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Pdx1-expressing dorsal and ventral foregut endoderm


Title: Pdx1-expressing dorsal and ventral foregut endoderm.
Abstract: Disclosed herein are cell cultures comprising dorsal and/or ventral PDX1-positive foregut endoderm cells and methods of producing the same. Also disclosed herein are cell populations comprising substantially purified dorsal and/or ventral PDX1-positive foregut endoderm cells as well as methods for enriching, isolating and purifying dorsal and/or ventral PDX1-positive foregut endoderm cells from other cell types. Methods of identifying differentiation factors capable of promoting the differentiation of dorsal and/or ventral PDX1-positive foregut endoderm cells, are also disclosed. ...




USPTO Applicaton #: #20100233755 - Class: 435 29 (USPTO) - 09/16/10 - Class 435 
Inventors: Kevin Allen D'amour, Alan D. Agulnick, Susan Eliazer, Emmanuel E. Baetge

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The Patent Description & Claims data below is from USPTO Patent Application 20100233755, Pdx1-expressing dorsal and ventral foregut endoderm.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 11/588,693, filed Oct. 27, 2006, entitled “PDX1-EXPRESSING DORSAL AND VENTRAL FOREGUT ENDODERM,” which is a nonprovisional application of and claims priority to U.S. Provisional Patent Application No. 60/730,917, entitled PDX1-EXPRESSING DORSAL AND VENTRAL FOREGUT ENDODERM, filed Oct. 27, 2005, the disclosures of which are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

- Top of Page


The present invention relates to the fields of medicine and cell biology. In particular, the present invention relates to compositions comprising mammalian foregut endoderm cells and compositions comprising dorsal and/or ventral PDX1-positive foregut endoderm cells and methods of making, isolating and using such cells.

BACKGROUND

Human pluripotent stem cells, such as embryonic stem (ES) cells and embryonic germ (EG) cells, were first isolated in culture without fibroblast feeders in 1994 (Bongso et al., 1994) and with fibroblast feeders (Hogan, 1997). Later, Thomson, Reubinoff and Shamblott established continuous cultures of human ES and EG cells using mitotically inactivated mouse feeder layers (Reubinoff et al., 2000; Shamblott et al., 1998; Thomson et al., 1998).

Human ES and EG cells (hESCs) offer unique opportunities for investigating early stages of human development as well as for therapeutic intervention in several disease states, such as diabetes mellitus and Parkinson's disease. For example, the use of insulin-producing β-cells derived from hESCs would offer a vast improvement over current cell therapy procedures that utilize cells from donor pancreases for the treatment of diabetes. However, presently it is not known how to generate an insulin-producing β-cell from hESCs. As such, current cell therapy treatments for diabetes mellitus, which utilize islet cells from donor pancreases, are limited by the scarcity of high quality islet cells needed for transplant. Cell therapy for a single Type I diabetic patient requires a transplant of approximately 8×108 pancreatic islet cells. (Shapiro et al., 2000; Shapiro et al., 2001a; Shapiro et al., 2001b). As such, at least two healthy donor organs are required to obtain sufficient islet cells for a successful transplant. Human embryonic stem cells offer a source of starting material from which to develop substantial quantities of high quality differentiated cells for human cell therapies.

Two properties that make hESCs uniquely suited to cell therapy applications are pluripotence and the ability to maintain these cells in culture for prolonged periods. Pluripotency is defined by the ability of hESCs to differentiate to derivatives of all 3 primary germ layers (endoderm, mesoderm, ectoderm) which, in turn, form all somatic cell types of the mature organism in addition to extraembryonic tissues (e.g. placenta) and germ cells. Although pluripotency imparts extraordinary utility upon hESCs, this property also poses unique challenges for the study and manipulation of these cells and their derivatives. Owing to the large variety of cell types that may arise in differentiating hESC cultures, the vast majority of cell types are produced at very low efficiencies. Additionally, success in evaluating production of any given cell type depends critically on defining appropriate markers. Achieving efficient, directed differentiation is of great importance for therapeutic application of hESCs.

In order to use hESCs as a starting material to generate cells that are useful in cell therapy applications, it would be advantageous to overcome the foregoing problems. For example, in order to achieve the level of cellular material required for islet cell transplantation therapy, it would be advantageous to efficiently direct hESCs toward the pancreatic islet/β-cell lineage at the very earliest stages of differentiation.

In addition to efficient direction of the differentiation process, it would also be beneficial to isolate and characterize intermediate cell types along the differentiation pathway towards the pancreatic islet/β-cell lineage and to use such cells as appropriate lineage precursors for further steps in the differentiation.

SUMMARY

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OF THE INVENTION

Embodiments of the present invention relate to cell cultures of PDX1-negative foregut endoderm cells (foregut endoderm cells). In some embodiments, the foregut endoderm expresses the HNF1b and FOXA1 markers but does not substantially express PDX1. Other embodiments of the present invention relate to cell cultures of PDX1-positive, dorsally-biased, foregut endoderm cells (dorsal PDX1-positive foregut endoderm cells). In some embodiments, the PDX1-positive, dorsally-biased, foregut endoderm cells express one or more markers selected from Table 3 and/or one or more markers selected from Table 4. Additional embodiments, relate to cell cultures of PDX1-positive, ventrally-biased, foregut endoderm cells (ventral PDX1-positive foregut endoderm cells). In some embodiments, the PDX1-positive, ventrally-biased, foregut endoderm cells express one or more markers selected from Table 3 but do not substantially express a marker selected from Table 4 as compared to the expression of the same marker in PDX1-positive, dorsally-biased, foregut endoderm cells.

Additional embodiments of the present invention relate to enriched, isolated and/or purified cell populations comprising PDX1-negative foregut endoderm cells. Other embodiments relate to PDX1-positive, dorsally-biased, foregut endoderm cells. Still other embodiments relate to enriched, isolated and/or purified cell populations comprising PDX1-positive, ventrally-biased, foregut endoderm cells.

Aspects of the present invention also relate to methods or processes for the production of cell cultures of PDX1-negative foregut endoderm cells from definitive endoderm cells. Such processes include reducing or eliminating TGFβ superfamily growth factor signaling in a cell culture or cell population of definitive endoderm cells. In some embodiments, reducing or eliminating TGFβ superfamily growth factor signaling is mediated by diluting or removing an exogenously added TGFβ superfamily growth factor, such as activin A, from the cell culture or cell population of definitive endoderm. In some embodiments, differentiation of definitive endoderm cells to foregut endoderm cells is enhanced by providing the definitive endoderm cell culture or cell population with an FGF-family growth factor and/or a hedgehog pathway inhibitor. In some embodiments, the definitive endoderm cells are derived from stem cells. Preferably, the stem cells are embryonic stem cells. Even more preferably, the stem cells are human embryonic stem cells (hESCs). In some embodiments, the PDX1-negative foregut endoderm cells are differentiated to PDX1-positive endoderm cells (pancreatic endoderm cells) by the addition of a retinoid, such as retinoic acid. Other aspects relate to methods or processes for the production of cell cultures of PDX1-positive, dorsally-biased, foregut endoderm cells. Such processes include providing definitive endoderm cells with retinoic acid. In some embodiments, the definitive endoderm cells are derived from stem cells. Preferably, the stem cells are embryonic stem cells. Even more preferably, the stem cells are human embryonic stem cells (hESCs). Further aspects of the present invention relate to methods or processes for the production of cell cultures of PDX1-positive, ventrally-biased, foregut endoderm cells. Such processes include providing definitive endoderm cells with an FGF-family growth factor. In some embodiments, the definitive endoderm cells are derived from stem cells. Preferably, the stem cells are embryonic stem cells. Even more preferably, the stem cells are hESCs.

Additional embodiments of the present invention relate to methods of enriching, isolating and/or purifying PDX1-negative foregut endoderm cells. In such embodiments, PDX1-negative foregut endoderm cells are separated from other cells in the cell population by using an antibody, ligand or other molecule that binds to a molecule that is expressed on the cell surface of PDX1-negative foregut endoderm cells, such as a cell surface molecule. Other embodiments of the present invention relate to methods of enriching, isolating and/or purifying PDX1-positive, dorsally-biased, foregut endoderm cells. In such embodiments, PDX1-positive, dorsally-biased, foregut endoderm cells are separated from other cells in the cell population by using an antibody, ligand or other molecule that binds to a molecule that is expressed on the cell surface of PDX1-positive, dorsally-biased, foregut endoderm cells, such as a cell surface molecule selected from Table 3 or a cell surface molecule selected from Table 4. Still other embodiments of the present invention relate to methods of enriching, isolating and/or purifying PDX1-positive, ventrally-biased, foregut endoderm cells. In such embodiments, PDX1-positive, ventrally-biased, foregut endoderm cells are separated from other cells in the cell population by using an antibody, ligand or other molecule that binds to a molecule that is expressed on the cell surface of PDX1-positive, ventrally-biased, foregut endoderm cells, such as a cell surface molecule selected from Table 3.

Embodiments of the present invention relate to additional methods of enriching, isolating and/or purifying PDX1-negative foregut endoderm cells. In such embodiments, pluripotent or multipotent cells that are precursors to PDX1-negative foregut endoderm cells are engineered to contain a fluorescent reporter gene under control of a promoter that endogenously controls the expression of a marker gene such as HNF1b or FOXA1. The fluorescently-tagged PDX1-negative foregut endoderm cells are then separated from other cells in the cell population by fluorescence activated cell sorting (FACS). Still other embodiments of the present invention relate to additional methods of enriching, isolating and/or purifying PDX1-positive, dorsally-biased, foregut endoderm cells. In such embodiments, pluripotent or multipotent PDX1-negative cells that are precursors to PDX1-positive cells are engineered to contain a fluorescent reporter gene under control of a promoter that endogenously controls the expression of a marker gene selected from Table 3 or Table 4. The fluorescently-tagged PDX1-positive, dorsally-biased, foregut endoderm cells are then separated from other cells in the cell population by fluorescence activated cell sorting (FACS). Yet other embodiments of the present invention relate to additional methods of enriching, isolating and/or purifying PDX1-positive, ventrally-biased, foregut endoderm cells. In such embodiments, pluripotent or multipotent PDX1-negative cells that are precursors to PDX1-positive cells are engineered to contain a fluorescent reporter gene under control of a promoter that endogenously controls the expression of a marker gene selected from Table 3. The fluorescently-tagged PDX1-positive, ventrally-biased, foregut endoderm cells are then separated from other cells in the cell population by FACS.

Further embodiments of the present invention relate to methods of identifying a differentiation factor capable of promoting the differentiation of human PDX1-negative foregut endoderm cells in a cell population comprising human cells. The method includes the steps of obtaining a cell population comprising human PDX1-negative foregut endoderm cells, providing a candidate differentiation factor to the cell population, determining expression of a marker, such as HNF1b, FOXA1 or PDX1, in the cell population at a first time point and determining expression of the same marker in the cell population at a second time point. In such embodiments, the second time point is subsequent to the first time point and the second time point is subsequent to providing the cell population with the candidate differentiation factor. If expression of the marker in the cell population at the second time point is increased or decreased as compared to the expression of the marker in the cell population at the first time point, then the candidate differentiation factor is capable of promoting the differentiation of the human PDX1-negative foregut endoderm cells. Still further embodiments of the present invention relate to methods of identifying a differentiation factor capable of promoting the differentiation of human PDX1-positive, dorsally-biased, foregut endoderm cells in a cell population comprising human cells. The method includes the steps of obtaining a cell population comprising human PDX1-positive, dorsally-biased, foregut endoderm cells, providing a candidate differentiation factor to the cell population, determining expression of a marker, such as a marker selected from Table 3 or a marker selected from Table 4, in the cell population at a first time point and determining expression of the same marker in the cell population at a second time point. In such embodiments, the second time point is subsequent to the first time point and the second time point is subsequent to providing the cell population with the candidate differentiation factor. If expression of the marker in the cell population at the second time point is increased or decreased as compared to the expression of the marker in the cell population at the first time point, then the candidate differentiation factor is capable of promoting the differentiation of the human PDX1-positive, dorsally-biased, foregut endoderm cells. Yet further embodiments of the present invention relate to methods of identifying a differentiation factor capable of promoting the differentiation of human PDX1-positive, ventrally-biased, foregut endoderm cells in a cell population comprising human cells. The method includes the steps of obtaining a cell population comprising human PDX1-positive, ventrally-biased, foregut endoderm cells, providing a candidate differentiation factor to the cell population, determining expression of a marker, such as a marker selected from Table 3, in the cell population at a first time point and determining expression of the same marker in the cell population at a second time point. In such embodiments, the second time point is subsequent to the first time point and the second time point is subsequent to providing the cell population with the candidate differentiation factor. If expression of the marker in the cell population at the second time point is increased or decreased as compared to the expression of the marker in the cell population at the first time point, then the candidate differentiation factor is capable of promoting the differentiation of the human PDX1-positive, ventrally-biased, foregut endoderm cells.

In certain jurisdictions, there may not be any generally accepted definition of the term “comprising.” As used herein, the term “comprising” is intended to represent “open” language which permits the inclusion of any additional elements. With this in mind, additional embodiments of the present inventions are described with reference to the numbered paragraphs below:

1. A cell culture comprising human cells wherein at least about 26% of said human cells are pancreatic-duodenal homoebox factor-1 (PDX1) positive, dorsally-biased, foregut endoderm cells that express at least one marker selected from Table 3, said PDX1-positive, dorsally-biased, foregut endoderm cells being multipotent cells that can differentiate into cells of the dorsal pancreatic bud.

2. The cell culture of paragraph 1, wherein said marker selected from Table 3 is a marker expressed on the cell surface.

3. The cell culture of paragraph 2, wherein said marker expressed on the cell surface is selected from the group consisting of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2 and SLC27A2.

4. The cell culture of paragraph 1, wherein said PDX1-positive, dorsally-biased, foregut endoderm cells express at least one marker selected from Table 4.

5. The cell culture of paragraph 4, wherein said marker selected from Table 4 is a marker expressed on the cell surface.

6. The cell culture of paragraph 5, wherein said marker expressed on the cell surface is selected from the group consisting of ADORA2A, CD47, EPB41L1, MAG, SFRP5, SLC16A10, SLC16A2, SLC1A3, SLC30A4, SLICK, SLITRK4 and XPR1.

7. The cell culture of paragraph 1, wherein at least about 30% of said human cells are PDX1-positive, dorsally-biased, foregut endoderm cells.

8. The cell culture of paragraph 1, wherein at least about 40% of said human cells are PDX1-positive, dorsally-biased, foregut endoderm cells.

9. The cell culture of paragraph 1, wherein at least about 50% of said human cells are PDX1-positive, dorsally-biased, foregut endoderm cells.

10. The cell culture of paragraph 1, wherein at least about 60% of said human cells are PDX1-positive, dorsally-biased, foregut endoderm cells.

11. The cell culture of paragraph 1, wherein at least about 75% of said human cells are PDX1-positive, dorsally-biased, foregut endoderm cells.

12. The cell culture of paragraph 1, wherein human feeder cells are present in said culture, and wherein at least about 2% of human cells other than said human feeder cells are PDX1-positive, dorsally biased, foregut endoderm cells.

13. The cell culture of paragraph 1, 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, dorsally biased, foregut endoderm cells.

14. The cell culture of paragraph 1, wherein said cell culture is substantially free of cells selected from the group consisting of visceral endodermal cells, parietal endodermal cells and neural cells.

15. The cell culture of paragraph 1 further comprising a retinoid.

16. The cell culture of paragraph 15, wherein said retinoid is retinoic acid (RA).

17. The cell culture of paragraph 16 further comprising B27.

18. A cell culture comprising human cells wherein at least about 2% of said human cells are pancreatic-duodenal homoebox factor-1 (PDX1) positive, ventrally-biased, foregut endoderm cells that express at least one marker selected from Table 3, said PDX1-positive, ventrally-biased, foregut endoderm cells being multipotent cells that can differentiate into cells of the ventral pancreatic bud.

19. The cell culture of paragraph 18, wherein said marker selected from Table 3 is a marker expressed on the cell surface.

20. The cell culture of paragraph 19, wherein said marker expressed on the cell surface is selected from the group consisting of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2 and SLC27A2.

21. The cell culture of paragraph 18, wherein said PDX1-positive, ventrally-biased, foregut endoderm cells do not substantially express one or more markers selected from Table 4.

22. The cell culture of paragraph 18, wherein at least about 5% of said human cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

23. The cell culture of paragraph 18, wherein at least about 10% of said human cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

24. The cell culture of paragraph 18, wherein at least about 25% of said human cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

25. The cell culture of paragraph 18, wherein at least about 50% of said human cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

26. The cell culture of paragraph 18, wherein at least about 75% of said human cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

27. The cell culture of paragraph 18, wherein human feeder cells are present in said culture, and wherein at least about 2% of human cells other than said human feeder cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

28. The cell culture of paragraph 18, 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, ventrally-biased, foregut endoderm cells.

29. The cell culture of paragraph 18, wherein said cell culture is substantially free of cells selected from the group consisting of visceral endodermal cells, parietal endodermal cells and neural cells.

30. The cell culture of paragraph 18 further comprising a retinoid.

31. The cell culture of paragraph 30, wherein said retinoid is retinoic acid (RA).

32. The cell culture of paragraph 31 further comprising B27.

33. A cell population comprising cells wherein at least about 90% of said cells are human PDX1-positive, dorsally-biased, foregut endoderm cells that express at least one marker selected from Table 3, said PDX1-positive, dorsally-biased, foregut endoderm cells being multipotent cells that can differentiate into cells of the dorsal pancreatic bud.

34. The cell population of paragraph 33, wherein said marker selected from Table 3 is a marker expressed on the cell surface.

35. The cell population of paragraph 34, wherein said marker expressed on the cell surface is selected from the group consisting of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2 and SLC27A2.

36. The cell population of paragraph 33, wherein said PDX1-positive, dorsally-biased, foregut endoderm cells express at least one marker selected from Table 4.

37. The cell population of paragraph 36, wherein said marker selected from Table 4 is a marker expressed on the cell surface.

38. The cell population of paragraph 37, wherein said marker expressed on the cell surface is selected from the group consisting of ADORA2A, CD47, EPB41L1, MAG, SFRP5, SLC16A10, SLC16A2, SLC1A3, SLC30A4, SLICK, SLITRK4 and XPR1.

39. The cell population of paragraph 33, wherein at least about 95% of said cells are PDX1-positive, dorsally-biased, foregut endoderm cells.

40. The cell population of paragraph 33, wherein at least about 98% of said cells are PDX1-positive, dorsally-biased, foregut endoderm cells.

41. The cell population of paragraph 33, wherein the expression of PDX1 is greater than the expression of a marker selected from the group consisting of AFP, SOX7, SOX1, ZIC1 and NFM in said PDX1-positive, dorsally-biased, foregut endoderm cells.

42. A cell population comprising cells wherein at least about 90% of said cells are human PDX1-positive, ventrally-biased, foregut endoderm cells that express at least one marker selected from Table 3, said PDX1-positive, ventrally-biased, foregut endoderm cells being multipotent cells that can differentiate into cells of the ventral pancreatic bud.

43. The cell population of paragraph 42, wherein said marker selected from Table 3 is a marker expressed on the cell surface.

44. The cell population of paragraph 43, wherein said marker expressed on the cell surface is selected from the group consisting of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2 and SLC27A2.

45. The cell population of paragraph 42, wherein said PDX1-positive, ventrally-biased, foregut endoderm cells do not substantially express one or more markers selected from Table 4.

46. The cell population of paragraph 42, wherein at least about 95% of said cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

47. The cell population of paragraph 42, wherein at least about 98% of said cells are PDX1-positive, ventrally-biased, foregut endoderm cells.

48. The cell population of paragraph 42, wherein the expression of PDX1 is greater than the expression of a marker selected from the group consisting of AFP, SOX7, SOX1, ZIC1 and NFM in said PDX1-positive, ventrally-biased, foregut endoderm cells.

49. A method of producing PDX1-positive, dorsally-biased, 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 26% of said PDX1-negative definitive endoderm cell population to PDX1-positive, dorsally-biased, foregut endoderm cells that express at least one marker selected from Table 3, wherein said PDX1-positive, dorsally-biased, foregut endoderm cells are multipotent cells that can differentiate into cells of the dorsal pancreatic bud.

50. The method of paragraph 49, wherein said PDX1-positive, dorsally-biased, foregut endoderm cells also express at least one marker selected from Table 4.

51. The method of paragraph 50 further comprising the step of allowing sufficient time for PDX1-positive, dorsally-biased, foregut endoderm cells to form, wherein said sufficient time for PDX1-positive, dorsally-biased, foregut endoderm cells to form has been determined by detecting the presence of a marker from Table 4 in dorsally-biased foregut endoderm cells in said cell population.

52. The method of paragraph 50, wherein the expression of said marker selected from Table 3 or Table 4 is determined by quantitative polymerase chain reaction (Q-PCR).

53. The method of paragraph 50, wherein the expression of said marker selected from Table 3 or Table 4 is determined by immunocytochemistry.

54. The method of paragraph 49, 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, dorsally-biased, foregut endoderm cells.

55. The method of paragraph 49, wherein said retinoid is RA.

56. The method of paragraph 55, wherein RA is provided in a concentration ranging from about 0.5 μM to about 50 μM.

57. The method of paragraph 56, wherein RA is provided in a concentration ranging from about 1 μM to about 20 μM.

58. The method of paragraph 57, wherein RA is provided in a concentration of about 2 μM.

59. The method of paragraph 55, wherein RA is provided when said culture is about 5-days-old.

60. The method of paragraph 49 further comprising providing B27 to said culture.

61. The method of paragraph 60, wherein said B27 is provided in a concentration ranging from about 0.1% to about 20% of the total medium.

62. The method of paragraph 61, wherein B27 is provided in a concentration ranging from about 0.5% to about 2% of the total medium.

63. The method of paragraph 62, wherein B27 is provided in a concentration of about 0.5% of the total medium.

64. The method of paragraph 60, wherein B27 is provided at approximately the same time as said retinoid.

65. The method of paragraph 49 further comprising providing activin A to said culture.

66. The method of paragraph 65, wherein activin A is provided in a concentration ranging from about 10 ng/ml to about 200 ng/ml.

67. The method of paragraph 66, wherein activin A is provided in a concentration ranging from about 20 ng/ml to about 100 ng/ml.

68. The method of paragraph 67, wherein activin A is provided in a concentration of about 25 ng/ml.

69. The method of paragraph 49, wherein said PDX1-positive, dorsally-biased, foregut endoderm cells are grown in CMRL medium.

70. The method of paragraph 69, wherein said CMRL medium comprises RA at about 2 μM, activin A at about 25 ng/ml and B27 at about 0.5% of the total medium.

71. The method of paragraph 49, wherein said step of obtaining a cell population comprising PDX1-negative definitive endoderm cells 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 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.

72. A PDX1-positive, dorsally-biased, foregut endoderm cell produced by the method of paragraph 49.

73. A method of producing PDX1-positive, ventrally-biased, 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 an FGF-family growth factor in an amount sufficient to promote differentiation of at least a portion of said PDX1-negative definitive endoderm cell population to PDX1-positive, ventrally-biased, foregut endoderm cells that express at least one marker selected from Table 3, wherein said PDX1-positive, ventrally-biased, foregut endoderm cells are multipotent cells that can differentiate into cells of the ventral pancreatic bud.

74. The method of paragraph 73, wherein said cell population is differentiated in the absence of RA.

75. The method of paragraph 73, wherein said PDX1-positive, ventrally-biased, foregut endoderm cells do not express one or more markers selected from Table 4.

76. The method of paragraph 75 further comprising the step of allowing sufficient time for PDX1-positive, ventrally-biased, foregut endoderm cells to form, wherein said sufficient time for PDX1-positive, ventrally-biased, foregut endoderm cells to form has been determined by detecting the presence of a marker from Table 3 in ventrally-biased foregut endoderm cells in said cell population.

77. The method of paragraph 76, wherein the expression of said marker selected from Table 3 is determined by quantitative polymerase chain reaction (Q-PCR).

78. The method of paragraph 76, wherein the expression of said marker selected from Table 3 is determined by immunocytochemistry.




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stats Patent Info
Application #
US 20100233755 A1
Publish Date
09/16/2010
Document #
12729084
File Date
03/22/2010
USPTO Class
435 29
Other USPTO Classes
435366, 435347
International Class
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Drawings
64


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