METHOD OF GENERATING NATURAL KILLER CELLS AND DENDRITIC CELLS FROM HUMAN EMBRYONIC STEM CELL-DERIVED HEMANGIOBLASTS

• US 20200131475A1
• Filed: 10/31/2019
• Published: 04/30/2020
• Est. Priority Date: 12/04/2009
• Status: Active Application

• Current Assignee: Astellas Institute for Regenerative Medicine (Astellas Pharma Incorporated)
• Original Assignee: Astellas Institute for Regenerative Medicine (Astellas Pharma Incorporated)
• Inventors: Kimbrel, Erin, Lu, Shi-Jiang
• Application Number:
  US16/669,931
• Publication Number:
  US 20200131475A1
• Time in Patent Office: Days
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 2239/48 Blood cells, e.g. leukemia or lymphoma
• CPC Class Codes: A61K 35/15 Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells presenting a specific antigen A61K39/00; therapeutic combinations of antibodies, or fragments thereof, and blood-derived cells A61K39/00
• CPC Class Codes: A61K 39/4613 Natural-killer cells [NK or NK-T]
• CPC Class Codes: A61K 39/4644 Cancer antigens
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• 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 2501/2303 Interleukin-3 (IL-3)
• CPC Class Codes: C12N 2501/2304 Interleukin-4 (IL-4)
• CPC Class Codes: C12N 2501/26 Flt-3 ligand (CD135L, flk-2 ligand)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0639 Dendritic cells, e.g. Langherhans cells in the epidermis
• CPC Class Codes: C12N 5/0646 Natural killers cells [NK], NKT cells

Abstract

This invention provides methods of generating natural killer (NK) cells and dendritic cells (DCs). The methods utilize human hemangioblasts as intermediate cells to generate the NK cells and DCs. In various embodiments, the methods do not require the use of stromal feeder layers.

Claims

1-38. -38. (canceled)

39. A method of producing natural killer (NK) cells, the method comprising culturing a hemangioblast under feeder free conditions in a first culture media comprising at least two growth factors selected from the group consisting of SCF, Flt3-ligand (FL), and IL3, thereby producing an NK cell.

40. The method of claim 39, wherein the first culture media is a liquid media.

41. The method of claim 39, wherein the first culture media is a semi-solid media.

42. The method of claim 41, wherein the semi-solid media comprises methylcellulose.

43. The method of claim 42, wherein the semi-solid media lacks erythropoietin.

44. The method of claim 39, wherein the first culture media further comprises at least one additional growth factor selected from the group consisting of IL2, IL6, IL7, and IL15.

45. The method of claim 39, further comprising culturing the hemangioblast under feeder free conditions in a second culture media comprising at least one growth factor selected from the group consisting of IL7, IL15, SCF, and FL.

46. The method of claim 45, wherein the second culture media is a liquid media.

47. The method of claim 39, wherein the hemangioblast is obtained from a pluripotent stem cell.

48. The method of claim 47, wherein the pluripotent stem cell is a human embryonic stem cell or a human induced pluripotent stem cell.

49. The method of claim 47, wherein the hemangioblast is obtained from a pluripotent stem cell under feeder-free conditions.

50. The method of claim 47, wherein the hemangioblast is obtained from a pluripotent stem cell without culturing with erythropoietin.

51. The method of claim 39, wherein the NK cells are CD56+.

52. The method of claim 39, wherein the NK cells are CD56−

.

53. The method of claim 39, wherein the first culture media is serum-free.

54. A method of producing a human pluripotent stem cell-derived natural killer (NK) cell, the method comprising

(a) differentiating a human pluripotent stem cell to a precursor cell capable of differentiating to give rise to at least hematopoietic cell types, endothelial cell types, or mesodermal derivatives under feeder free conditions in at least one culture media comprising at least one growth factor selected from the group consisting of bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), and bFGF;

and

(b) differentiating the precursor cell to an NK cell under feeder free conditions in at least one culture media comprising at least two growth factors selected from the group consisting of SCF, Flt3-ligand (FL), and IL3.

55. The method of claim 54, wherein the human pluripotent stem cell is a human embryonic stem cell or a human induced pluripotent stem cell.

56. The method of claim 54, wherein the NK cell is CD56+.

57. The method of claim 54, wherein the at least one culture media of step (a) is serum-free.

58. The method of claim 54, wherein the at least one culture media of step (b) further comprises at least one additional growth factor selected from the group consisting of IL2, IL6, IL7, and IL15.

59. The method of claim 54, wherein the at least one culture media of steps (a) and (b) lack erythropoietin.

60. The method of claim 54, further comprising differentiating the precursor cell to an NK cell under feeder free conditions in step (b) in a second culture media comprising IL7, IL15, SCF, and FL.

61. The method of claim 54, wherein the precursor cell of step (a) is harvested before step (b).

62. A method of producing a natural killer (NK) cell, the method comprising

(a) differentiating an embryoid body to a precursor cell capable of differentiating to give rise to at least hematopoietic cell types, endothelial cell types, or mesodermal derivatives under feeder-free conditions in at least one culture media comprising at least two growth factors selected from the group consisting of TPO, vascular endothelial growth factor (VEGF), and Flt3-ligand (FL);

and

(b) differentiating the precursor cell to an NK cell under feeder-free conditions in at least one culture media comprising at least three growth factors selected from the group consisting of SCF, FL, IL3, IL7, IL2, and IL6.

63. The method of claim 62, wherein the embryoid body is obtained from a pluripotent stem cell.

64. The method of claim 63, wherein the pluripotent stem cell is a human embryonic stem cell or a human induced pluripotent stem cell.

65. The method of claim 62, wherein the NK cell is CD56+.

66. The method of claim 62, wherein the at least one culture media of step (a), (b), or both are serum-free.

67. The method of claim 62, wherein the at least one culture media of steps (a) and (b) lack erythropoietin.

68. The method of claim 62, wherein the precursor cell of step (a) is harvested before step (b).

69. A method of producing a natural killer (NK) cell, the method comprising

(a) culturing an embryoid body in a culture media comprising bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), and bFGF;

and

(b) culturing the product of step (a) in a culture media comprising at least two growth factors selected from the group consisting of TPO, VEGF, and Flt3-ligand (FL);

(c) culturing the product of (b) in a culture media comprising at least three growth factors selected from the group consisting of SCF, FL, IL3, IL7, IL2, and IL6, thereby generating an NK cell;

wherein the culturing is conducted under feeder-free conditions throughout steps (a)-(c) of the method.

70. The method of claim 69, wherein the embryoid body is obtained from a pluripotent stem cell.

71. The method of claim 69, wherein the pluripotent stem cell is a human embryonic stem cell or a human induced pluripotent stem cell.

72. The method of claim 69, wherein the NK cell is CD56+.

73. The method of claim 69, wherein the culture media of step (a), (b), and/or (c) are serum-free.

74. The method of claim 69, wherein the culture media of steps (a), (b), and (c) lack erythropoietin.

75. A method of producing a human pluripotent stem cell-derived natural killer (NK) cell, the method comprising

differentiating a human pluripotent stem cell to an NK cell under feeder-free conditions in a series of culture media, wherein (i) a first culture media is a serum-free culture media comprising at least two growth factors selected from the group consisting of bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), and bFGF;

(ii) a second culture media is a serum-free culture media comprising at least two growth factors selected from the group consisting of TPO, VEGF, Flt3-ligand (FL), bFGF, IL2, IL7, and IL15; and

(iii) a third culture media comprises at least three growth factors selected from the group consisting of SCF, FL, IL3, IL7, IL2, and IL6, thereby generating an NK cell.

76. The method of claim 75, wherein the human pluripotent stem cell is a human embryonic stem cell or a human induced pluripotent stem cell.

77. The method of claim 75, wherein the NK cell is CD56+.

78. The method of claim 75, wherein the first culture media, the second culture media, and the third culture media lack erythropoietin.