Pdx1 Expressing Endoderm

• US 20090011502A1
• Filed: 04/26/2005
• Published: 01/08/2009
• Est. Priority Date: 04/27/2004
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Novocell, Inc. (Vertex Pharmaceuticals Incorporated)
• Inventors: Eliazer, Susan, D'Amour, Kevin Allen, Agulnick, Alan D., Baetge, Emmanuel E.
• Granted Patent: US 8,647,873 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• 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 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• 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

Abstract

Disclosed herein are cell cultures comprising PDX1-positive endoderm cells and methods of producing the same. Also disclosed herein are cell populations comprising substantially purified PDX1-positive endoderm cells as well as methods for enriching, isolating and purifying PDX1-positive endoderm cells from other cell types. Methods of identifying differentiation factors capable of promoting the differentiation of endoderm cells, such as PDX1-positive foregut endoderm cells and PDX1-negative definitive endoderm cells, are also disclosed.

Claims

1-28. -28. (canceled)

29. A method of producing PDX1-positive foregut endoderm cells, said method comprising the steps of:

obtaining a cell population comprising PDX1-negative definitive endoderm cells; and

providing said cell population with a retinoid in an amount sufficient to promote differentiation of at least a portion of said PDX1-negative definitive endoderm cells to PDX1-positive foregut endoderm cells, wherein said PDX1-positive foregut endoderm cells are multipotent cells that can differentiate into cells, tissues or organs derived from the anterior portion of the gut tube.

30. The method of claim 29 further comprising the step of allowing sufficient time for PDX1-positive foregut endoderm cells to form, wherein said sufficient time for PDX1-positive foregut endoderm cells to form has been determined by detecting the presence of PDX1-positive foregut endoderm cells in said cell population.

31. The method of claim 29, wherein at least about 2% of said PDX1-negative definitive endoderm cells differentiate into PDX1-positive foregut endoderm cells.

32. The method of claim 29, wherein at least about 5% of said PDX1-negative definitive endoderm cells differentiate into PDX1-positive foregut endoderm cells.

33. The method of claim 29, wherein at least about 10% of said PDX1-negative definitive endoderm cells differentiate into PDX1-positive foregut endoderm cells.

34. The method of claim 29, wherein at least about 25% of said PDX1-negative definitive endoderm cells differentiate into PDX1-positive foregut endoderm cells.

35. The method of claim 29, wherein detecting the presence of PDX1-positive foregut endoderm cells in said cell population comprises detecting the expression of PDX1.

36. The method of claim 35, wherein the expression of PDX1 is greater than the expression of a marker selected from the group consisting of alpha-fetoprotein (AFP), SOX7, SOX1, ZIC1 and NFM in said PDX1-positive foregut endoderm cells.

37. The method of claim 35, wherein the expression of said PDX1 is determined by quantitative polymerase chain reaction (Q-PCR).

38. The method of claim 35, wherein the expression of PDX1 is determined by immunocytochemistry.

39. The method of claim 29, wherein said retinoid is retinoic acid (RA).

40. The method of claim 39, wherein RA is provided in a concentration ranging from about 0.01 μ

M to about 50 μ

M.

41. The method of claim 39, wherein RA is provided in a concentration ranging from about 0.04 μ

M to about 20 μ

M.

42. The method of claim 39, wherein RA is provided in a concentration ranging from about 0.1 μ

M to about 10 μ

M.

43. The method of claim 39, wherein RA is provided in a concentration ranging from about 0.2 μ

M to about 2.5 μ

M.

44. The method of claim 39, wherein RA is provided in a concentration ranging from about 0.5 μ

M to about 1.5 μ

M.

45. The method of claim 39, wherein RA is provided in a concentration of about 1 μ

M.

46. The method of claim 39, wherein RA is provided when said culture is about 4-days-old.

47. The method of claim 29 further comprising providing to said culture a factor selected from the group consisting of FGF-10, FGF-4, activin A, activin B, B27, conditioned medium, and combinations of said factors in an amount sufficient to enhance the production of PDX1-positive foregut endoderm cells.

48. The method of claim 47, wherein said factor is selected from the group consisting of FGF-10, FGF-4, activin A and activin B.

49. The method of claim 48, wherein said factor is provided in a concentration ranging from about 10 ng/ml to about 500 ng/ml.

50. The method of claim 48, wherein said factor is provided in a concentration ranging from about 20 ng/ml to about 200 ng/ml.

51. The method of claim 48, wherein said factor is provided in a concentration ranging from about 25 ng/ml to about 75 ng/ml.

52. The method of claim 48, wherein said factor is provided in a concentration about 50 ng/ml.

53. The method of claim 48, wherein said factor is provided at approximately the same time as said retinoid.

54. The method of claim 47 wherein said factor is B27.

55. The method of claim 54, wherein B27 is provided in a concentration ranging from about 0.1% to about 20% of the total medium.

56. The method of claim 54, wherein B27 is provided in a concentration ranging from about 0.2% to about 5% of the total medium.

57. The method of claim 54, wherein B27 is provided in a concentration ranging from 0.5% to about 2% of the total medium.

58. The method of claim 54, wherein B27 is provided in a concentration of about 1% of the total medium.

59. The method of claim 54, wherein B27 is provided at approximately the same time as said retinoid.

60. The method of claim 47 wherein said factor is conditioned medium.

61. The method of claim 60, wherein conditioned medium is provided in a concentration ranging from about 10% to about 100% of the total medium.

62. The method of claim 60, wherein conditioned medium is provided in a concentration ranging from about 20% to about 80% of the total medium.

63. The method of claim 60, wherein conditioned medium is provided in a concentration ranging from about 40% to about 60% of the total medium.

64. The method of claim 60, wherein conditioned medium is provided in a concentration of about 50% of the total medium.

65. The method of claim 60, wherein conditioned medium is provided at approximately the same time as said retinoid.

66. The method of claim 60, wherein conditioned medium is prepared by contacting differentiated human embryonic stem cells (hESCs) with a cell culture medium for about 24 hours.

67. The method of claim 66, wherein said hESCs are differentiated for about 5 days in a cell culture medium selected from the group consisting of RPMI supplemented with 3% serum, low serum RPMI supplemented with activin A and low serum RPMI supplemented with BMP4.

68. A PDX1-positive foregut endoderm cell produced by the method of claim 29.

110. The method of claim 29, wherein obtaining said cell population comprising PDX1-negative definitive endoderm cells further comprises providing a growth factor of the TGFβ

superfamily to a population of human embryonic stem cells.

111. The method of claim 110, wherein said growth factor of the TGFβ

superfamily is activin A.

112. The method of claim 111, wherein said activin A is provided to said population of human embryonic stem cells at a concentration of about 100 ng/ml.

113. The method of claim 110, wherein said growth factor of the TGFβ

superfamily is removed from said cell culture prior to providing said retinoid.

114. The method of claim 113, wherein FGF-10 is provided to said cell culture.

115. The method of claim 114, wherein said FGF-10 is provided to said cell culture at a concentration of about 50 ng/ml.

116. The method of claim 114, wherein said retinoid is retinoic acid (RA).

117. The method of claim 116, wherein RA is provided in a concentration ranging from about 0.2 μ

M to about 2.5 μ

M.

69-109. -109. (canceled)