G-CSF, GM-CSF)
• CPC Class Codes: C12N 2501/23 Interleukins [IL]
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2502/28 Vascular endothelial cells
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2510/04 Immortalised cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• CPC Class Codes: C12N 5/0635 B lymphocytes
• CPC Class Codes: C12N 5/0636 T lymphocytes
• CPC Class Codes: C12N 5/0641 Erythrocytes
• CPC Class Codes: C12N 5/069 Vascular Endothelial cells
• CPC Class Codes: C12N 5/0692 Stem cells; Progenitor cells; Precursor cells

Abstract

The present invention relates to novel immortalized precursor cell populations derived from embryonic stem cell populations and methods to produce such cell populations. Also disclosed is an assay to identify regulatory compounds capable of controlling cell growth for therapeutic and experimental use.

Claims

1. A cell population selected from the group consisting of (a) a HOX11 precursor cell population comprising a cell surface molecule selected from the group consisting of Fcγ

RII, Fcγ

RIII, Thy-1, CD44, VLA-4α

, LFA-1β and

combinations thereof;

(b) a HOX11 precursor cell population comprising a cell surface molecule selected from the group consisting of Fcγ

RII, Fcγ

RIII, CD44, VLA-4α

, LFA-1β and

combinations thereof;

(c) a HOX11 precursor cell population comprising a cell surface molecule selected from the group consisting of HSA, CD44, VLA-4α

, LFA-1β

, ICAM-1 and combinations thereof;

(d) a HOX11 precursor cell population comprising a cell surface molecule selected from the group consisting of CD45, Aa4.1, Sca-1, HSA, Fcγ

RII, Fcγ

RIII, Thy-1, Mac-1, Gr-1, CD44, VLA-4α

, LFA-1β and

combinations thereof; and

(e) a HOX11 precursor cell population comprising a cell surface molecule selected from the group consisting of CD45, Aa4.1, HSA, Fcγ

RII, Fcγ

RIII, Thy-1, Mac-1, Gr-1, CD44, VLA-4α

, LFA-1β

, ICAM-1 and combinations thereof.

2. The cell population of claim 1, wherein said cell population of group (a) is responsive to a growth factor selected from the group consisting of interleukin-3, interleukin-4, interleukin-6, interleukin-11, erythropoietin, C-kit ligand, leukocyte inhibitory factor and mixtures thereof.

3. The cell population of claim 1, wherein said cell population of group (c) is responsive to a growth factor selected from the group consisting of interleukin-3, erythropoietin, C-kit ligand, leukocyte inhibitory factor and mixtures thereof.

4. The cell population of claim 1, wherein said cell population of group (d) is responsive to a growth factor selected from the group consisting of interleukin-3, erythropoietin, GM-CSF and mixtures thereof.

5. The cell population of claim 1, wherein said cell population of group (a) and group (c) express β

H1, ζ and

β

major-globin RNA.

6. The cell population of claim 1, wherein said cell population comprises cells of a cellular lineage selected from the group consisting of erythroid lineage, endothelial lineage, and leukocyte lineage.

7. The cell population of claim 1, wherein said cell population is derived from an embryonic stem cell population.

8. The cell population of claim 1, wherein said cell population is transformed with a HOX11 gene.

9. The cell population of claim 1, wherein said cell population is derived from a transformed embryoid body cell population, said transformed embryoid body cell population being derived by culturing an embryonic stem cell population transformed with a HOX11 gene in an embryonic body cell medium.

10. The cell population of claim 9, wherein said embryoid body cell medium comprises about 15% serum selected from the group consisting of platelet-poor fetal bovine serum and pre-selected normal fetal calf serum.

11. The cell population of claim 9, wherein said embryoid body cell medium does not include leukocyte inhibitory factor.

12. The cell population of claim 9, wherein said step of culturing is performed from about 1 day to about 28 days.

13. The cell population of claim 9, wherein said step of culturing is performed from about 3 days to about 8 days.

14. The cell population of claim 1, wherein said cell population is derived from a transformed embryoid body cell population cultured under effective conditions in an effective medium comprising serum selected from the group consisting of platelet-poor fetal bovine serum and pre-selected normal fetal calf serum, and a growth factor selected from the group consisting of C-kit ligand, interleukin 3, erythropoietin, and combinations thereof.

15. The cell population of claim 1, wherein said cell population is derived from a transformed embryoid body cell population cultured under effective conditions in an effective medium comprising serum selected from the group consisting of platelet-poor fetal bovine serum and pre-selected normal fetal calf serum, and a combination of growth factors selected from the group consisting of C-kit ligand combined with erythropoietin, and interleukin 3 combined with erythropoietin.

16. The cell population of claim 1, wherein said cell population is produced by the method comprising:

a) introducing a HOX11 gene into an embryonic stem cell population to create a modified stem cell population;

b) culturing said modified stem cell population for about 6 days in an embryoid body cell medium under effective conditions to produce a transformed embryoid body cell population; and

c) incubating said modified embryoid body cell population in the presence of a combination of growth factors selected from the group consisting of C-kit ligand combined with erythropoietin, and interleukin 3 combined with erythropoietin to obtain said cell population.

17. The cell population of claim 1, wherein said cell population has the identifying characteristics of a cell population selected from the group consisting of EBHX-1, EBHX-2, EBHX-3, EBHX-4, EBHX-5, EBHX-6, EBHX-7, EBHX-8, EBHX-9, EBHX-10, EBHX-11, EBHX-12, EBHX-13, EBHX-14 and EBHX-15.

19. The cell population of claim 18, wherein said cell population comprises a cell surface molecule selected from the group consisting of Fcγ

RII, Fcγ

RIII, Thy-1, CD44, VLA-4α

, LFA-1β and

combinations thereof.

20. The cell population of claim 18, wherein said cell population express β

H1, ζ and

β

major-globin RNA.

21. The cell population of claim 18, wherein said cell population is EBHX-1.

18. A HOX11 precursor cell population that is responsive to a growth factor selected from the group consisting of interleukin-3, interleukin-4, interleukin-6, interleukin-11, erythropoietin, C-kit ligand, leukocyte inhibitory factor and mixtures thereof.

22. A HOX11 precursor cell population that is responsive to a growth factor selected from the group consisting of interleukin-3, erythropoietin, C-kit ligand, leukocyte inhibitory factor and mixtures thereof.

23. The cell population of claim 22, wherein said cell population comprises a cell surface molecule selected from the group consisting of HSA, CD44, VLA-4α

, LFA-1β

, ICAM-1 and combinations thereof.

24. The cell population of claim 22, wherein said cell population express β

H1, ζ and

β

major-globin RNA.

25. The cell population of claim 22, wherein said cell population is EBHX-11.

27. The cell population of claim 26, wherein said cell population comprises a cell surface molecule selected from the group consisting of CD45, Aa4.1, Sca-1, HSA, Fcγ

RII, Fcγ

RIII, Thy-1, Mac-1, Gr-1, CD44, VLA-4α

, LFA-1β and

combinations thereof.

28. The cell population of claim 26, wherein said cell population is EBHX-14.

26. A HOX11 precursor cell population that is responsive to a growth factor selected from the group consisting of interleukin-3, erythropoietin, GM-CSF and mixtures thereof.

29. A precursor cell population having the identifying characteristics of a cell population selected from the group consisting of EBHX-1, EBHX-2, EBHX-3, EBHX-4, EBHX-5, EBHX-6, EBHX-7, EBHX-8, EBHX-9, EBHX-10, EBHX-11, EBHX-12, EBHX-13, EBHX-14 and EBHX-15.

30. A method to produce an immortalized precursor cell population, comprising:

a) transforming an embryonic stem cell population with an immortalizing gene to create a transformed stem cell population;

b) culturing said transformed stem cell population under effective conditions to produce a transformed embryoid body cell population; and

c) incubating said transformed embryoid body cell population under conditions suitable to obtain an immortalized precursor cell population.

31. The method of claim 30, wherein said embryonic stem cell population is derived from a mammalian embryo.

32. The method of claim 30, wherein said immortalizing gene comprises human HOX11.

33. The method of claim 30, wherein said immortalizing gene is contained in the recombinant molecule MSCV-HOX11.

34. The method of claim 30, wherein said step of culturing comprises culturing said transformed embryonic stem cells in an embryoid body cell medium comprising about 15% serum selected from the group consisting of platelet-poor fetal bovine serum and pre-selected normal fetal calf serum.

35. The method of claim 30, wherein said step of culturing is performed for from about 3 days to about 8 days.

36. The method of claim 30, wherein said step of incubating comprises culturing said embryoid body cell population in a medium comprising serum selected from the group consisting of platelet-poor fetal bovine serum and pre-selected normal fetal calf serum, and a growth factor selected from the group consisting of C-kit ligand, interleukin 3, erythropoietin, and combinations thereof.

37. The method of claim 30, wherein said. step of incubating comprises culturing said embryoid, body cell population in a medium comprising serum selected from the group consisting of platelet-poor fetal bovine serum and pre-selected normal fetal calf serum, and a combination of growth factors selected from the group consisting of C-kit ligand combined with erythropoietin and interleukin 3 combined with erythropoietin.

39. The method of claim 38, wherein said HOX11 precursor cell population is selected from the group consisting of EBHX-1, EBHX-2, EBHX-3, EBHX-4, EBHX-5, EBHX-6, EBHX-7, EBHX-8, EBHX-9, EBHX-10, EBHX-11, EBHX-12, EBHX-13, EBHX-14, EBHX-15 and derivatives thereof.

40. The method of claim 38, wherein said step of contacting further comprises contacting said cell with a molecule selected from the group consisting of a known regulator of said regulatory factor and a putative regulator of said regulatory factor.

41. The method of claim 38, wherein said step of assessing comprises performing an assay selected from the group consisting of a proliferation assay and a differentiation assay.

42. The method of claim 41, wherein said proliferation assay is selected from the group consisting of cell count number, determining thymidine uptake by a cell and enzyme-based assays.

43. The method of claim 41, wherein said differentiation assay comprises a method selected from the group consisting of:

(a) determining globin expression;

(b) identifying cell surface markers;

(c) determining responsiveness to a growth factor;

(d) observing alterations in morphology; and

determining expression of genes associated with differentiation of hematopoietic cells.

38. A method to identify a regulatory factor that influences the growth or differentiation of a cell, comprising:

a) contacting a HOX11 precursor cell population with a regulatory factor selected from the group consisting of a putative regulatory factor, a known regulatory factor and mixtures thereof; and

b) assessing the responsiveness of said precursor cell population to said regulatory factor.

44. A method to identify a compound expressed during the development of a population of embryonic stem cells, comprising characterizing at least a portion of the cellular composition of at least one cell contained in a HOX11 precursor cell population, to identify a compound expressed during the development of a population of embryonic stem cells.

45. The method of claim 44, wherein said HOX11 precursor cell population is selected from the group consisting of EBHX-1, EBHX-2, EBHX-3, EBHX-4, EBHX-5, EBHX-6, EBHX-7, EBHX-8, EBHX-9, EBHX-10, EBHX-11, EBHX-12, EBHX-13, EBHX-14, EBHX-15 and derivatives thereof.

47. The method of claim 46, wherein said HOX11 precursor cell population is selected from the group consisting of EBHX-1, EBHX-2, EBHX-3, EBHX-4, EBHX-5, EBHX-6, EBHX-7, EBHX-8, EBHX-9, EBHX-10, EBHX-11, EBHX-12, EBHX-13, EBHX-14, EBHX-15 and derivatives thereof.

49. The method of claim 48, wherein said HOX11 precursor cell population is selected from the group consisting of EBHX-1, EBHX-2, EBHX-3, EBHX-4, EBHX-5, EBHX-6, EBHX-7, EBHX-8, EBHX-9, EBHX-10, EBHX-11, EBHX-12, EBHX-13, EBHX-14, EBHX-15 and derivatives thereof.

46. A method to produce an antibody, comprising administering to an animal an effective amount of a protein derived from a HOX11 precursor cell population and recovering an antibody capable of selectively binding to said protein.

48. A method to repopulate a hematopoietic cell population in an animal, comprising administering to an animal a suitable number of cells of a HOX11 precursor cell population.

50. A method to identify a neutralizing reagent, comprising:

a) contacting a HOX11 precursor cell population with a known regulatory factor to produce a controlled cell population;

b) combining said controlled cell population with a neutralizing reagent selected from the group consisting of a known neutralizing compound of said regulatory factor and a putative neutralizing compound of said regulatory factor; and

c) assessing the responsiveness of said precursor cell population to said neutralizing compound.

51. An endothelial cell population produced by the method comprising,

a) transforming an EB cell population with a nucleic acid molecule encoding a Polyoma Middle T antigen to form Polyoma Middle T EB cells;

and

b) culturing said Polyoma Middle T EB cells under conditions suitable to obtain an endothelial cell population.

52. The endothelial cell population of claim 51, wherein said embryoid body cell population is obtained by culturing an ES cell population for no more than about 8 days.

53. The endothelial cell population of claim 51, wherein said embryoid body cell population is obtained by culturing an ES cell population for no more than about 6 days.

54. The endothelial cell population of claim 51, wherein said embryoid body cell population is obtained by culturing an ES cell population for about 4 days.

55. The endothelial cell population of claim 51, wherein said nucleic acid molecule encodes a full-length Polyoma Middle T antigen.

56. The endothelial cell population of claim 51, wherein said step of culturing is performed in the presence of endothelial cell growth supplement.

57. The endothelial cell population of claim 51, wherein said step of culturing is performed in the presence of from about 50 μ

g/ml to about 1000 μ

g/ml of endothelial cell growth supplement.

58. The endothelial cell population of claim 51, wherein said step of culturing is performed at a cell density of from about 1×

10⁵ cells to about 5×

10⁵ cells per milliliter of culture medium.

59. The endothelial cell population of claim 51, wherein said step of culturing is performed for about 2 months.

60. The endothelial cell population of claim 51, wherein said endothelial cell population comprises a cell that can take up diI-acetylated-low density lipoproteins.

61. The endothelial cell population of claim 51, wherein said endothelial cell population comprises a cell that expresses a protein selected from the group consisting of CD31 and Flk-1.

62. The endothelial cell population of claim 51, wherein said endothelial cell population has the identifying characteristics of D4T.

63. An endothelial cell population having the identifying characteristics of D4T.

64. A conditioned medium comprising a cell culture supernatant recovered from a culture of an endothelial cell population produced by the method comprising,

a) transforming an EB cell population with a nucleic acid molecule encoding a Polyoma Middle T antigen to form Polyoma Middle T EB cells;

and

b) culturing said Polyoma Middle T EB cells under conditions suitable to obtain an endothelial cell population.

65. The conditioned medium of claim 64, wherein said endothelial cell population has the identifying characteristics of D4T.

66. The conditioned medium of claim 64, wherein said cell culture supernatant is produced by the method comprising culturing said endothelial cell population in a medium suitable for the growth of said endothelial cell population to a cell density at which said population is substantially confluent.

67. The conditioned medium of claim 64, wherein said cell culture supernatant is produced by a method comprising culturing said endothelial cell population for about:

72 hours in a medium suitable for the growth of said endothelial cell population.

68. The conditioned medium of claim 64, wherein said cell culture supernatant further comprises endothelial cell growth factor.

69. The conditioned medium of claim 64, wherein said conditioned medium is capable of enhancing the growth of a BLAST cell population about 2-fold when said conditioned medium is added to a culture of about 5×

10⁴ cells from a 3 day EB cell population, about 5-fold when said conditioned medium is added to a culture of about 1.7×

10⁴ cells from a 3 day EB cell population and about 23-fold when said conditioned medium is added to a culture of about 6×

10³ cells from a 3 day EB cell population, wherein said culture is performed under conditions suitable for the development of a BLAST cell population.

70. A method to identify a hematopoietic cell growth factor, comprising isolating a compound from a conditioned medium comprising a cell culture supernatant recovered from a culture of an endothelial cell population produced by the method comprising,

a) transforming an EB cell population with a nucleic acid molecule encoding a Polyoma Middle T antigen to form Polyoma Middle T EB cells;

and

b) culturing said Polyoma Middle T EB cells under conditions suitable to obtain an endothelial cell population.

71. The method of claim 70, wherein said endothelial cell population is has the identifying characteristics of D4T.

72. The method of claim 70, wherein said step of isolating comprises using a technique selected from the group consisting of antibody binding studies, gel electrophoresis and chromatography.

73. The method of claim 70, wherein said method further comprises testing the ability of said isolated compound to stimulate the growth of a hematopoietic cell population.

74. A method to enhance a population of precursor cells, comprising contacting a precursor cell population with a cell culture supernatant recovered from a culture of an endothelial cell population produced by the method comprising,

a) transforming an EB cell population with a nucleic acid molecule encoding a Polyoma Middle T antigen to form Polyoma Middle T EB cells;

and

b) culturing said Polyoma Middle T EB cells under conditions suitable to obtain an endothelial cell population.

75. The method of claim 74, wherein said precursor cell population is selected from the group consisting of a totipotent cell population, a pluripotent cell population and a stem cell lineage restricted precursor cell.

76. An enhanced precursor cell population, comprising a precursor cell population contacted with a cell culture supernatant recovered from a culture of an endothelial cell population produced by the method comprising,

a) transforming an EB cell population with a nucleic acid molecule encoding a Polyoma Middle T antigen to form Polyoma Middle T EB cells;

and

b) culturing said Polyoma Middle T EB cells under conditions suitable to obtain an endothelial cell population,

wherein said precursor cell population, when contacted with said cell supernatant results in the formation of an enhanced precursor population.

77. The cell population of claim 76, wherein said enhanced precursor cell population comprises about 2-fold more cells than the precursor cell population.

78. The cell population of claim 76, wherein said enhanced precursor cell population comprises about 5-fold more cells than the precursor cell population.

79. The cell population of claim 76, wherein said enhanced precursor cell population comprises about 20-fold more cells than the precursor cell population.

80. The cell population of claim 76, wherein said precursor cell population comprises a human precursor cell population.

81. A method to identify a hematopoietic cell growth factor, comprising isolating a compound from an endothelial cell population produced by the method comprising,

a) transforming an EB cell population with a nucleic acid molecule encoding a Polyoma Middle T antigen to form Polyoma Middle T EB cells;

and

b) culturing said Polyoma Middle T EB cells under conditions suitable to obtain an endothelial cell population.

82. The method of claim 81, wherein said endothelial cell population is has the identifying characteristics of D4T.

83. The method of claim 81, wherein said compound is identified by a method selected from the group consisting of analysis of RNA expression and expression cloning.