Definitive endoderm

• US 20070154984A1
• Filed: 12/23/2004
• Published: 07/05/2007
• Est. Priority Date: 12/23/2003
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: Agulnick, Alan, D'Amour, Kevin, Baetge, Emmanuel
• Granted Patent: US 7,704,738 B2
• Time in Patent Office: Days
• US Class Current: 435/69.100
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/068 Stem cells; Progenitors

Abstract

Disclosed herein are cell cultures comprising definitive endoderm cells and methods of producing the same. Also disclosed herein are cell populations comprising substantially purified definitive endoderm cells as well as methods for enriching, isolating and purifying definitive endoderm cells from other cell types.

Claims

1-35. -35. (canceled)

36. A method of producing definitive endoderm cells, said method comprising the steps of:

obtaining a cell population comprising pluripotent human cells;

providing said cell population with at least one growth factor of the TGFβ

superfamily in an amount sufficient to promote differentiation of said pluripotent cells to definitive endoderm cells, said definitive endoderm cells being multipotent cells that can differentiate into cells of the gut tube or organs derived therefrom; and

allowing sufficient time for definitive endoderm cells to form, wherein said sufficient time for definitive endoderm cells to form has been determined by detecting the presence of definitive endoderm cells in said cell population.

37. The method of claim 36, wherein at least about 10% of said pluripotent cells differentiate into definitive endoderm cells.

38. The method of claim 36, wherein at least about 50% of said pluripotent cells differentiate into definitive endoderm cells.

39. The method of claim 36, wherein at least about 70% of said pluripotent cells differentiate into definitive endoderm cells.

40. The method of claim 36, wherein at least about 80% of said pluripotent cells differentiate into definitive endoderm cells.

41. The method of claim 36, wherein detecting the presence of definitive endoderm cells in said cell population comprises detecting the expression of at least one marker selected from the group consisting of SOX17 and CXCR4 and at least one marker from the group consisting of OCT4, AFP, TM, SPARC and SOX7 in cells of said cell population, wherein the expression of a marker selected from the group consisting of SOX17 and CXCR4 is greater than the expression of a marker selected from the group consisting of OCT4, AFP, TM, SPARC and SOX7 in said definitive endoderm cells.

42. The method of claim 36, wherein detecting the presence of definitive endoderm cells in said cell population comprises detecting the expression of at least one marker selected from the group consisting of SOX17 and CXCR4 and at least one marker from the group consisting of AFP, TM, and SOX7 in cells of said cell population, wherein the expression of a marker selected from the group consisting of SOX17 and CXCR4 is greater than the expression of a marker selected from the group consisting of AFP, TM, and SOX7 in said definitive endoderm cells.

43. The method of claim 42, wherein the expression of at least one of said markers is determined by Q-PCR.

44. The method of claim 42, wherein the expression of at least one of said markers is determined by immunocytochemistry.

45. The method of claim 36, wherein detecting the presence of definitive endoderm cells in said cell population comprises detecting the expression of at least one marker selected from the group consisting of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 and at least one marker from the group consisting of OCT4, AFP, TM, SPARC and SOX7 in cells of said cell population, wherein the expression of a marker selected from the group consisting of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 is greater than the expression of a marker selected from the group consisting of OCT4, AFP, TM, SPARC and SOX7 in said definitive endoderm cells.

46. The method of claim 36, wherein said at least one growth factor is of the Nodal/Activin subgroup of the TGFβ

superfamily.

47. The method of claim 46, wherein said at least one growth factor is selected from the group consisting of Nodal Activin A, Activin B and combinations thereof.

48. The method of claim 47, wherein said at least one growth factor is Nodal.

49. The method of claim 47, wherein said at least one growth factor is Activin A.

50. The method of claim 47, wherein said at least one growth factor is Activin B.

51. The method of claim 36, wherein a plurality of growth factors of the TGFβ

superfamily is provided.

52. The method of claim 51, wherein said plurality of growth factors comprises Nodal Activin A and Activin B.

53. The method of claim 36, wherein said at least one growth factor is provided in a concentration of at least about 10 ng/ml.

54. The method of claim 36, wherein said at least one growth factor is provided in a concentration of at least about 100 ng/ml.

55. The method of claim 36, wherein said at least one growth factor is provided in a concentration of at least about 500 ng/ml.

56. The method of claim 36, wherein said at least one growth factor is provided in a concentration of at least about 1000 ng/ml.

57. The method of claim 36, wherein said at least one growth factor is provided in a concentration of at least about 5000 ng/ml.

58. The method of claim 36, wherein said cell population is grown in a medium comprising less than about 10% serum.

59. The method of claim 36, wherein said pluripotent cells comprise stem cells.

60. The method of claim 59, wherein said pluripotent cells comprise embryonic stem cells.

61. The method of claim 60, wherein said embryonic stem cells are derived from a tissue selected from the group consisting of the morula, the ICM of an embryo and the gonadal ridges of an embryo.

62. A definitive endoderm cell produced by the method of claim 36.

63. A method of producing a cell population enriched in definitive endoderm cells, said method comprising the steps of:

differentiating cells in a population of pluripotent human cells so as to produce definitive endoderm cells, said definitive endoderm cells being multipotent cells that can differentiate into cells of the gut tube or organs derived therefrom;

providing to said cell population a reagent which binds to a marker expressed in said definitive endoderm cells but which is not substantially expressed in other cell types present in said cell population; and

separating said definitive endoderm cells bound to said reagent from said other cell types present in said cell population, thereby producing a cell population enriched in definitive endoderm cells.

64. The method of claim 63, wherein the differentiating step further comprises obtaining a cell population comprising pluripotent human cells, providing said cell population with at least one growth factor of the TGFβ

superfamily in an amount sufficient to promote differentiation of said pluripotent cells to definitive endoderm cells, said definitive endoderm cells being multipotent cells that can differentiate into cells of the gut tube or organs derived therefrom, and allowing sufficient time for definitive endoderm cells to form, wherein said sufficient time for definitive endoderm cells to form has been determined by detecting the presence of definitive endoderm cells in said cell population.

65. The method of claim 63, wherein detecting comprises detecting the expression of at least one marker selected from the group consisting of SOX17 and CXCR4 and at least one marker from the group consisting of OCT4, AFP, TM, SPARC and SOX7 in cells of said cell population, wherein the expression of a marker selected from the group consisting of SOX17 and CXCR4 is greater than the expression of a marker selected from the group consisting of OCT4, AFP, TM, SPARC and SOX7 in said definitive endoderm cells.

66. The method of claim 63, wherein detecting comprises detecting the expression of at least one marker selected from the group consisting of SOX17 and CXCR4 and at least one marker from the group consisting of AFP, TM, and SOX7 in cells of said cell population, wherein the expression of a marker selected from the group consisting of SOX17 and CXCR4 is greater than the expression of a marker selected from the group consisting of AFP, TM, and SOX7 in said definitive endoderm cells.

67. The method of claim 63, wherein detecting comprises detecting the expression of at least one marker selected from the group consisting of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 and at least one marker from the group consisting of OCT4, AFP, TM, SPARC and SOX7 in cells of said cell population, wherein the expression of a marker selected from the group consisting of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 is greater than the expression of a marker selected from the group consisting of OCT4, AFP, TM, SPARC and SOX7 in said definitive endoderm cells.

68. The method of claim 63, wherein at least about 95% of said cells are definitive endoderm cells.

69. The method of claim 63, wherein at least about 98% of said cells are definitive endoderm cells.

70. The method of claim 63, wherein said marker is CXCR4.

71. The method of claim 63, wherein said reagent is an antibody

72. The method of claim 71, wherein said antibody has affinity for CXCR4.

73. An enriched population of definitive endoderm cells produced by the method of claim 63.

74-75. -75. (canceled)