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 FcγRII, FcγRIII, 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 FcγRII, FcγRIII, 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, FcγRII, FcγRIII, 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, FcγRII, FcγRIII, 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 βH1, 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.
- 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.
- 19. The cell population of claim 18, wherein said cell population comprises a cell surface molecule selected from the group consisting of FcγRII, FcγRIII, Thy-1, CD44, VLA-4α, LFA-1β and combinations thereof.
- 20. The cell population of claim 18, wherein said cell population express βH1, ζ and β major-globin RNA.
- 21. The cell population of claim 18, wherein said cell population is EBHX-1.
- 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 βH1, ζ and β major-globin RNA.
- 25. The cell population of claim 22, wherein said cell population is EBHX-11.
- 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.
- 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, FcγRII, FcγRIII, 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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 μg/ml to about 1000 μg/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.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of U.S. patent application Ser. No. 08/343,686 for “Novel Embryonic Cell Populations and Methods to Isolate Such Populations”, filed Nov. 21, 1994, incorporated herein by this reference in its entirety. The present application is also a continuation-in-part of PCT patent application Ser. No.______ for “Novel Embryonic Cell Populations and Methods to Isolate Such Populations”, filed Nov. 20, 1995, incorporated herein by this reference in its entirety.
GOVERNMENT RIGHTS
[0002] This invention was made in part with government support under HL 48834-02, awarded by the National Institutes of Health. The government has certain rights to this invention.
Divisions (2)
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Number |
Date |
Country |
Parent |
09255127 |
Feb 1999 |
US |
Child |
09767893 |
Jan 2001 |
US |
Parent |
08570211 |
Dec 1995 |
US |
Child |
09255127 |
Feb 1999 |
US |
Continuation in Parts (2)
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Number |
Date |
Country |
Parent |
08343686 |
Nov 1994 |
US |
Child |
08570211 |
Dec 1995 |
US |
Parent |
PCT/US95/14495 |
Nov 1995 |
US |
Child |
08570211 |
Dec 1995 |
US |