CN101048495A - PDX1 expressing endoderm - Google Patents

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.

Description

Express the entoderm of PDX1

Related application

Present patent application is the 11/021st of submission on December 23rd, 2004, the part continuation application of No. 618 U.S. Patent applications, title is " definitive entoderm (definitive endoderm) ", this application requires to enjoy the right of priority of following three temporary patent applications based on 35U.S.C. § 119 (e): the 60/566th of submission on April 27th, 2004, No. 293 U.S. Provisional Patent Application, title are " entoderm of expressing PDX1 "; The 60/587th, No. 942 U.S. Provisional Patent Application that on July 14th, 2004 submitted to, title is " being used to separate the chemokine cell surface receptor of definitive entoderm "; The 60/586th, No. 566 U.S. Provisional Patent Application that on July 9th, 2004 submitted to, title is " being used to separate the chemokine cell surface receptor of definitive entoderm ".

Technical field

The present invention relates to medical science and cytobiology field, especially comprise the method for composition and generation, separation and this type of cell of use of Mammals PDX1-positive entoderm cell.

Background of invention

People's multipotent stem cells (pluripotent stem cell), as embryonic stem cell (ES) and embryonic genital cell (EG), at first from the culture of no inoblast feeder layer, separated (Bongso et al. in 1994,1994), from the culture that contains the inoblast feeder layer, separated in 1997 (Hogan, 1997).Thomson, Reubinoff and Shamblott have set up and have adopted the mouse feeder layer cultured continuously people ES of mitotic division inactivation and method (Reubinoffet al., 2000 of EG cell subsequently; Shamblott et al., 1998; Thomson et al., 1998).

People ES and EG cell (hESC) provide chance scarcely ever for the human early development of research with for therapeutic intervention some diseases state (as diabetes and Parkinson's disease).For example, in the process that adopts cell therapy treatment diabetes, use can have very much progress than the cell therapy diabetes that are used at present from donor pancreas derived from the β cell of the generation Regular Insulin of hSEC.But also do not know at present how to obtain producing the β cell of Regular Insulin from hSEC.Therefore, being used to cell therapy from the islet cells of donor pancreas treatment diabetes at present is subject to lack and transplants required high quality islet cells.Cell therapy to single type i diabetes people need transplant about 8 * 10 ⁸Islet cells (Shapiro et al., 2000; Shapiro et al., 2001a; Shapiro et al., 2001b).Once successfully transplanting required islet cells needs the organ of two healthy donors to provide at least.And a large amount of high quality noble cells that human embryo stem cell is used for human cell treatment for exploitation provides the source of parent material.

Versatility and can keep the hSEC cell long period and cultivate these two character and make it be particularly suitable for cell therapy.Versatility is meant that hESC can be divided into the derivative of all 3 kinds of main germinal layers (entoderm, mesoderm and ectoderm), and then also forms the ability of all somatocyte types of ripe organ organize (as placenta) and sexual cell except that the embryo outside.Although versatility has given hSEC special purposes, this character brings challenges also for the research and the control of these cells and derivative thereof.Owing in differentiation hSEC substratum, can produce a large amount of cell types, so most of cell type generation efficient is very low.And the generation key of successfully estimating arbitrary designated cell type depends on the mark that definition is suitable.Efficiently, Ding Xiang differentiation is used extremely important to the treatment of hESCs.

Address the above problem utilizing hSEC to produce the cell that is used for cell therapy highly beneficial as parent material.For example, for obtaining the level of the required cell material of islet cell transplantation treatment, advantageously the very early time in differentiation makes hESC be orientated pancreas islet/β clone efficiently.

Except the efficiently and directionally of atomization, in pancreas islet/β clone atomization, separating and identifying the intermediate cell type also advantageously in addition, and utilizing these cells as the suitable precursor clone of further breaking up.

Brief summary of the invention

Embodiment of the present invention relate to composition that comprises the endoderm cell who expresses PDX1 (the PDX1-positive) and the method that produces said composition.Other embodiments relate to cell mass that is rich in the PDX1-positive entoderm cell and the method that produces this cell mass.Other embodiment relates to the method that the expression that strengthens endoderm cell PDX1 and discriminating are used for further breaking up the factor of PDX1 feminine gender and/or PDX1-positive entoderm.In some embodiments of composition that runs through the application's description and method, the PDX1-positive entoderm cell is the positive anterior intestine of PDX1-/midgut endoderm cell.In the certain preferred embodiments of composition that runs through the application's description and method, the PDX1-positive entoderm cell is the positive anterior intestine endoderm cell of PDX1-.In other preferred embodiments, the PDX1-positive entoderm cell is the PDX1-positive entoderm cell of anterior intestine end.

Embodiments more of the present invention relate to the cell culture that comprises the positive anterior intestine endoderm cell of PDX1-, wherein the positive anterior intestine endoderm cell of PDX1-is multipotential cell (multipotentcell), can be divided into derived from intestinal tube forward cell, tissue or organ.In some embodiments, described cell culture comprises people's cell.In this person's cell culture, the positive anterior intestine endoderm cell of PDX1-comprise account for people's cell in the culture at least about 2%, at least about 5%, at least about 10% or at least about 25%.In some embodiments, calculate described at least about 2%, at least about 5%, at least about 10% or at least about the feeder cell of not considering in the described culture in 25% o'clock.In the embodiment of some cell cultures of Miao Shuing, PDX1-is positive, and the anterior intestine endoderm cell can express the mark that is selected from homology frame A13 (HOXA13) gene, homology frame C6 (HOXC6) gene and SOX17 herein.In other embodiments, the cell culture that comprises the positive anterior intestine endoderm cell of PDX1-is substantially free of internal organ endoderm cell, body wall endoderm cell and/or neurocyte.In some embodiments, cell culture also comprises one or more of following material: the retinoid compound, and as vitamin A acid (RA), FGF-10 or B27.

Other embodiments of the present invention relate to the positive anterior intestine endoderm cell group of enrichment, isolating or pure basically PDX1-, and wherein the positive anterior intestine endoderm cell of PDX1-is a multipotential cell, can be divided into derived from intestinal tube forward cell, tissue or organ.In some embodiments, the positive anterior intestine endoderm cell of PDX1-is derived from multipotential cell, as human embryo stem cell.Other embodiments of the present invention relate to the cell mass that comprises cell, be the positive anterior intestine endoderm cell of PDX1-wherein at least about 90% cell, wherein the positive anterior intestine endoderm cell of PDX1-is a multipotential cell, can be divided into derived from intestinal tube forward cell, tissue or organ.In preferred embodiments, the cell at least about 95% is the positive anterior intestine endoderm cell of PDX1-in the cell mass.In the preferred embodiment, the ratio of the positive anterior intestine endoderm cell of PDX1-in cell mass is at least about 98%.

Other embodiment of the present invention relates to the method that produces the positive anterior intestine endoderm cell of PDX1-, and this method is by providing anterior intestine differentiation factor (for example retinoid) to carry out for cell culture or the cell mass that comprises the definitive entoderm cell (the negative definitive entoderm cell of PDX1-) of not expressing PDX1 basically.Retinoid (for example vitamin A acid) can about 0.01 to 50 μ M concentration add.In some embodiments, by provide FGF-10 and/or B27 to increase of the differentiation of the negative definitive entoderm cell of PDX1-for cell culture or cell mass to the PDX1-positive cell.FGF-10 can the extremely concentration interpolation of about 1000ng/ml of about 5ng/ml.In some embodiments, B27 adds in cell culture or the cell mass to about 20% concentration with about 0.1%.FGF-10 and/or B27 can roughly be added in cell culture or the cell mass simultaneously with retinoid, and perhaps every kind of factor also can be added respectively at interval in several hours.In some embodiments, retinoid is added in the negative definitive entoderm cell culture of 4 days PDX1-of about cultivation.In some embodiments, retinoid is added in the negative definitive entoderm cell culture of 5 days PDX1-of about cultivation.

Other embodiment also relates to utilizes the anterior intestine differentiation factor further to promote the method that the positive anterior intestine endoderm cell of PDX1-produces in cell culture or the cell mass, and described cell culture or cell mass contact with retinoid (as vitamin A acid).In these embodiments, for providing activin A and/or activin B, described cell culture or cell mass can promote the negative definitive entoderm of PDX1-to the endoblastic differentiation of the positive anterior intestine of PDX1-.Activin A and/or activin B can 5ng/ml to 1000ng/ml concentration provide.Other embodiments relate to increases the method that the positive anterior intestine endoderm cell of PDX1-produces in cell culture or the cell mass, it carries out keeping or growth conditionsization by some cell type before the wherein said substratum by differentiation PDX1 negative cells in containing the substratum of retinoid.These cell types include but not limited to embryonic stem cell or other multipotential cells, and these cells break up in the substratum that contains serum or somatomedin TGF beta superfamily member (as activin A, activin B, Nodal and/or bone morphogenic protein BMP-2).In some embodiments, conditioned medium adds in cell culture or the cell mass with about 1% to about 100% of whole substratum.TGF beta superfamily somatomedin and/or conditioned medium can roughly add in cell culture or the cell mass simultaneously with retinoid, and perhaps add respectively at the interval that every kind of factor also can several hours.

Embodiment of the present invention relate to the method that produces the cell mass that is rich in the positive anterior intestine endoderm cell of PDX1-.In some embodiments, these methods comprise the step that obtains the multipotential cell group, and wherein at least one cell comprises at least one and copied by nucleic acid of PDX1 promotor control among the multipotential cell group.In some embodiments, described nucleic acid comprises the sequence of encoding green fluorescent protein or its biological active fragment.In other embodiments, the other step of this method comprises makes described multipotential cell differentiation so that produce the positive anterior intestine endoderm cell of PDX1-, and the PDX1-positive cell is separated with the PDX1 negative cells, wherein the positive anterior intestine endoderm cell of this PDX1-is a multipotential cell, can be divided into derived from intestinal tube forward cell, tissue or organ.In some embodiments of method described herein, differentiation step also is included as the somatomedin that described multipotential cell group provides at least a TGF beta superfamily, present in an amount at least sufficient to promote of the differentiation of described multipotential cell to the negative definitive entoderm cell of PDX1-, and, present in an amount at least sufficient to promote the negative definitive entoderm cell of this PDX1-to break up to the PDX1-of anterior intestine positive entoderm cell for the negative definitive entoderm cell of this PDX1-provides the anterior intestine differentiation factor.

Embodiments more of the present invention relate to the method for PDX1 gene product expression in the definitive entoderm cell of promote expressing SOX-17 (the SOX-17 positive), and it contacts with the differentiation factor of the amount that is enough to promote the PDX1 gene product expression by making these cells.In some embodiments, described differentiation factor is selected from RA, FGF-10 and B27.

Other embodiment of the present invention relates to evaluation can promote the method for the negative definitive entoderm cell of PDX1-to the differentiation factor of the positive anterior intestine endoderm cell differentiation of PDX1-.In these methods, the negative definitive entoderm cell of PDX1-is contacted with candidate's differentiation factor, and determine with cell mass before candidate's differentiation factor contact in the expression of PDX1 compare, whether the expression of PDX1 increases in contact back cell mass with candidate's differentiation factor.The increase that PDX1 expresses in the cell mass shows that candidate's differentiation factor can promote the differentiation of the negative definitive entoderm cell of PDX1-to the positive anterior intestine endoderm cell of PDX1-.In some embodiments, the expression of PDX1 is determined by quantitative polyase chain reaction (Q-PCR).Some embodiments of aforesaid method also comprise determines HOXA13 and/or HOXC6 expression of gene in contact the front and back cell mass with candidate's differentiation factor.In some embodiments, candidate's differentiation factor is a small molecules, and retinoid for example is as vitamin A acid.In other embodiment, candidate's differentiation factor is a polypeptide, and somatomedin for example is as FGF-10.

Other embodiment of the present invention relates to the differentiation factor that evaluation can promote the positive anterior intestine endoderm cell differentiation of PDX1-.In these methods, the positive anterior intestine endoderm cell of PDX1-is contacted with candidate's differentiation factor, and determine with cell mass before candidate's differentiation factor contact in the expression of mark compare, the expression of mark is increase or reduction in contact back cell mass with candidate's differentiation factor.The increase of marker expression or reduction show that candidate's differentiation factor can promote the positive anterior intestine endoderm cell's of PDX1-differentiation.In some embodiments, marker expression detects by Q-PCR.In some embodiments, candidate's differentiation factor is a small molecules, and retinoid for example is as vitamin A acid.In other embodiment, candidate's differentiation factor is a polypeptide, and somatomedin for example is as FGF-10.

In some authorities, term " comprises " may not have any definition of being accepted usually." to comprise " be open to the term of Shi Yonging herein, can comprise any other element.Consider this point, other embodiments of the present invention are described with reference to the paragraph of following numbering:

1. the cell culture that comprises people's cell, be pancreas-positive anterior intestine endoderm cell of the duodenum homology frame factor-1 (PDX1) at least about described people's cell of 2% wherein, described PDX1-is positive, and the anterior intestine endoderm cell is the multipotential cell that can be divided into derived from intestinal tube forward cell, tissue or organ.

2. the cell culture of paragraph 1 is the positive anterior intestine endoderm cell of PDX1-at least about described people's cell of 5% wherein.

3. the cell culture of paragraph 1 is the positive anterior intestine endoderm cell of PDX1-at least about described people's cell of 10% wherein.

4. the cell culture of paragraph 1 is the positive anterior intestine endoderm cell of PDX1-at least about described people's cell of 25% wherein.

5. wherein there are people's feeder cell in the cell culture of paragraph 1 in described culture, and wherein except described people's feeder cell, the people's cell at least about 2% is the positive anterior intestine endoderm cell of PDX1-.

6. the cell culture of paragraph 1, wherein said PDX1-is positive, and the anterior intestine endoderm cell expresses homology frame A13 (HOXA13) gene.

7. the cell culture of paragraph 1, wherein said PDX1-is positive, and the anterior intestine endoderm cell expresses homology frame C6 (HOXC6) gene.

8. the cell culture of paragraph 1, wherein said PDX1-is positive, and the anterior intestine endoderm cell expresses SOX17.

9. the cell culture of paragraph 1, wherein the expression of PDX1 is higher than the expression of the mark that is selected from α-fetoprotein (AFP), SOX7, SOX1, ZIC1 and NFM in the positive anterior intestine endoderm cell of described PDX1-.

10. the cell culture of paragraph 1, wherein said cell culture is substantially free of the cell that is selected from internal organ endoderm cell, body wall endoderm cell and neurocyte.

11. the cell culture of paragraph 1, the negative definitive entoderm cell of per approximately 10 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of a PDX1-in the wherein said cell culture.

12. the cell culture of paragraph 1, the negative definitive entoderm cell of per approximately 5 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of a PDX1-in the wherein said cell culture.

13. the cell culture of paragraph 1, the negative definitive entoderm cell of per approximately 4 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of a PDX1-in the wherein said cell culture.

14. the cell culture of paragraph 1 also comprises embryonic stem cell.

15. the cell culture of paragraph 14, wherein said embryonic stem cell is derived from the tissue that is selected from morula, embryo's inner cell mass (ICM) and embryo sexual fold.

16. the cell culture of paragraph 1 also comprises retinoid.

17. the cell culture of paragraph 16, wherein said retinoid are vitamin A acid (RA).

18. the cell culture of paragraph 1 also comprises FGF-10.

19. the cell culture of paragraph 1 also comprises B27.

20. the cell culture of paragraph 1 also comprises RA and FGF-10.

21. the cell culture of paragraph 20 also comprises B27.

22. comprise the cell mass of cell, be the positive anterior intestine endoderm cell of people PDX1-wherein at least about 90% described cell, described PDX1-is positive, and the anterior intestine endoderm cell is the multipotential cell that can be divided into derived from intestinal tube forward cell, tissue or organ.

23. the cell mass of paragraph 22 is the positive anterior intestine endoderm cell of PDX1-at least about 95% described cell wherein.

24. the cell mass of paragraph 22 is the positive anterior intestine endoderm cell of PDX1-at least about 98% described cell wherein.

25. the cell mass of paragraph 22, wherein said PDX1-is positive, and the anterior intestine endoderm cell expresses the HOXA13 gene.

26. the cell mass of paragraph 22, wherein said PDX1-is positive, and the anterior intestine endoderm cell expresses the HOXC6 gene.

27. the cell mass of paragraph 22, wherein said PDX1-is positive, and the anterior intestine endoderm cell expresses SOX17.

28. the cell mass of paragraph 22, wherein the expression of PDX1 is higher than the expression of the mark that is selected from AFP, SOX7, SOX1, ZIC1 and NFM in the positive anterior intestine endoderm cell of described PDX1-.

29. produce the positive anterior intestine endoderm cell's of PDX1-method, described method comprises following steps: the cell mass that obtains to comprise the negative definitive entoderm cell of PDX1-; For providing, described cell mass is enough to promote that wherein said PDX1-is positive, and the anterior intestine endoderm cell is the multipotential cell that can be divided into derived from intestinal tube forward cell, tissue or organ to the retinoid of the negative definitive entoderm cell of the described PDX1-of small part to the amount of the positive anterior intestine endoderm cell differentiation of PDX1-.

30. the method for paragraph 29, also comprise giving the step that the enough time forms for the positive anterior intestine endoderm cell of PDX1-, the enough time that the positive anterior intestine endoderm cell of the wherein said PDX1-of confession forms is determined by the existence that detects the positive anterior intestine endoderm cell of PDX1-in the described cell mass.

31. the method for paragraph 29 is the positive anterior intestine endoderm cell of PDX1-at least about the negative definitive entoderm cytodifferentiation of 2% described PDX1-wherein.

32. the method for paragraph 29 is the positive anterior intestine endoderm cell of PDX1-at least about the negative definitive entoderm cytodifferentiation of 5% described PDX1-wherein.

33. the method for paragraph 29 is the positive anterior intestine endoderm cell of PDX1-at least about the negative definitive entoderm cytodifferentiation of 10% described PDX1-wherein.

34. the method for paragraph 29 is the positive anterior intestine endoderm cell of PDX1-at least about the negative definitive entoderm cytodifferentiation of 25% described PDX1-wherein.

35. the method for paragraph 29, the existence that wherein detects the positive anterior intestine endoderm cell of PDX1-in described cell mass comprises the expression that detects PDX1.

36. the method for paragraph 35, wherein the expression of PDX1 is higher than the expression of the mark that is selected from α-fetoprotein (AFP), SOX7, SOX1, ZIC1 and NFM in the positive anterior intestine endoderm cell of described PDX1-.

37. the method for paragraph 35, the expression of wherein said PDX1 is measured by quantitative polyase chain reaction (Q-PCR).

38. the method for paragraph 35, the expression of wherein said PDX1 is measured by immunocytochemistry.

39. the method for paragraph 29, wherein said retinoid is RA.

40. the method for paragraph 39, wherein RA provides to the concentration range of about 50 μ M with about 0.01 μ M.

41. the method for paragraph 39, wherein RA provides to the concentration range of about 20 μ M with about 0.04 μ M.

42. the method for paragraph 39, wherein RA provides to the concentration range of about 10 μ M with about 0.1 μ M.

43. the method for paragraph 39, wherein RA provides to the concentration range of about 2.5 μ M with about 0.2 μ M.

44. the method for paragraph 39, wherein RA provides to the concentration range of about 1.5 μ M to be about 0.5 μ M.

45. the method for paragraph 39, wherein RA provides with the concentration of about 1 μ M.

46. the method for paragraph 39, wherein RA provides when described cultivation is carried out about 4 days.

47. the method for paragraph 29, also comprise the factor of the amount that the generation that is enough to strengthen the positive anterior intestine endoderm cell of PDX1-is provided in described culture, the described factor is selected from the combination of FGF-10, FGF-4, activin A, activin B, B27, conditioned medium and the described factor.

48. the method for paragraph 47, the wherein said factor are selected from FGF-10, FGF-4, activin A and activin B.

49. the method for paragraph 48, the wherein said factor provides to the concentration range of about 500ng/ml with about 10ng/ml.

50. the method for paragraph 48, the wherein said factor provides to the concentration range of about 200ng/ml with about 20ng/ml.

51. the method for paragraph 48, the wherein said factor provides to the concentration range of about 75ng/ml with about 25ng/ml.

52. the method for paragraph 48, the concentration of the about 50ng/ml of the wherein said factor provides.

53. the method for paragraph 48, the wherein said factor and described retinoid roughly provide simultaneously.

54. the method for paragraph 47, the wherein said factor is B27.

55. the method for paragraph 54, wherein B27 provides with about 0.1% to about 20% concentration range of whole substratum.

56. the method for paragraph 54, wherein B27 provides with about 0.2% to about 5% concentration range of whole substratum.

57. the method for paragraph 54, wherein B27 provides with about 0.5% to about 2% concentration range of whole substratum.

58. the method for paragraph 54, wherein B27 provides with about 1% concentration of whole substratum.

59. the method for paragraph 54, wherein B27 and described retinoid roughly provide simultaneously.

60. the method for paragraph 47, the wherein said factor is a conditioned medium.

61. the method for paragraph 60, wherein conditioned medium provides with about 10% to about 100% concentration range of whole substratum.

62. the method for paragraph 60, wherein conditioned medium provides with about 20% to about 80% concentration range of whole substratum.

63. the method for paragraph 60, wherein conditioned medium provides with about 40% to about 60% concentration range of whole substratum.

64. the method for paragraph 60, wherein conditioned medium provides with about 50% concentration of whole substratum.

65. the method for paragraph 60, wherein conditioned medium and described retinoid roughly provide simultaneously.

66. the method for paragraph 60, wherein conditioned medium contacts about 24 hours by the human embryo stem cell (hESC) that makes differentiation and prepares with cell culture medium.

67. the method for paragraph 66, wherein said hESC is being selected from the RPMI that adds 3% serum, is adding the low serum RPMI of activin A and was adding in the substratum of low serum RPMI of BMP4 differentiation about 5 days.

68. the positive anterior intestine endoderm cell of the PDX1-that the method for paragraph 29 produces.

69. produce the method for the positive anterior intestine endoderm cell's of enrichment PDX1-cell mass, said method comprising the steps of: obtain the multipotential cell group, at least one cell comprises the nucleic acid that is subjected to the control of PDX1 promotor of at least one copy among the wherein said multipotential cell group, and described nucleic acid comprises the sequence of encoding green fluorescent protein (GFP) or its biological active fragment; Break up described multipotential cell so that produce the positive anterior intestine endoderm cell of PDX1-, described PDX1-is positive, and the anterior intestine endoderm cell is the multipotential cell that can be divided into derived from intestinal tube forward cell, tissue or organ; And the positive anterior intestine endoderm cell of described PDX1-is separated with the PDX1-negative cells.

70. the method for paragraph 69, the cell mass of wherein said enrichment comprise the positive anterior intestine endoderm cell of PDX1-at least about 95%.

71. the method for paragraph 69, the cell mass of wherein said enrichment comprise the positive anterior intestine endoderm cell of PDX1-at least about 98%.

72. the method for paragraph 69, wherein said differentiation step also is included as to provide among the described multipotential cell group is enough to promote the somatomedin of described multipotential cell at least a TGF beta superfamily of the amount of the negative definitive entoderm cytodifferentiation of PDX1-, and is enough to promote the retinoid of the negative definitive entoderm cell of described PDX1-to the amount of the positive anterior intestine endoderm cell differentiation of PDX1-for the negative definitive entoderm cell of described PDX1-provides.

73. the method for paragraph 72, wherein said retinoid is RA.

74. the positive anterior intestine endoderm cell's of the PDX1-that the method for paragraph 69 produces enriched populations.

75. increase the method for the expression of PDX1 gene product in expressing the definitive entoderm cell of SOX17, described method comprises makes described definitive entoderm cell contact with the differentiation factor of the amount that is enough to increase described PDX1 gene product expression.

76. the method for paragraph 75, wherein said differentiation factor is a retinoid.

77. the method for paragraph 76, wherein said differentiation factor is RA.

78. the method for paragraph 75, wherein said differentiation factor are selected from the combination of FGF-10, FGF-4, activin A, activin B, B27, conditioned medium and the described factor.

79. identify to promote the method for the negative definitive entoderm cell of PDX1-, said method comprising the steps of: the group who obtains to comprise the negative definitive entoderm cell of PDX1-to the differentiation factor of the positive anterior intestine endoderm cell differentiation of PDX1-; The group of the negative definitive entoderm cell of the described PDX1-of comprising is contacted with candidate's differentiation factor; And with described cell mass before described candidate's differentiation factor contacts in PDX1 expression ratio, determine with described cell mass after described candidate's differentiation factor contacts in the expression of PDX1 whether increase, wherein the increase of expressing at PDX1 described in the described cell mass shows that described candidate's differentiation factor can promote the negative definitive entoderm cell of described PDX1-to the positive anterior intestine endoderm cell differentiation of PDX1-, and described PDX1-is positive, and the anterior intestine endoderm cell is the multipotential cell that can be divided into derived from intestinal tube forward cell, tissue or organ.

80. the method for paragraph 79, wherein said PDX1 expresses and detects by Q-PCR.

81. the method for paragraph 79 also is included in the step that contacts HOXA13 expression of gene in the described cell mass of front and back mensuration with described candidate's differentiation factor.

82. also being included in, the method for paragraph 79 adds the method that HOXC6 expression of gene in the described cell mass is detected in described candidate's differentiation factor front and back.

83. the method for paragraph 79, wherein said candidate's differentiation factor is a small molecules.

84. the method for paragraph 83, wherein said small molecules is a retinoid.

85. the method for paragraph 84, wherein said retinoid is RA.

86. the method for paragraph 79, wherein said candidate's differentiation factor is a polypeptide.

87. the method for paragraph 79, wherein said candidate's differentiation factor is a somatomedin.

88. the method for paragraph 79, wherein said candidate's differentiation factor is FGF-10.

89. identify the method for the differentiation factor that can promote the positive anterior intestine endoderm cell differentiation of PDX1-, said method comprising the steps of: the group who obtains to comprise the positive anterior intestine endoderm cell of PDX1-; The positive anterior intestine endoderm cell's of the described PDX1-of comprising group is contacted with candidate's differentiation factor; And with described cell mass before described candidate's differentiation factor contacts in mark expression ratio, determine to be after described candidate's differentiation factor contact that the expression of identical mark is to increase or reduce in the described cell mass, wherein increase or the reduction in marker expression described in the described cell mass shows that described candidate's differentiation factor can promote the positive anterior intestine endoderm cell's of PDX1-differentiation.

90. the method for paragraph 81, wherein said marker expression is measured by Q-PCR.

91. the method for paragraph 81, wherein said candidate's differentiation factor is a small molecules.

92. the method for paragraph 81, wherein said candidate's differentiation factor is a polypeptide.

93. the method for paragraph 81, wherein said candidate's differentiation factor is a somatomedin.

94. comprise the carrier of the reporter gene that is operably connected to the PDX1 control region.

95. the carrier of paragraph 94, wherein said reporter gene is EGFP.

96. the cell of the carrier of paragraph 94.

97. comprise the cell of the reporter gene that is operably connected to the PDX1 control region.

98. the cell of paragraph 97, the wherein said reporter gene that is operably connected to described PDX1 control region is integrated into karyomit(e).

99. the cell of paragraph 97, wherein said reporter gene is EGFP.

100. the cell of paragraph 97, wherein said cell is polyenergic.

101. the cell of paragraph 100, wherein said cell is hESC.

102. the cell of paragraph 97, wherein said cell are the definitive entoderm cells.

103. the cell of paragraph 97, wherein said cell are PDX1-positive anterior intestine endoderm cells.

104. the conditioned medium by following steps preparations: make fresh cell culture medium contact about 24 hours with the hESC group of differentiation, wherein said hESC is being selected from the RPMI that adds 3% serum, is adding the low serum RPMI of activin A and add in the cell culture medium of low serum RPMI of BMP4 and broke up about 5 days; And the hESC group who from described substratum, removes described differentiation.

105. the conditioned medium of paragraph 104, wherein said fresh cell culture medium is RPMI.

106. the conditioned medium of paragraph 105, wherein said RPMI are low serum RPMI.

107. the method for conditioning substratum, said method comprising the steps of: make fresh cell culture medium contact about 24 hours with the hESC group of differentiation, wherein said hESC is being selected from the RPMI that adds 3% serum, is adding the low serum RPMI of activin A and was adding in the cell culture medium of low serum RPMI of BMP4 differentiation about 5 days; And the hESC group who from described substratum, removes described differentiation.

108. the method for paragraph 107, wherein said fresh cell culture medium is RPMI.

109. the conditioned medium of paragraph 108, wherein said RPMI are low serum RPMI.

Should be appreciated that above-mentioned method and composition is relevant with the cell of vitro culture.But the cell composition of above-mentioned vitro differentiation can be used for purposes in the body.

Other embodiments of the present invention can be referring to following patent application: the 60/532nd, No. 004 U.S. Provisional Patent Application that on December 23rd, 2003 submitted to, and title is " definitive entoderm "; The 60/566th, No. 293 U.S. Provisional Patent Application that on April 27th, 2004 submitted to, title are " entoderm of expressing PDX1 "; The 60/586th, No. 566 U.S. Provisional Patent Application that on July 9th, 2004 submitted to, title is " being used to separate the chemokine cell surface receptor of definitive entoderm "; The 11/021st, No. 618 U.S. Patent application that on December 23rd, 2004 submitted to, title is " definitive entoderm ".

Description of drawings

Fig. 1 is the imaginary differentiation pathway synoptic diagram that produces beta cell from hESC.The first step of approach is responsible for ES cell to definitive entoderm, also represents the first step before the further differentiation incident of pancreas entoderm, internal secretion entoderm or pancreas islet/beta cell.Second step of approach shows that the definitive entoderm of the SOX17-positive/PDX1-feminine gender is to the endoblastic conversion of the positive anterior intestine of PDX1-.These factors that work in transforming are represented with italics in mediation.The correlating markings thing of definition target cell is represented with underscore.

Fig. 2 is the synoptic diagram of people SOX17 cDNA, has shown the position of conserved regions and has highlighted the zone that is used for by the immune step of GENOVAC.

Fig. 3 concerns dendrogram, shows that SOX17 and SOX7 relation are nearest, and is far away slightly with the SOX18 relation.SOX17 protein is more much better than than other member's of the SOX group F subtribe in the same species dependency in allied species

Fig. 4 is a Western trace of making probe with the anti-SOX17 antibody of rabbit, this trace proves that this antibody has specificity to crossing the SOX17 that expresses in the inoblast (1 road), and the immunoreactivity of shortage and EGFP (2 road) or immediate SOX family member SOX7 (3 road).

Fig. 5 A-B shows a large amount of AFP ⁺Be total to the SOX17 of labeled cell ⁺The Photomicrograph of cell cluster.This and other SOX17 ⁺Bunch (B) forms significantly contrast, because at other SOX17 ⁺Can only observe seldom in bunch or just do not have AFP at all ⁺Cell.

Fig. 6 A-C is the Photomicrograph that shows body wall entoderm and SOX17.Figure A shows that the immunocytochemistry of the human thrombomodulin adjusting albumen (TM) on body wall endoderm cell surface in the hES cell culture that breaks up at random detects.Figure B shows the identical zone with figure A, to TM and SOX17 double-tagging.Figure C is the figure that differs with the same area of DAPI mark nuclear.Attention: the complete correspondence of the nuclear of DAPI mark and SOX17 mark.

Fig. 7 A-B shows the SOX17 genetic expression of quantitative PCR (Q-PCR) detection and the bar graph of the anti-SOX17 positive cell that the SOX17-specific antibody detects.Figure A shows with respect to undifferentiated control medium (SR20) activin A increases SOX17 genetic expression, and vitamin A acid (RA) strongly inhibited SOX17 expresses.Figure B is presented at SOX17 ⁺The similar multiple that has reflected similar pattern and these variations on the cell quantity shows that Q-PCR detects SOX17 genetic expression and can reflect variation in the individual cells level.

Fig. 8 A is a bar graph, shows that activin A exists the hESC culture that is breaking up down to keep low-level AFP genetic expression, and the strong rise of the cell of differentiation demonstration AFP at random in 10% calf serum (FBS).The difference of expression level approximately is 7 times.

Fig. 8 B-C is two displaing micro pictures, shows that activin A is also very remarkable to the individual cells level that is suppressed at that AFP expresses, because with respect to single 10%FBS (top) that uses, can only observe considerably less and little AFP down in the condition (bottom) that activin A handles ⁺Cell cluster.

Fig. 9 A-B shows to use the quantitative AFP of flow cytometer ⁺The contrast picture of cell count.This figure proof exists at activin A under the condition of (left figure) or disappearance (right figure), and the change multiple of AFP genetic expression (Fig. 8 A) is fully corresponding to AFP ⁺The quantity of cell has further been supported to use Q-PCR to analyze and has been indicated the variation that takes place on the individual cells level.

Figure 10 A-F shows to make hESC be exposed to nodal, activin A and activin B (NAA) SOX17 after 5 days ⁺Cell number significantly increases the displaing micro picture of (A-C).By comparing each region S OX17 ⁺Cell accounts for the relative abundance of whole cell numbers (shown in the painted nuclear of DAPI (D-F)), and the cell of about 30-50% shows immunoreactivity to SOX17 after handling 5 days with NAA as can be seen.

Figure 11 is the bar graph of SOX17 genetic expression among the hESC that breaking up of the dose-dependent increase of proof activin A (0,10,30 or 100ng/ml).It is very high to handle the expression that increases after 3 days when adhere to cultivating, and lasts till 1,3 and 5 day of suspension culture subsequently always.

Figure 12 A-C is the bar graph of proof activin A to the influence of the expression of MIXL1 (figure A), GATA4 (figure B) and HNF3b (figure C).Also observe the dose-dependent increase definitive entoderm of activin A 3 kinds of other marks: MIXL1, GATA4 and HNF3b.The multiple that the expression corresponding with activin dosage increases shows all 4 kinds of gene (SOX17 of specialization of activin A coexpression with viewed closely similar to SOX17 ⁺, MIXL1 ⁺, GATA4 ⁺And HNF3b ⁺) cell mass.

Figure 13 A-C is the bar graph of proof activin A to the influence of the expression of AFP (figure A), SOX7 (figure B) and SPARC (figure C).The expression of internal organ entoderm mark AFP is to the dependent reduction of activin A show dose.Primitive endoderm mark (SOX7) and body wall entoderm mark (SPARC) remain unchanged or show inhibition at some time points, show that activin A does not play these extraembryonic endoderm cell types of specialization.This has further supported the increase of the increase of the following fact: SOX17, MIXL1, GATA4 and HNF3b expression owing to the definitive entoderm cell number of corresponding activin A.

Figure 14 A-B shows the bar graph of activin A to the influence of the expression of ZIC1 (figure A) and Brachyury (figure B).The expression proof activin A that neural mark ZIC1 is stable does not have dose-dependent effect to Neural Differentiation.The expression of Brachyury reduces demonstration 100ng/ml activin A processing and has significantly suppressed the mesoderm differentiation.This may be from the result of mesendoderm precursor to the increase of specialization of definitive entoderm.With respect to untreated control cultures, low-level activin A (10 and 30ng/ml) handles the expression that keeps brachyury than the later stage in differentiation.

Figure 15 A-B shows that the body wall entoderm that the response activin is handled breaks up the displaing micro picture that reduces.Having only differentiation phase under the condition of serum, body wall entoderm Tm ^HiThe zone is present in (A) in all cultures, and when comprising activin A, to TM ⁺The differentiation of cell is considerably less, and the immunoreactive total intensity of TM is lower.

Figure 16 A-D is the displaing micro picture that shows the marker expression of response activin A and activin B processing.HESC handled 4 days continuously with activin A and activin B, with SOX17, AFP and TM antibody three heavy labels.Figure A-SOX17; Figure B-AFP; Figure C-TM; Figure DPhase/DAPI.Notice that many SOX17 positive cells are accompanied by AFP (B) and the immunoreactive disappearance fully of TM (C).

Figure 17 is presented at externally definitive entoderm and the endoblastic displaing micro picture of internal organ to occur from hESC.Pass through AFP ^Hi/ SOX17 ^Lo/-Identify internal organ entoderm zone, and definitive entoderm shows antipodal Mode S OX17 ^Hi/ AFP ^Lo/-Because these two zones are adjacent to each other, so selected this zone.But, from AFP ^HiObserve SOX17 through regular meeting in the complete isolate of the arbitrary region of cell ^Hi/ AFP ^Lo/-The zone shows the independent origin from the endoblastic definitive entoderm of internal organ.

Figure 18 describes the part of TGF 'beta ' family and the chart of acceptor.The factor that activates AR Smads and BR Smads can the process that produces definitive entoderm from human embryo stem cell, work (referring to: J Cell Physiol.187:265-76).

Figure 19 shows as what the SOX17 through the TGF β factor result of single or combination expressed to induce time dependent bar graph.

Figure 20 is the SOX17 that shows as the TGF β factor result through making up ⁺The time dependent bar graph of cell number.

Figure 21 shows as what the SOX17 through the TGF β factor result of combination expressed to induce time dependent bar graph.

Figure 22 induces SOX17 with showing activin A dose-dependently ⁺The bar graph that cell number increases.

Figure 23 is a bar graph, shows that adding Wnt3a in the culture of activin A and activin B processing impels the expression level of SOX17 to be higher than only with activin A and activin B inductive expression level.

Figure 24 A-C shows that low FBS condition strengthens the bar graph to the definitive entoderm differentiation.In containing the substratum of 2%FBS, handle (2AA) hESC with activin A and activin B, the expression level that can cause SOX17 than in containing the substratum of 10%FBS through the expression level high 2 to 3 times (figure A) of the SOX17 of same treatment (10AA).Inducing of definitive entoderm mark MIXL1 (figure B) is also influenced in the same manner, the rejection ratio to AFP (internal organ entoderm) in 2%FBS stronger under the 10%FBS condition (figure C).

Figure 25 A-D shows SOX17 in the culture ⁺Fissional displaing micro picture.The SOX17 immunoreactive cell is present in hESC clone's dividing edge (C, D), by proliferating cell nuclear antigen (PCNA) mark (figure B), but is not total to mark (figure C) by OCT4.In addition, at SOX17 ⁺Cell (arrow) and OCT4 ⁺All can see mitotic division figure (D) clearly with DAPI mark nuclear in the cell, undifferentiated hESCs (arrow head).

Figure 26 is the bar graph that is presented at the relative expression's level of CXCR4 among the hESC that is breaking up under the different culture medium condition.

Figure 27 A-D shows that how one group of definitive entoderm mark share the bar graph of closely similar expression pattern with CXCR4 under the identical differentiation that shows as Figure 26 is handled.

Figure 28 A-E is a bar graph, show the mesoderm mark (BRACHYURY, MOX1), the ectoderm mark (SOX1, ZIC1) and internal organ entoderm mark (SOX7) under the same treatment that shows as Figure 26, how to show with CXCR4 and express opposite relation.

Figure 29 A-F is the displaing micro picture that is presented at the relative different of SOX17 immunoreactive cell under three kinds of culture medium condition of showing among Figure 26-28.

Figure 30 A-C is the fluidic cell point diagram, proves CXCR4 ⁺Cell number increases with the increase of the concentration of the activin A that is added into division culture medium.

Figure 31 A-D is a bar graph, shows from high dosage activin A to handle (A100-CX+) isolating CXCR4 ⁺Cell is than the further enrichment definitive entoderm mark of parental generation group (A100).

Figure 32 is a bar graph, shows to use the isolating CXCR4 of fluorescence activated cell sorting (FACS) ⁺And CXCR4 ^-Genetic expression among gene expression of cells and the parental generation group.This proves CXCR4 ⁺Cell has comprised all CXCR4 genetic expression among each parental cell group, CXCR4 substantially ^-Cell mass comprises seldom or does not have CXCR4 genetic expression.

Figure 33 A-D is a bar graph, proves from high dosage activin A to handle isolating CXCR4 ⁺Mesoderm in the cell (BRACHYURY, MOX1), (SOX1, ZIC1) and the disappearance of internal organ entoderm (SOX7) genetic expression, the expression of these non-definitive entoderm marks is suppressed ectoderm.

Figure 34 A-M is the bar graph that shows the expression pattern of the marker gene can be used for identifying the definitive entoderm cell.Figure G-L has shown the expression analysis of definitive entoderm mark FGF 17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 respectively.Figure A-F shown previously described clone marker gene SOX17, SOX7, SOX17/SOX7, TM, ZIC1 and MOX1 respectively) expression analysis.Figure M has shown the expression analysis of CXCR4.For each figure of figure A-M, the genetic expression from the human embryo stem cell of purifying is shown in the tabulation of mark hESC; 2NF represents that cell through the 2%FBS processing, does not add activin; 0.1A100 the expression cell is handled through 0.1%FBS and 100ng/ml activin A; 1A100 represents that cell is through 1%FBS and 100ng/ml activin A processing; 2A100 represents that cell is through 2%FBS and 100ng/ml activin A processing.

Figure 35 is the chart that is presented under the condition that contains or do not contain activin PDX1 gene relative expression in the hESC culture of cultivating 4 days and 6 days, wherein at the 4th day interpolation vitamin A acid (RA) and fibroblast growth factor (FGF-10).

Figure 36 A-F is the chart that is presented under the condition that contains or do not contain activin marker gene relative expression in the hESCs culture of cultivating 4 days and 6 days, wherein at the 4th day interpolation vitamin A acid (RA) and fibroblast growth factor (FGF-10).Each figure has shown the level relatively that following marker gene is expressed: (A) SOX17; (B) SOX7; (C) AFP; (D) SOX1; (E) ZIC1; (F) NFM.

Figure 37 A-C is the chart that is presented under the condition that contains or do not contain activin marker gene relative expression in the hESC culture of cultivating 4 days and 8 days, wherein in the combination of the 4th day interpolation vitamin A acid (RA), fibroblast growth factor (FGF-10) and fibroblast growth factor (FGF-4).Each figure has shown the level relatively that following marker gene is expressed: (A) PDX1; (B) SOX7 and (C) NFM.

Figure 38 A-G is the chart that is presented at the relative expression of marker gene in the definitive entoderm cell culture that contacts with 50ng/ml FGF-10, wherein adds 1 μ M, 0.2 μ M or 0.04 μ M vitamin A acid (RA) and 50ng/ml FGF-10 at the 4th day and unites use.Each figure has shown the level relatively that following marker gene is expressed: (A) PDX1; (B) HOXA3; (C) HOXC6; (D) HOXA13; (E) CDX1; (F) SOX1 and (G) NFM.

Figure 39 A-E is presented at and contains or do not contain activin, exist vitamin A acid (RA), fibroblast growth factor (FGF-10) and following among a kind of: serum substitute (SR), cultivate the relative expression of marker gene in the hESC culture of 4 days and 8 days under the condition of the combination that calf serum (FBS) or B27 constitute.Each hurdle has shown the level relatively that following marker gene is expressed: (A) PDX1; (B) SOX7; (C) AFP; (D) ZIC1 and (E) NFM.

The relative expression's of the marker gene of pancreas (PDX1, HNF6) and liver (HNF6) chart in hESC culture when Figure 40 A-B is (just before the adding RA) back that shows 6 days and 9 days (being exposed to RA after 3 days).Comprise the comparison of different conditions, under the condition of 25ng/ml (A25) or 50ng/ml (A50), added the activin B of 10ng/ml (a10), 25ng/ml (a25) or 50ng/ml (a50).The condition that does not contain any activin A and activin B (NF) is as the negative control that produces definitive entoderm and PDX1-positive entoderm.Each figure has shown the level relatively that following marker gene is expressed: (A) PDX1 and (B) HNF6.

Figure 41 A-C be presented under the condition that contains 100ng/ml (A100), 50ng/ml (A50) or do not contain (NF) activin A when cultivating 5 days (just before adding vitamin A acid) and add RA after the relative expression of marker gene in the hESCs culture of 2,4 and 6 days (being respectively the 7th, 9 and 11 day).The per-cent of direct mark shows the FBS dosage of differentiation during 3 to 5 days under each post figure.Since the 7th day, handle the cell of (R) with RA and in comprising the RPMI substratum of 0.5%FBS, grow.The concentration of RA was 2 μ M at the 7th day, and the 9th day is 1 μ M, and the 11st day is 0.2 μ M.Each figure has shown the level relatively that following marker gene is expressed: (A) PDX1; (B) ZIC1 and (C) SOX7.

Figure 42 A-B demonstration is at first induced definitive entoderm (the 5th day) with activin A processing in low FBS, induce PDX1-to express entoderm with fresh (A25R) substratum that comprises 25ng/ml activin A and RA or various conditioned medium (MEFCM, CM#2, CM#3 and CM#4) and RA then.Detected marker expression at the 5th, 6,7,8 and 9 day.Each figure has shown the level relatively that following marker gene is expressed: (A) PDX1 and (B) CDX1.

Figure 43 has shown at first in low FBS to handle with activin A and has induced definitive entoderm, handles with fresh culture then, and this fresh culture comprises the RA of activin A and vitamin A acid (A25R) or different amounts in the conditioned medium that dilutes with fresh culture.The cumulative volume of substratum all is 5ml.

Figure 44 is the Western trace, shows PDX1 immunoprecipitation of the definitive entoderm cell of RA-processing when 3 days (d8) and 4 days (d9) after adding RA and 50ng/ml activin A.

Figure 45 is a summary chart, the result of the positive anterior intestine endoderm cell's fluorescence activated cell sorting of PDX1-(FACS) of the EGFP reporter gene under the control of PDX1 promotor that shown genetic marker.

Figure 46 is the chart of the relative PDX1 expression level of group after proofreading and correct with respect to house-keeping gene of viable cell (Live), EGFP-negative cells (Neg) and the EGFP-positive cell (GFP+) of demonstration sorting.

Figure 47 is the viable cell (Live) that shows sorting, EGFP-negative cells (Neg), faciation that half has the EGFP-positive cell group of minimum EGFP strength of signal (Lo) and an EGFP-positive cell group that half has the highest EGFP strength of signal (Hi) the relative PDX1 expression level after for the house-keeping gene correction.

Figure 48 A-E shows back 5 kinds of pancreas entoderm marks of the relative expression's level faciation of viable cell (Live), EGFP-negative cells (Neg) and the EGFP-positive cell (GFP+) of sorting is proofreaied and correct to(for) house-keeping gene.Each figure: A-NKX2.2; B-GLUT2; C-HNF3 β; D-KRT19 and E-HNF4 α.

Figure 49 is relative expression's level that the group of viable cell (Live), EGFP-negative cells (Neg) and the EGFP-positive cell (GFP+) of demonstration sorting proofreaies and correct back two kinds of non-pancreas entoderm marks at relative house-keeping gene.Each figure: A-ZIC1 and B-GFAP.

Describe in detail

The developmental important stage of early stage people that is called primitive gut formation betides 2～3 weeks of after fertilization. Primitive gut forms extremely important, because in this stage three kinds of main germinal layers specialization first time and systematism (Lu et al., 2001; Schoenwolf and Smith, 2000). Ectoderm is responsible for health appearance and whole neural final formation, and mesoderm derives the heart, blood, bone, skeletal muscle and other connective tissues. The entoderm of determining is defined as being responsible for germinal layer (Grapin-Botton and Melton, 2000 that organ (such as lung, liver, thymus gland, parathyroid gland, thyroid gland, gall-bladder and pancreas) that whole intestinal tube (comprising esophagus, stomach, small intestine and large intestine) and intestinal tube derive forms; Kimelman and Griffin, 2000; Tremblay et al., 2000; Wells and Melton, 1999; Wells and Melton, 2000)). Definitive entoderm is that important difference is arranged with the complete isolated cell that is called primitive endoderm. Primitive endoderm mainly is responsible for the formation of embryo outside organization, mainly is the chamber wall of placenta yolk bag and the extracellular matrix material of internal organ entoderm part and Reichert ' s film.

Form the phase at primitive gut, the definitive entoderm forming process originates in cell migration, and wherein mesendoderm cell (cell that forms mesoderm or entoderm ability is arranged) migration is passed and is called former structure. Definitive entoderm passes the cell of former front portion and joint (the specialization structure in former forefront zone) derived from some migrations. When migration took place, definitive entoderm at first concentrated on the forefront intestinal tube, stops with the formation of intestinal tube rear end subsequently.

PDX1 gene expression in the growth course

PDX1 (being also referred to as STF-1, IDX-1 and IPF-1) is that pancreas and beak duodenum (rostral duodenum) are grown essential transcription factor. PDX1 at first expresses at the pancreas entoderm, the pancreas entoderm from the rear portion before gut entoderm, produce exocrine and endocrine cell, in mouse, start from E8.5. Thereafter, PDX1 is defined in beta cell and some δ-cells. This expression pattern also keeps after growing up. Grow the also duodenum entoderm expression of the pancreas in contiguous formation of early stage PDX1, in duodenal enterocyte and enteroendocrine cell, stomach hole and choledochus, gall-bladder and bile duct, express then. Express when limited at pancreas, this is expressed zone and mainly is limited to the beak duodenum.

PDX1 positive cell and relative step

Embodiment of the present invention relate to the new definite bright method that produces the PDX1-positive entoderm cell, wherein the PDX1-positive entoderm cell is multipotential cell, can be divided into cell, tissue or the organ of anterior intestine/midgut zone (the positive anterior intestine of PDX1-/middle gut entoderm) derived from intestinal tube. " pluripotency " used herein or " multipotential cell " relate to such cell type, and it can produce other cell types of limited quantity. " anterior intestine/midgut " used herein duodenum 12 pipe front portion and middle part cell comprise the cell of anterior intestine/midgut connecting portion.

Some preferred embodiments of the present invention relate to the method that produces the positive anterior intestine endoderm cell of PDX1-. In some embodiments, these PDX1 positive entoderm cells are multipotential cells, can be divided into cell, tissue or organ derived from intestinal tube front portion (gut entoderm before the PDX1-positive).

Other preferred embodiments relate to the method for the PDX1-positive entoderm cell that produces the intestinal tube rear portion. In some embodiments, these PDX1 positive entoderm cells are multipotential cells, can be divided into cell, tissue or organ derived from the rear portion in the anterior intestine zone of intestinal tube.

The positive anterior intestine endoderm cell of PDX1-, for example those can be used for producing the fully β cell of the generation insulin of differentiation according to the cell that method described herein produces. In embodiments more of the present invention, basically do not express definitive entoderm cell (the negative definitive entoderm cell of PDX1-of PDX1 by differentiation; Also specify the shape entoderm herein) the positive anterior intestine endoderm cell of generation PDX1-. Can use method described herein or other any known method differentiation multipotential cells (such as embryonic stem cell) to prepare the negative definitive entoderm cell of PDX1-. The title of submitting on December 23rd, 2004 is that the 11/021st, No. 618 U.S. Patent application of " definitive entoderm " described a kind of method that produces the negative definitive entoderm of PDX1-from the multipotential cell convenience and high-efficiency.

The method that produces the positive anterior intestine endoderm cell of PDX1-provides the foundation for efficiently producing pancreatic tissue (such as acinar cells, vessel cell and islet cells) from multipotential cell. In some preferred embodiment, the negative definitive entoderm cell of the positive anterior intestine endoderm cell of people PDX1-derived from human PDX1-, and these cells are derived from hESC. The positive anterior intestine endoderm cell of these people PDX1-is used to produce the beta cell of functional generation insulin then. Be the insulin β cell of the generation that obtains effective dose, wish that each differentiation step has differentiated efficient before being divided into pancreas islet/beta cell terminal point. Because the negative definitive entoderm cell of PDX1-especially wishes to have differentiated efficient in this step to an early stage step (as shown in Figure 1) of differentiation representative generation functional islets/beta cell of the positive anterior intestine endoderm cell of PDX1-.

Considering needs the negative definitive entoderm cell of PDX1-to effective differentiation of the positive anterior intestine endoderm cell of PDX1-, aspects more of the present invention relate to in-vitro method to be learned, and it can cause about 2%～25% the negative definitive entoderm cell of PDX1-to the positive anterior intestine endoderm cell conversion of PDX1-. Typically, the method comprises with what determine and uses culture medium and growth factor condition with of short duration specific mode. Separate and/or the positive anterior intestine endoderm cell of purifying PDX1-by using in reagent other cells from cell mass with the positive anterior intestine endoderm cell of PDX1-specific bond, further the positive anterior intestine endoderm cell of enrichment PDX1-in cell mass. Perhaps, the positive anterior intestine endoderm cell of PDX1-can use the reporter gene mark such as green fluorescent protein (GFP), expresses so that can detect PDX1. This fluorescently-labeled cell can be used fluorescence activated cell sorting (FACS) purifying. The more deep aspect of the present invention relates to cell culture and comprises the cell mass of the positive anterior intestine endoderm cell's of PDX1-enrichment, and identify gut entoderm before the PDX1-positive and before the PDX1-positive the useful factor the gut entoderm differentiation.

Be to measure the quantity of the positive anterior intestine endoderm cell of PDX1-in cell culture or the cell mass, need a kind of in culture or cell mass the method from this cell type of other cell differentiation. Correspondingly, certain embodiments of the present invention relate to cell sign thing and detection and determine the method for these marker expression, and the existence of these marks, disappearance and/or relative expression's level are the positive anterior intestine endoderm cell's of PDX1-indications. " expression " used herein refers to level or the quantity of generation and material or the material generation of material or material. Therefore, the expression that detects the special sign thing refers to measure the relative or absolute magnitude of expressing mark, or only detects existence or the disappearance of mark. " mark " used herein refers to any molecule of can be observed or detecting. For example, mark is including, but not limited to polypeptide product, non-genomic product polypeptide, glycoprotein, carbohydrate, glycolipid, fat, lipoprotein or the little molecule (such as the molecule of molecular weight less than 10,000amu) of, nucleotides (such as the transcription product of specific gene), gene.

In embodiments more of the present invention, measure existence, disappearance and/or the expression of mark with quantitative PCR (Q-PCR). For example, some genetic marker, the amount of the transcription product that produces such as PDX1, SOX17, SOX7, SOX1, ZIC1, NFM, α-fetoprotein (AFP), homology frame A13 (HOXA13), homology frame C6 (HOXC6) and/or other marks described herein is determined with Q-PCR. In other embodiments, detect the albumen that said gene is expressed with ImmunohistochemistryMethods Methods. In other embodiments, use amount and the relative scale of Q-PCR and these marks of two kinds of method Identification and detections of SABC.

Use differentiation described herein and detection method just may in cell culture or cell mass, identify the positive anterior intestine endoderm cell of PDX1-and the ratio of determining these cells. For example, in some embodiments of the present invention, the positive anterior intestine endoderm cell of the PDX1-of generation or cell mass express the level of PDX1 gene than PDX1-negative cells or cell mass height at least about 2 orders of magnitude. In other embodiments, the positive anterior intestine endoderm cell of the PDX1-of generation and cell mass are expressed the level of PDX1 gene than PDX1-negative cells or high 2 the above orders of magnitude of cell mass. In other embodiments, the positive anterior intestine endoderm cell of the PDX1-of generation or cell mass are expressed the level of one or more marks that are selected from PDX1, SOX17, HOXA13 or HOXC6 than the negative definitive entoderm cell of PDX1-high about 2 or 2 above orders of magnitude.

Composition described herein and method have several useful characteristics. For example, the method that comprises cell culture and the cell mass of PDX1-positive entoderm and produce this cell culture and cell mass is conducive to simulate the commitment that the people grows. In addition, composition described herein and method also can be used for the therapeutic intervention of morbid state (such as diabetes). For example, because gut entoderm is as the source of limited quantity tissue before the PDX1-positive, it can be used to the exploitation of pure tissue or cell type.

Produce the negative definitive entoderm (definitive entoderm) of PDX1-from multipotential cell

Produce cell culture and/or the cell mass that comprises the positive anterior intestine endoderm cell of PDX1-from multipotential cell, at first will produce the negative definitive entoderm (being also referred to as " definitive entoderm ") of PDX1-. The differentiation multipotential cell comprises the cell mass of the cell culture of definitive entoderm and enrichment with generation method has simply been described below, detailed step sees that the title of submitting on December 23rd, 2004 is the 11/021st, No. 618 U.S. Patent application of " definitive entoderm ". In certain methods, be stem cell as the multipotential cell of parent material. In some method, the definitive entoderm cell culture originates from embryonic stem cell with the cell mass that comprises the enrichment of definitive entoderm cell. " embryo's " used herein refers to organic a series of stage of development, starts from single embryonated egg, ends at multi-cellular structure, and this multi-cellular structure no longer comprises multipotency or totipotent cell except the gamogenesis extracellular of growing. Except the embryo from Gamete Fusion, term " embryo's " also refers to the embryo from SCNT. The preferred method that obtains the definitive entoderm cell utilizes human embryo stem cell to produce definitive entoderm as parent material. This multipotential cell can be the cell that is derived from mulberry body, embryo's inner cell mass or embryo sexual fold. Can use method well known in the art in culture medium, to keep the substantially undifferentiated multipotency state of human embryo stem cell. Such as the 5th, 453,357,5,670,372,5,690,926,5,843,780,6,200,806 and 6,251, in No. 671 United States Patent (USP)s these methods have been described.

In the certain methods that produces the definitive entoderm cell, hESC maintains on the feeder layer. In the method, can use any energy so that hESC maintains the feeder layer of multipotency state. A kind of feeder layer of cultivation human embryo stem cell commonly used is the l cell layer. Recently, HF's feeder layer has been used for cultivating hESC (referring to No. 2002/0072117 U.S. Patent application). What produce definitive entoderm just can be kept multipotency hESC by the choosing method permission without feeder layer. In No. 2003/0175956 U.S. Patent application, the method for keeping multipotency hESC under the no feeder layer condition has been described.

Human embryo stem cell used herein can be kept in the culture medium that contains or do not contain serum. Keep in the step at some embryonic stem cells, use serum substitute. In other steps, use for example free serum culture technology of No. 2003/0190748 U.S. Patent application description.

Stem cell is gone down to posterity by routine in culture medium and keeps the multipotency state, until need to be divided into definitive entoderm. In the certain methods, the promotion by in stem cell culture, adding effective dose to the growth factor of the TGF beta superfamily of definitive entoderm differentiation to obtain the differentiation to definitive entoderm. The growth factor that produces the useful TGF beta superfamily of definitive entoderm is selected from Nodal/ activin (activin) or BMP subgroup. In some preferred differentiation methods, growth factor is selected from Nodal, activin A, activin B and BMP4. In addition, growth factor Wnt3a and other Wnt family members are conducive to the generation of definitive entoderm cell. In some differentiation step, can use the combination of any above-mentioned growth factor.

For the certain methods of multipotential stem cell to the definitive entoderm Cell Differentiation, add above-mentioned growth factor and make that the concentration of growth factor is enough to impel at least a portion stem cell to the definitive entoderm Cell Differentiation in the culture medium in the cell. In the certain methods, the concentration of above-mentioned growth factor is at least about 5ng/ml, at least about 10ng/ml, at least about 25ng/ml, at least about 50ng/ml, at least about 75ng/ml, at least about 100ng/ml, at least about 200ng/ml, at least about 300ng/ml, at least about 400ng/ml, at least about 500ng/ml, at least about 1000 ng/ml, at least about 2000ng/ml, at least about 3000ng/ml, at least about 4000ng/ml, at least about 5000ng/ml or more than about 5000ng/ml in the cell culture medium.

In some method of definitive entoderm Cell Differentiation, add earlier above-mentioned growth factor at multipotential stem cell, and then from cell culture, remove. For example, growth factor can added about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days or afterwards removal in about 10 days. In a preferred method, growth factor is being added afterwards removal in about 4 days.

The culture of definitive entoderm can be grown in the culture medium of the serum that contains minimizing or serum-free. Under some condition of culture, the serum-concentration scope is from about 0.05%v/v to about 20%v/v. For example, in some differentiation step, serum-concentration can be lower than about 0.05% (v/v) in the culture medium, be lower than about 0.1% (v/v), be lower than about 0.2% (v/v), be lower than about 0.3% (v/v), be lower than about 0.4% (v/v), be lower than about 0.5% (v/v), be lower than about 0.6% (v/v), be lower than about 0.7% (v/v), be lower than about 0.8% (v/v), be lower than about 0.9% (v/v), be lower than about 1% (v/v), be lower than about 2% (v/v), be lower than about 3% (v/v), be lower than about 4% (v/v), be lower than about 5% (v/v), be lower than about 6% (v/v), be lower than about 7% (v/v), be lower than about 8% (v/v), be lower than about 9% (v/v), be lower than about 10% (v/v), be lower than about 15% (v/v) or be lower than about 20% (v/v). In the certain methods, definitive entoderm is grown under serum-free or the condition at serum substitute. In the additive method, the definitive entoderm cell is grown containing under the condition of B27. In the method, the concentration range of B27 additive is from about 0.1%v/v to about 20%v/v.

The monitoring multipotential cell is to the differentiation of the negative definitive entoderm of PDX1-(definitive entoderm)

Can monitor the hESC culture to the differentiation process of definitive entoderm by the expression of measuring the definitive entoderm specific markers. In certain methods, by the existence that detects mark or the expression that lacks definite certain mark. Perhaps, the expression of some mark is determined by the level of measuring the mark of cell in cell culture or the cell mass. In the method, the detection of marker expression can be qualitatively, also can be quantitative. Use quantitative PCR (Q-PCR) is that quantitative a kind of method is carried out in the expression of the mark of marker gene generation. The method of carrying out Q-PCR is being known in the art. Also available additive method well known in the art carries out quantitatively the expression of marker gene. For example, use for the special antibody of interested marker gene product, detect the expression of marker gene product. In some method, determine the expression of definitive entoderm specific markers gene and lack hECS and the remarkable expression of other cell type specific markers genes.

Further describe such as following examples, the reliable mark of definitive entoderm is the SOX17 gene. Therefore, with the definitive entoderm cellular expression SOX17 marker gene of method generation described herein, thereby produce the SOX17 gene outcome. Other mark of definitive entoderm is MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1. Because the level of definitive entoderm cellular expression SOX17 marker gene is higher than the level of expressing the SOX7 marker gene, and this is original and the endoblastic feature of internal organ (seeing Table 1), so in some steps, the expression of SOX17 and SOX7 is all monitored. In other steps, the special SOX17 of hESC and the expression of OCT4 marker gene have been monitored. In addition, because the expression of SOX17 marker gene is than the expression height of AFP, SPARC or thrombomodulin (TM) marker gene, so the expression of these genes is also monitored in the definitive entoderm cell.

Another mark of definitive entoderm is the CXCR4 gene. CXCR4 gene code cell surface chemokine receptors, the part of this acceptor is chemoattractant SDF-1. The Main Function that contains the cell of CXCR4 acceptor in adult is considered to hematopoietic cell to migration, lymphocyte transportation and various B cell and the huge differentiation [Kim that bites blood cell line of marrow, C. and Broxmeyer, H.J.Leukocyte Biol.65,6-15 (1999)]. The CXCR4 acceptor also enters the co-receptor [Feng, Y., et al.Science, 272,872-877 (1996)] of T cell as HIV-1. At [McGrath, K.E.et al.Dev.Biology 213,442-456 (1999)] carry out a series of widely research in, described the early development of mouse and adult period chemokine receptors CXCR4 and its unique part SDF-1[Kim, C. and Broxmeyer, H., J.Leukocyte Biol.65,6-15 (1999)] expression. The interaction of CXCR4/SDF1 is very obvious in growth, because confirm, destroys arbitrary gene [Nagasawa et al.Nature, 382,635-638 (1996) among CXCR4 and the SDF1 in transgenic mice; Ma, Q., et al Immunity, 10,463-471 (1999)], all can cause mouse in late embryo stage death. The people such as McGrath utilize the method proof of RNase protection and in situ hybridization combination to make the abundantest chemokine receptors mRNA that detects at early stage (E7.5) CXCR4 of Gastrulation embryo. In Gastrulation embryonic period, embryonic phase, the CXCR4/SDF-1 signal may mainly participate in inducing the migration of former germinal layer cell, and definitive entoderm at this moment, mesoderm and extraembryonic mesoderm are expressed. In the E7.2-7.8 mice embryonic, CXCR4 and α-fetoprotein are mutual exclusions, show the disappearance [McGrath, K.E.et al.Dev.Biology 213,442-456 (1999)] of expressing in the internal organ entoderm.

Because by the definitive entoderm cellular expression CXCR4 marker gene of differentiation multipotential cell generation, so can pass through to monitor the expression of CXCR4 to follow the trail of the generation of definitive entoderm cell. In addition, the definitive entoderm cell that produces with method described herein is also expressed other marks of definitive entoderm, and these marks include but not limited to SOX17, MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1. Because expressing the level of CXCR4 marker gene, definitive entoderm is higher than the level of expressing the SOX7 gene, so can monitor the expression of CXCR4 and two kinds of genes of SOX7. In the additive method, the expression of CXCR4 marker gene and OCT4 marker gene is all monitored. In addition, because the level of definitive entoderm cellular expression CXCR4 marker gene is higher than the level of expressing AFP, SPARC or thrombomodulin (TM) marker gene, also can monitor the expression of these genes.

The expression that should be appreciated that CXCR4 among the endoderm cell does not hinder the expression of SOX17. The definitive entoderm cell that produces with method described herein is great expression SOX17 and CXCR4, but not great expression AFP, TM, SPARC or PDX1.

The enrichment of definitive entoderm, separation and/or purifying

Definitive entoderm cell with any said method produces can carry out enrichment, separation and/or purifying to the affinity tag of these cell-specifics by utilizing. Example to the affinity tag of definitive entoderm cell-specific is antibody, part or other binding reagents special to marker molecules, polypeptide for example, wherein said marker molecules is present on the definitive entoderm cell surface, and basically is not present on other cell types that can find in the cell culture that produces with method described herein. In the certain methods, use the antibody of being combined with CXCR4 as affinity tag, with enrichment, isolated or purified definitive entoderm cell. In additive method, use chemotactic factor (CF) SDF-1 or based on other molecules of SDF-1 as affinity tag. These molecules include but not limited to SDF-1 fragment, SDF-1 fusions or SDF-1 analogies.

Dispersal risk and the method for utilizing antibody to carry out cell separation are being known in the art, and these methods can be used for antibody described herein and definitive entoderm cell. In a method, in connection with the antibody coupling of CXCR4 to magnetic bead, then with cell culture in the definitive entoderm Cell binding, wherein this cell culture processes to reduce the adhesion of iuntercellular and substrate through enzyme. Subsequently but cell/antibody/magnetic bead mixture is placed moving field, for separating of the definitive entoderm cell of being combined with magnetic bead and unconjugated cell. When definitive entoderm cell in the culture and other cells have carried out after the physical separation, antibody is in conjunction with destroyed, and cell is planted again in suitable tissue culture medium (TCM).

Also available additive method obtains definitive entoderm cell culture enrichment, that separate or purifying or group. For example, in some embodiments, CXCR4 antibody and the cell culture that contains definitive entoderm are hatched, wherein this culture is through processing to reduce the adhesion of iuntercellular and substrate. Washing, centrifugal and re-suspended cell. The cell suspension is hatched with two anti-(antibody of the FITC coupling that for example can be combined with primary antibodie). The washing, centrifugal and in buffer solution re-suspended cell. Use then fluorescence activated cell sorting (FACS) that the cell suspension is analyzed and sorting. Be collected in the CXCR4-positive cell after the CXCR4-negative cells separates, thereby separated this cell type. If need, can use another affine method or increase the sorting number of times cellular component that separates is further purified, utilization be the identical or different mark that is specific to the definitive entoderm cell.

In additive method, utilize part or other molecule enrichments be combined with CXCR4, separate and/or purifying definitive entoderm cell. In the certain methods, this molecule is SDF-1 or its fragment, fusions or analogies.

In the method for optimizing, after the induced dry-cell culture breaks up to definitive entoderm system, from other non-definitive entoderm cell enrichments, separation and/or purifying definitive entoderm cell. Should be appreciated that above-mentioned enrichment, separation and purification step can be used for any stage of this culture differentiation.

Except the step of just having described, can also separate the definitive entoderm cell with other cell separation technologies. In addition, the method enrichment or the separation definitive entoderm cell that go down to posterity and cultivate by under the growth conditions that promotes the survival of definitive entoderm cell selective or selective amplification, carrying out series.

Use method described herein, definitive entoderm cell mass enrichment, that separate and/or purifying and/or tissue can produce from multipotential cell culture or cell mass external, have for example carried out stem cell culture or the group of at least part of differentiation. In certain methods, cell breaks up at random. But in a preferred method, the main directed differentiation of cell is definitive entoderm. Some preferred enrichments, separation and/or purification process relate to external from human embryo stem cell generation definitive entoderm. Use method described herein, the definitive entoderm content in cell mass or the cell culture is compared with untreated cell mass or cell culture, at least enrichment about 2 to about 1000 times.

The composition that comprises the negative definitive entoderm of PDX1-(definitive entoderm)

The cell composition that said method produces comprises the cell culture that comprises definitive entoderm and the cell mass of enrichment definitive entoderm. For example, can produce the cell culture that comprises the definitive entoderm cell, wherein the cell at least about 50～80% is the definitive entoderm cell in the culture. Because the efficient of differentiation method can (these parameters include but not limited to by changing some parameter, the control of time of Growth of Cells condition, growth factor concentration and incubation step) adjust, differentiation method described herein can cause about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or surpass about 95% multipotential cell to the conversion of definitive entoderm. In adopting the method for separating the definitive entoderm cell, for example, by using the affinity reagent in conjunction with the CXCR4 acceptor, can obtain basically pure definitive entoderm cell mass.

Produce gut entoderm before the PDX1-positive from the negative definitive entoderm of PDX1-

PDX1-positive anterior intestine endoderm cell culture and the group who comprises the positive anterior intestine endoderm cell of PDX1-described herein produced by the negative definitive entoderm of PDX1-, and wherein the negative definitive entoderm of this PDX1-is produced by multipotential cell as mentioned above. A preferred method utilizes human embryo stem cell as parent material. In one embodiment, hESC at first is converted into the negative definitive entoderm cell of PDX1-, is converted into then the positive anterior intestine endoderm cell of PDX1-. But should be appreciated that the parent material that produces for the positive anterior intestine endoderm cell of PDX1-is not limited to the definitive entoderm cell that produces with the multipotential cell differentiation method. And the negative definitive entoderm cell of any PDX1-all can be used for method described herein, and is irrelevant with their source.

In some embodiments of the present invention, the cell culture or the cell mass that comprise the negative definitive entoderm cell of PDX1-can be used for further breaking up to the cell culture that comprises PDX1-positive anterior intestine endoderm cell and/or the cell mass of enrichment. For example, can use cell culture or the cell mass of the definitive entoderm cell that comprises people PDX1-feminine gender, the SOX17-positive. In some embodiments, cell culture or cell mass may comprise the differentiation factor that keeps from front differentiation step (namely breaking up the step that multipotential cell is the definitive entoderm cell), such as activin, nodal and/or BMP. In other embodiments, before adding the factor that is used for PDX1-feminine gender, the positive anterior intestine endoderm cell differentiation to PDX1-of the positive definitive entoderm cell of SOX17-, from cell culture or cell mass, remove the factor in the differentiation step of front. In other embodiments, use the cell mass of enrichment PDX1-feminine gender, the positive definitive entoderm cell of SOX17-as the source that produces the positive anterior intestine endoderm cell of PDX1-.

Promote cell to the differentiation factor (anterior intestine differentiation factor) of the positive anterior intestine endoderm cell differentiation of PDX1-by adding in the cell culture that comprises PDX1-feminine gender, the positive definitive entoderm cell of SOX17-, the negative definitive entoderm cell of the PDX1-in the culture medium can be divided into the PDX1-positive entoderm cell. In some embodiments of the present invention, the anterior intestine differentiation factor is retinoids, for example retinoic acid (RA). In some embodiments, retinoids and fibroblast growth factor (for example FGF-4 and FGF-10) coupling. In other embodiments, retinoids and growth factor TGF beta superfamily member and/or conditioned medium coupling.

" conditioned medium " refers to minimal medium the culture medium that changes take place relatively. For example, the conditioning of culture medium may cause from the initial level of culture medium and add or removal molecule, for example nutrients and/or growth factor. In some embodiments, in culture medium, grow or keep the certain hour section by the cell that makes certain type under certain condition and make culture medium condition. For example, make culture medium condition by making hESC in the culture medium of uniform temperature, definite composition, increase, break up or keep the regular hour. It will be recognized by one of ordinary skill in the art that numerous combinations of cell, type of culture medium, duration and environmental condition can be used for producing the conditioned medium near infinite arrangement. In embodiments more of the present invention, by comprise about 1% to the culture medium of about 20% serum-concentration growth or the multipotential cell of keeping differentiation come conditioned culture media. In other embodiments, the multipotential cell by making differentiation comprise about 1ng/ml to the culture medium of about 1000ng/ml activin A growth or keep conditioned culture media. In other embodiments, the multipotential cell by making differentiation is grown to the culture medium of about 1000ng/ml BMP4 or is kept conditioned culture media comprising about 1ng/ml. In preferred embodiments, grow in the culture medium (such as RPMI) that comprises about 25ng/ml activin A and about 2 μ M RA by the hESC that makes differentiation or keep the preparation condition culture medium.

In embodiments more of the present invention, the cell that is used for conditioned culture media (these culture mediums are used for strengthening the negative definitive entoderm of PDX1-to the endoblastic differentiation of the positive anterior intestine of PDX1-) be contain about 0% to the culture medium (scheme such as RPMI) of about 20% serum and/or one or more TGF beta superfamily growth/differentiation factors from the cell of multipotential cell (such as hESC) differentiation above 5 days. Add the concentration range of differentiation factor (for example activin A and BMP4) from about 1ng/ml to about 1000ng/ml. In certain embodiments of the present invention, the cell that is used for conditioned culture media breaks up above 5 days from hESC at low serum RPMI. According to some embodiments, low serum RPMI refers to contain the culture medium of low serum, and wherein serum-concentration increases in the certain hour section gradually. For example, in one embodiment, low serum RPMI comprises the hyclone (FBS) of concentration about 0.2%, the about 0.5%FBS of Growth of Cells second day, the 3rd to 5 day about 2%FBS of Growth of Cells at the Growth of Cells first day. In another embodiment, it is about 0% that low serum RPMI first day comprises serum-concentration, about 0.2%, the 3～6 day about 2% of second day. In certain preferred aspects, add one or more differentiation factors, scheme such as activin A and BMP4 among the low serum RPMI. Except the cell for the preparation of conditioned culture media, low serum RPMI also can be used as the negative definitive entoderm cell of PDX1-to the culture medium of the positive anterior intestine endoderm cell differentiation of PDX1-.

Those of ordinary skill in the art should be appreciated that for the preparation of the culture medium of conditioned medium RPMI just, and prerequisite is that this culture medium does not disturb the positive anterior intestine endoderm cell's of PDX1-growth or keeps. Should also be clear that the cell for conditioned culture media can be different type. Come in the embodiment of conditioned culture media at the cell with fresh differentiation, this cell can break up in being different from the culture medium of RPMI, and prerequisite is that this culture medium can not suppress the growth of this cell or keeps. In addition, the technical staff should be appreciated that conditioning duration or for the preparation of duration of the cell of conditioning be not must be respectively 24 hours or 5 days because other time span also is enough to the effect that obtains to report herein.

Usually, retinoids and fibroblast growth factor (member of growth factor TGF beta superfamily), conditioned medium or the arbitrarily combination coupling of these anterior intestine differentiation factors can cause the negative definitive entoderm of the PDX1-stronger than alone retinoids to the endoblastic differentiation of the positive anterior intestine of PDX1-. In preferred embodiments, add RA and FGF-10 in the negative definitive entoderm cell culture of PDX1-. In another preferred embodiment, the negative definitive entoderm cell of PDX1-breaks up in the culture medium that contains conditioned medium, activin A, activin B and RA.

With regard to some embodiments of differentiation method described herein, above-mentioned anterior intestine differentiation factor is added in the cell, make the concentration of anterior intestine differentiation factor in cell culture or the cell mass be enough to promote the negative definitive entoderm cell culture of at least part of PDX1-or cell mass to the positive anterior intestine endoderm cell differentiation of PDX1-. When relating to cell culture and/or cell mass, term " part " expression cell culture or cell mass be non-vanishing quantity arbitrarily, and scope is from individual cells to whole cell culture or cell mass.

In embodiments more of the present invention, adding retinoids in the cell of culture makes its concentration at least about 0.01 μ M, at least about 0.02 μ M, at least about 0.04 μ M, at least about 0.08 μ M, at least about 0.1 μ M, at least about 0.2 μ M, at least about 0.3 μ M, at least about 0.4 μ M, at least about 0.5 μ M, at least about 0.6 μ M, at least about 0.7 μ M, at least about 0.8 μ M, at least about 0.9 μ M, at least about 1 μ M, at least about 1.1 μ M, at least about 1.2 μ M, at least about 1.3 μ M, at least about 1.4 μ M, at least about 1.5 μ M,, at least about 1.6 μ M, at least about 1.7 μ M, at least about 1.8 μ M, at least about 1.9 μ M, at least about 2 μ M, at least about 2.1 μ M, at least about 2.2 μ M, at least about 2.3 μ M, at least about 2.4 μ M, at least about 2.5 μ M, at least about 2.6 μ M, at least about 2.7 μ M, at least about 2.8 μ M, at least about 2.9 μ M, at least about 3 μ M, at least about 3.5 μ M, at least about 4 μ M, at least about 4.5 μ M, at least about 5 μ M, at least about 10 μ M, at least about 20 μ M, at least about 30 μ M, at least about 40 μ M or at least about 50 μ M. " retinoids (retinoid) " used herein refers to any derivative of retinol, retinene or retinoic acid and these compounds. In preferred embodiments, retinoids is retinoic acid.

In other embodiments of the present invention, there is the differentiation factor of one or more fibroblast growth families in the cell culture. For example, in some embodiments, in the cell culture concentration of FGF-4 at least about 10ng/ml, at least about 25ng/ml, at least about 50ng/ml, at least about 75ng/ml, at least about 100ng/ml, at least about 200ng/ml, at least about 300 ng/ml, at least about 400ng/ml, at least about 500ng/ml or at least about 1000ng/ml. In the further embodiment of the present invention, the concentration of FGF-10 is at least about 10 ng/ml, at least about 25ng/ml, at least about 50ng/ml, at least about 75ng/ml, at least about 100ng/ml, at least about 200ng/ml, at least about 300ng/ml, at least about 400ng/ml, at least about 500ng/ml or at least about 1000ng/ml in the cell culture. In some embodiments, selecting one from FGF-4 and FGF-10 adds in the cell culture with RA. In the preferred embodiment, RA concentration is 1 μ M in the cell culture, and FGF-10 concentration is 50ng/ml.

In some embodiments of the present invention, in cell culture, there be growth factor and/or the conditioned medium of TGF beta superfamily. These differentiation factors can with RA and/or anterior intestine differentiation factor (including but not limited to FGF-4 and FGF-10) coupling in other. For example, in some embodiments, can have activin A and/or activin B in the cell culture, its concentration is at least about 5 ng/ml, at least about 10ng/ml, at least about 25ng/ml, at least about 50ng/ml, at least about 75ng/ml, at least about 100ng/ml, at least about 200ng/ml, at least about 300ng/ml, at least about 400ng/ml, at least about 500ng/ml or at least about 1000ng/ml. In another embodiment of the present invention, existence condition culture medium in the cell culture, its concentration be total culture medium at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100%. In some embodiments, be added with activin A, activin B and conditioned medium and RA in the cell culture. In preferred embodiments, the negative definitive entoderm Cell Differentiation of PDX1-is the positive anterior intestine endoderm cell of PDX1-in the culture that comprises 1 μ M RA, about 25ng/ml activin A and low serum RPMI culture medium (about 24 hours of the hESC conditioning that this culture medium has been broken up, wherein the hESC of differentiation broke up about 5 days) in comprising the low serum RPMI of 100 ng/ml activin As. In another preferred embodiment, also there are activin B and/or FGF-10 in the culture, its concentration is respectively 25 ng/ml and 50ng/ml.

In certain embodiments of the present invention, above-mentioned anterior intestine differentiation factor is removed from cell culture after interpolation. For example, the anterior intestine differentiation factor can be removed in about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days or about 10 days after interpolation.

PDX1-is positive, and the anterior intestine endoderm cell can grow in containing the culture medium that reduces serum-concentration. The serum-concentration scope can be from about 0.05% (v/v) to about 20% (v/v). In some embodiments, the positive anterior intestine endoderm cell of PDX1-is grown under the condition that contains serum substitute. For example, in certain embodiments, the serum-concentration of culture medium can be lower than about 0.05% (v/v), be lower than about 0.1% (v/v), be lower than about 0.2% (v/v), be lower than about 0.3% (v/v), be lower than about 0.4% (v/v), be lower than about 0.5% (v/v), be lower than about 0.6% (v/v), be lower than about 0.7% (v/v), be lower than about 0.8% (v/v), be lower than about 0.9% (v/v), be lower than about 1% (v/v), be lower than about 2% (v/v), be lower than about 3% (v/v), be lower than about 4% (v/v), be lower than about 5% (v/v), be lower than about 6% (v/v), be lower than about 7% (v/v), be lower than about 8% (v/v), be lower than about 9% (v/v), be lower than about 10% (v/v), be lower than about 15% (v/v) or be lower than about 20% (v/v). In some embodiments, the positive anterior intestine endoderm cell of PDX1-can grow under the condition of serum-free. In other embodiments, the positive anterior intestine endoderm cell of PDX1-grows containing under the condition of serum substitute.

In other embodiments, the positive anterior intestine endoderm cell of PDX1-grows in the presence of B27. In these embodiments, B27 can add to the culture medium from about 0.1% (v/v) to the concentration range of about 20% (v/v) or greater than the concentration of 20% (v/v). In certain embodiments, about 0.1% (v/v) of the concentration of B27 in the culture medium, about 0.2% (v/v), about 0.3% (v/v), about 0.4% (v/v), about 0.5% (v/v), about 0.6% (v/v), about 0.7% (v/v), about 0.8% (v/v), about 0.9% (v/v), about 1% (v/v), about 2% (v/v), about 3% (v/v), about 4% (v/v), about 5% (v/v), about 6% (v/v), about 7% (v/v), about 8% (v/v), about 9% (v/v), about 10% (v/v), about 15% (v/v) or about 20% (v/v). Perhaps, the concentration of the B27 additive of interpolation can be calculated according to the multiple of commercial B27 storage liquid concentration. For example, Invitrogen (Carlsbad, CA) provide 50 * B27 storage liquid. Add this storage liquid of capacity in the growth medium of enough volumes and can produce the culture medium that contains aequum B27. For example, add 10ml 50 * B27 storage liquid in the 90ml growth medium and will obtain having added the growth medium of 5 * B27. In the culture medium concentration of B27 additive can be about 0.1 *, about 0.2 *, about 0.3 *, about 0.4 *, about 0.5 *, about 0.6 *, about 0.7 *, about 0.8 *, about 0.9 *, about 1 *, about 1.1 *, about 1.2 *, about 1.3 *, about 1.4 *, about 1.5 *, about 1.6 *, about 1.7 *, about 1.8 *, about 1.9 *, about 2 *, about 2.5 *, about 3 *, about 3.5 *, about 4 *, about 4.5 *, about 5 *, about 6 *, about 7 *, about 8 *, about 9 *, about 10 *, about 11 *, about 12 *, about 13 *, about 14 *, about 15 *, about 16 *, about 17 *, about 18 *, about 19 *, about 20 * and be higher than about 20 *.

The negative definitive entoderm of monitoring PDX1-is to the endoblastic differentiation of the positive anterior intestine of PDX1-

As the situation of multipotential cell to the definitive entoderm Cell Differentiation, the positive definitive entoderm of PDX1-feminine gender, SOX17-process of gut entoderm differentiation before the PDX1-positive can be monitored by the expression of measuring the distinctive mark of these cell types. This monitoring is so that can determine under different condition (for example one or more differentiation factor concentration and environmental conditions) to be enough to produce the time of gut entoderm needs before the aequum PDX1-positive. In the preferred embodiment, deciding by the expression that detects PDX1 is enough to produce the required time of gut entoderm before the aequum PDX1-positive. In embodiments more of the present invention, the expression that the existence by detecting mark or disappearance are measured some mark. Perhaps, the level that exists by mark in the cell of measuring cell culture or cell mass is measured the expression of some mark. In these embodiments, the measurement of marker expression can be qualitative or quantitative. As mentioned above, the preferred method that quantitatively detects the marker expression that is produced by marker gene is to use Q-PCR. In specific embodiments, Q-PCR is used to the disappearance expressed by the expression of the distinctive marker gene of gut entoderm before the quantitative detection PDX1-positive and the distinctive marker gene of other cell types, monitors PDX1-feminine gender, the positive definitive entoderm culture of SOX17-to the process of the positive anterior intestine endoderm cell differentiation of PDX1-. Also can quantitatively detect marker gene with other known methods of this area expresses. For example, can utilize the expression that the special antibody of interested marker gene product is detected the marker gene product. In some embodiments of the present invention, measured the disappearance of the remarkable expression of the expression of the distinctive marker gene of gut entoderm before the PDX1-positive and the negative definitive entoderm of PDX-1, hESC and other cell types distinctive marker genes.

Such as further describing of following embodiment, PDX1 is the marker gene relevant with gut entoderm before the PDX1-positive. Therefore, measured in some embodiments of the present invention the expression of PDX1. In other embodiments, also measured the expression of other marks that gut entoderm is expressed before the PDX1-positive, these marks include but not limited to SOX17, HOXA13 and/or HOXC6. Because some other cell type (being internal organ entoderm and some neuroderm) are also expressed PDX1, some embodiments of the present invention relate to the disappearance of the proof gene expression relevant with internal organ entoderm and/or neuroderm or basically lack. For example, in some embodiments, the expression of the mark (including but not limited to SOX7, AFP, SOX1, ZIC1 and/or NFM) of expressing at internal organ entoderm and/or neuroderm is determined.

In some embodiments, the positive anterior intestine endoderm cell of the PDX1-culture that produces with method described herein is substantially free of the cell of expressing SOX7, AFP, SOX1, ZIC1 or NFM marker gene. In certain embodiments, the positive anterior intestine endoderm cell of the PDX1-culture that produces with step described herein is substantially free of internal organ entoderm, body wall entoderm and/or nerve cell.

The endoblastic enrichment of the positive anterior intestine of PDX1-separates and/or purifying

According to other aspects of the invention, the positive anterior intestine endoderm cell of PDX1-can be by enrichment, separation and/or purifying. In embodiments more of the present invention, the positive anterior intestine endoderm cell's of enrichment PDX1-cell mass produces by separate this cell from cell culture.

In embodiments more of the present invention, PDX1-is positive, and the anterior intestine endoderm cell uses fluorescence labeling, uses then fluorescence activated cell sorting (FACS) to separate with unlabeled cells. In this class embodiment, the nucleotides mark PDX1-positive cell that can express the fluorescent marker gene with nucleotides or another coding of encoding green fluorescent protein (GFP). For example, in some embodiments, the coding GFP of at least one copy or the nucleotides of its biological active fragment are imported into the downstream of multipotential cell (preferably human embryo stem cell) PDX1 promoter, so that the expression of GFP gene outcome or its biological active fragment is subjected to the control of PDX1 promoter. In some embodiments, the be encoded nucleotides of GFP or its biological active fragment of the whole coding region of nucleotides of coding PDX1 is replaced. In other embodiments, the nucleotides of coding GFP or its biological active fragment carries out (in frame) fusion in the frame with at least a portion of the nucleotides of coding PDX1, thereby produces fusion. In this class embodiment, fusion keeps the fluorescence ability similar to GFP.

As previously mentioned, fluorescently-labeled cell (for example above-mentioned multipotential cell) is divided into definitive entoderm, is gut entoderm before the PDX1-positive then. The anterior intestine endoderm cell expresses the fluorescent marker gene because PDX1-is positive, and the PDX1-negative cells is not expressed, and these two kinds of cell types can be separated. In some embodiments, the cell suspension that comprises the mixture of fluorescently-labeled PDX1-positive cell and unlabelled PDX1-negative cells with the FACS sorting. The PDX1 positive cell is collected after the PDX1-negative cells separates, thereby separates this cell type. If need, can use the identical or different mark increase sorting number of times special to gut entoderm before the PDX1-positive, to be further purified the cell composition of separation.

Except the step of just having described, the positive anterior intestine endoderm cell of the PDX1-also other technologies of available cell separation separates. In addition, the positive anterior intestine endoderm cell of PDX1-also can use and carry out method enrichment or the separation definitive entoderm cell that series goes down to posterity and cultivates under the growth conditions of the selective survival of the positive anterior intestine endoderm cell of the above-mentioned PDX1-of promotion or selective amplification.

Should be appreciated that above-mentioned enrichment, separation and purification step can be used for any differential period of this class culture.

Use method described herein, the positive anterior intestine endoderm cell's of PDX1-enrichment, that separate and/or purifying group and/or tissue can obtain from the positive definitive entoderm cell culture of PDX1-feminine gender, SOX17-or the cell mass that proceeds to the small part differentiation external. In some embodiments, cell breaks up at random. But in preferred embodiments, the main directed differentiation of cell is the positive anterior intestine endoderm cell of PDX1-. Some preferred enrichments, separation and/or purification process relate to external from the positive anterior intestine endoderm cell of human embryo stem cell generation PDX1-.

Use method described herein, compare with untreated cell mass or cell culture, the positive anterior intestine endoderm cell of the PDX1-in cell mass or cell culture content can be enriched to less about 2 to about 1000 times. In some embodiments, compare with untreated cell mass or cell culture, PDX1-is positive, and the anterior intestine endoderm cell can be enriched to less about 5 to about 500 times. In other embodiments, compare with untreated cell mass or cell culture, PDX1-is positive, and the anterior intestine endoderm cell can be enriched to less about 10 to about 200 times. In other embodiments, compare with untreated cell mass or cell culture, PDX1-is positive, and the anterior intestine endoderm cell can be enriched to less about 20 to about 100 times. In other embodiments, compare with untreated cell mass or cell culture, PDX1-is positive, and the anterior intestine endoderm cell can be enriched to less about 40 to about 80 times. In some embodiment, compare with untreated cell mass or cell culture, PDX1-is positive, and the anterior intestine endoderm cell can be enriched to less about 2 to about 20 times.

Comprise the endoblastic composition of the positive anterior intestine of PDX1-

Embodiments more of the present invention relate to the cell composition that comprises the positive anterior intestine endoderm cell of PDX1-, for example cell culture or cell mass, wherein the positive anterior intestine endoderm cell of PDX1-is multipotential cell (gut entoderm before the PDX1-positive), can be divided into cell, tissue or organ derived from the intestinal tube front portion. According to some embodiment, gut entoderm is mammalian cell before the PDX1-positive, and in preferred embodiments, the definitive entoderm cell is people's cell.

Other embodiments of the present invention relate to and comprise the composition that one or more are selected from the cell of the negative definitive entoderm cell of hESC, PDX1-, the positive anterior intestine endoderm cell of PDX1-and mesoblastemic cell type, for example cell culture or cell mass. In some embodiments, in the culture hESC be less than about 5%, be less than about 4%, be less than about 3%, be less than about 2% or be less than whole cells of about 1%. In other embodiments, the negative definitive entoderm cell of PDX1-is less than about 90% in the culture, be less than about 85%, be less than about 80%, be less than about 75%, be less than about 70%, be less than about 65%, be less than about 60%, be less than about 55%, be less than about 50%, be less than about 45%, be less than about 40%, be less than about 35%, be less than about 30%, be less than about 25%, be less than about 20%, be less than about 15%, be less than about 12%, be less than about 10%, be less than about 8%, be less than about 6%, be less than about 5%, be less than about 4%, be less than about 3%, be less than about 2% or be less than whole cells of about 1%. In other embodiments, in the culture mesoblastema be less than about 90%, be less than about 85%, be less than about 80%, be less than about 75%, be less than about 70%, be less than about 65%, be less than about 60%, be less than about 55%, be less than about 50%, be less than about 45%, be less than about 40%, be less than about 35%, be less than about 30%, be less than about 25%, be less than about 20%, be less than about 15%, be less than about 12%, be less than about 10%, be less than about 8%, be less than about 6%, be less than about 5%, be less than about 4%, be less than about 3%, be less than about 2% or be less than whole cells that account for of about 1%.

Other embodiments of the present invention relate to what method described herein produced and comprise the front gut entoderm of the PDX1-positive as composition, for example cell culture or the cell mass of main cell type. In some embodiments, produce with method described herein comprise at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 94%, at least about 93%, at least about 92%, at least about 91%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, at least about 54%, at least about 53%, at least about 52% or at least about 51% the positive anterior intestine endoderm cell's of PDX1-cell culture and/or cell mass. In the preferred embodiment, the cell of cell culture or cell mass comprises people's cell. In other embodiments, comprise at least about 50% with method generation described herein, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 24%, at least about 23%, at least about 22%, at least about 21%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2% or at least about the positive anterior intestine endoderm cell's of 1% PDX1-cell culture and/or cell mass. In the preferred embodiment, the cell of cell culture or cell mass comprises the human cell. In some embodiments, do not consider to remain in the feeder cells in the culture when calculating the ratio of the positive anterior intestine endoderm cell of PDX1-in cell culture or the cell mass.

Other embodiments of the present invention relate to the composition of the mixture that comprises the positive anterior intestine endoderm cell of PDX1-and the negative definitive entoderm cell of PDX1-, for example cell culture or cell mass. For example, can produce and comprise the negative definitive entoderm cell of approximately per 95 PDX1-correspondence at least about the positive anterior intestine endoderm cells' of 5 PDX1-cell culture or cell mass. In other embodiments, can produce and comprise the negative definitive entoderm cell of approximately per 5 PDX1-correspondence at least about the positive anterior intestine endoderm cells' of 95 PDX1-cell culture or cell mass. In addition, expection can obtain comprising cell culture or the cell mass of the positive anterior intestine endoderm cell of PDX1-and negative other ratios of definitive entoderm of PDX1-. For example, expection can obtain comprising approximately per 1,000,000 the negative definitive entoderm cell of PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, approximately per 100,000 the negative definitive entoderm cell of PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, approximately per 10,000 the negative definitive entoderm cell of PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, the negative definitive entoderm cell of approximately per 1000 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, the negative definitive entoderm cell of approximately per 500 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, the negative definitive entoderm cell of approximately per 100 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, the negative definitive entoderm cell of approximately per 10 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, the negative definitive entoderm cell of approximately per 5 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, the negative definitive entoderm cell of approximately per 4 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, the negative definitive entoderm cell of approximately per 2 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, the negative definitive entoderm cell of approximately per 1 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, the negative definitive entoderm cell of approximately per 1 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 2 PDX1-, the negative definitive entoderm cell of approximately per 1 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 4 PDX1-, the negative definitive entoderm cell of approximately per 1 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 5 PDX1-, the negative definitive entoderm cell of approximately per 1 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 10 PDX1-, the negative definitive entoderm cell of approximately per 1 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 20 PDX1-, the negative definitive entoderm cell of approximately per 1 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 50 PDX1-, the negative definitive entoderm cell of approximately per 1 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 100 PDX1-, the negative definitive entoderm cell of approximately per 1 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1000 PDX1-, the negative definitive entoderm cell of approximately per 1 PDX1-correspondence is at least about 10,000 positive anterior intestine endoderm cell of PDX1-, the negative definitive entoderm cell of approximately per 1 PDX1-correspondence is at least about 100,000 positive anterior intestine endoderm cell of PDX1-, the negative definitive entoderm cell of per 1 PDX1-of peace treaty correspondence is at least about 1,000,000 positive anterior intestine endoderm cell's of PDX1-composition.

In some embodiments of the present invention, cell-derived from people's multipotential cell, for example human pluripotent stem cells for generation of the negative definitive entoderm of the positive anterior intestine endoderm cell's of PDX1-PDX1-. In some embodiment, people's multipotential cell is derived from mulberry body, embryo's inner cell mass or embryo sexual fold. In some other embodiment, people's multipotential cell is derived from growing gonadal tissue or the germinal tissue of having crossed brephic multi-cellular structure.

Further embodiment of the present invention relates to and comprises people's cell composition of (comprising the front gut entoderm of the people PDX1-positive), for example cell culture or cell mass wherein are higher than the expression of AFP, SOX7, SOX1, ZIC1 and/or NFM mark at least about the expression of PDX1 mark in people's cell of 2%. In other embodiments, in people's cell of at least 5%, in people's cell of at least 10%, in people's cell of at least 15%, in people's cell of at least 20%, in people's cell of at least 25%, in people's cell of at least 30%, in people's cell of at least 35%, in people's cell of at least 40%, in people's cell of at least 45%, in people's cell of at least 50%, in people's cell of at least 55%, in people's cell of at least 60%, in people's cell of at least 65%, in people's cell of at least 70%, in people's cell of at least 75%, in people's cell of at least 80%, in people's cell of at least 85%, in people's cell of at least 90%, in people's cell of at least 95% or in people's cell of at least 98%, the expression of PDX1 mark is higher than AFP, SOX7, SOX1, the expression of ZIC1 and/or NFM mark. In some embodiments, do not consider feeder cells when calculating the ratio of people's cell (wherein the expression of PDX1 is higher than AFP, SOX7, SOX1, the expression of ZIC1 and/or NFM) among cell culture or the group.

Should be appreciated that embodiments more of the present invention relate to the composition that comprises the positive anterior intestine endoderm cell of people PDX1-, for example cell culture or cell mass, wherein from least about 2% to the expression that surpasses one or more expression that are selected from SOX17, HOXA13 or HOXC6 people's cell of about 98% and be higher than AFP, SOX7, SOX1, ZIC1 and/or NFM mark. In some embodiments, in people's cell of 5%, in people's cell of 10%, in people's cell of 15%, in people's cell of 20%, in people's cell of 25%, in people's cell of 30%, in people's cell of 35%, in people's cell of 40%, in people's cell of 45%, in people's cell of 50%, in people's cell of 55%, in people's cell of 60%, in people's cell of 65%, in people's cell of 70%, in people's cell of 75%, in people's cell of 80%, in people's cell of 85%, in people's cell of 90%, at least about in people's cell of 95% or at least about in 98% the cell, one or more are selected from SOX17, the expression of HOXA13 or HOXC6 is higher than AFP, SOX7, SOX1, the expression of ZIC1 and/or NFM. In some embodiments, do not consider feeder cells when calculating the ratio of people's cell (one or more expression that are selected from SOX17, HOXA13 or HOXC6 are higher than the expression of AFP, SOX7, SOX1, ZIC1 and/or NFM) among cell culture or the group.

Other embodiments of the present invention relate to the composition that comprises mammal endoderm cell (for example people endoderm cell), for example cell culture or cell mass wherein are higher than the expression of AFP, SOX7, SOX1, ZIC1 and/or NFM mark at least about the expression of PDX1 mark among 2% the endoderm cell. In other embodiments, in 5% endoderm cell, in 10% endoderm cell, in 15% endoderm cell, in 20% endoderm cell, in 25% endoderm cell, in 30% endoderm cell, in 35% endoderm cell, in 40% endoderm cell, in 45% endoderm cell, in 50% endoderm cell, in 55% endoderm cell, in 60% endoderm cell, in 65% endoderm cell, in 70% endoderm cell, in 75% endoderm cell, in 80% endoderm cell, in 85% endoderm cell, in 90% endoderm cell, at least about among 95% the endoderm cell or at least about among 98% the endoderm cell, the expression of PDX1 mark is higher than AFP, SOX7, SOX1, the expression of ZIC1 and/or NFM mark.

Other embodiments of the present invention relate to the composition that comprises mammal endoderm cell (for example people endoderm cell), for example cell culture or cell mass wherein are higher than the expression of AFP, SOX7, SOX1, ZIC1 and/or NFM mark at least about one or more expression that are selected from the mark of SOX17, HOXA13 and HOXC6 in 2% the embryonic cell. In other embodiments, in 5% endoderm cell, in 10% endoderm cell, in 15% endoderm cell, in 20% endoderm cell, in 25% endoderm cell, in 30% endoderm cell, in 35% endoderm cell, in 40% endoderm cell, in 45% endoderm cell, in 50% endoderm cell, in 55% endoderm cell, in 60% endoderm cell, in 65% endoderm cell, in 70% endoderm cell, in 75% endoderm cell, in 80% endoderm cell, in 85% endoderm cell, in 90% endoderm cell, at least about among 95% the endoderm cell or at least about among 98% the endoderm cell, one or more are selected from SOX17, the expression of the mark of HOXA13 and HOXC6 is higher than AFP, SOX7, SOX1, the expression of ZIC1 and/or NFM mark.

Use method described herein, can produce the composition that comprises the positive anterior intestine endoderm cell of PDX1-that is substantially free of other cell types. About the cell in cell culture or the cell mass, term " is substantially free of " ratio that specific cell type that expression cell culture or cell mass do not contain occupies and is less than 5% in all cells of cell culture or cell mass. In embodiments more of the present invention, the positive anterior intestine endoderm cell group of PDX1-or the cell culture that produce with method described herein are substantially free of remarkable cell of expressing AFP, SOX7, SOX1, ZIC1 and/or NFM marker gene.

In one embodiment of the invention, according to the expression of marker gene being described below the positive anterior intestine endoderm cell of PDX1-: the PDX1 height, AFP is low, SOX7 is low, SOX1 is low, ZIC1 is low and NFM is low.

Increase the expression of PDX1 in the positive definitive entoderm cell of SOX17-

Aspects more of the present invention relate to increases the method for expressing the PDX1 gene outcome in the cell culture comprise the positive definitive entoderm cell of SOX17-or the cell mass. In this class embodiment, in the positive definitive entoderm cell of SOX17-, add the differentiation factor that is enough to increase PDX1 gene product expression amount. The SOX17-that contacts with differentiation factor is positive, and the definitive entoderm cell can be PDX1-feminine gender or the PDX1-positive. In some embodiments, differentiation factor can be retinoids. Among some embodiment, make the positive definitive entoderm cell of SOX17-and the about 0.01 μ M of concentration range extremely the retinoids of about 50 μ M contact. In preferred embodiments, described retinoids is RA.

In other embodiments of the present invention, the expression of PDX1 gene outcome in the cell culture by making the positive definitive entoderm cell of SOX17-and the differentiation factor of fibroblast growth family contact to increase to comprise SOX17-positive definitive entoderm cell or the cell mass. This class differentiation factor can use separately or with the RA coupling. In some embodiments, make the positive definitive entoderm cell of SOX17-and the about 10ng/ml of concentration range extremely the fibroblast growth factor of about 1000ng/ml contact. In the preferred embodiment, the FGF growth factor is FGF-10.

In embodiments more of the present invention, the expression of PDX1 gene outcome in the cell culture by making the SOX17-positive cell contact to increase with B27 to comprise the positive definitive entoderm cell of SOX17-or the cell mass. This class differentiation factor can use separately or with FGF family differentiation factor and retinoids in one or both couplings. In some embodiments, make the positive definitive entoderm cell of SOX17-and about 0.1% (v/v) of concentration range extremely the B27 of about 20% (v/v) contact. In preferred embodiments, the positive definitive entoderm cell of SOX17-is contacted with B27 with RA, FGF-10.

The method that increases PDX1 gene product expression in the cell culture comprise the positive definitive entoderm cell of SOX17-or the cell mass can be carried out in the growth medium that reduces serum-concentration or serum-free. In some embodiments, the serum-concentration scope is from about 0.05% (v/v) to about 20% (v/v). In some embodiments, the positive definitive entoderm cell of SOX17-is grown containing under the condition of serum substitute.

Can promote the negative definitive entoderm cell of PDX1-to the evaluation of the factor of the positive anterior intestine endoderm cell differentiation of PDX1-

Other aspects of the present invention relate to identifies that one or more can promote the negative definitive entoderm cell of PDX1-to the method for the differentiation factor of the positive anterior intestine endoderm cell differentiation of PDX1-. In these class methods, obtain to comprise cell culture or the cell mass of the negative definitive entoderm cell of PDX1-, and measure the expression of PDX1 in cell culture or the cell mass. After measuring the PDX1 expression, the cell of cell culture or cell mass is contacted with candidate's differentiation factor. In some embodiments, when cell is contacted with candidate's differentiation factor or with the expression that detects soon PDX1 after candidate's differentiation factor contacts. One or more time points after making cell and candidate's differentiation factor contacts detect the PDX1 expression then. Compare with the expression of PDX1 before contacting candidate's differentiation factor, if the expression of PDX1 increases behind contact candidate differentiation factor, candidate's differentiation factor can be accredited as and can promote the negative definitive entoderm cell of PDX1-to the positive anterior intestine endoderm cell's of PDX1-differentiation.

In some embodiments, above-mentioned evaluation can promote the negative definitive entoderm cell of PDX1-also to comprise the expression of measuring HOXA13 in cell culture or the cell mass and/or HOXC6 gene to the method for the differentiation factor of the positive anterior intestine endoderm cell differentiation of PDX1-. In this class embodiment, make cell contact the expression that HOXA13 and/or HOXC6 gene are measured respectively in front and back with candidate's differentiation factor. With contact differentiation factor before the expression of PDX1 and HOXA13 compare, if behind contact candidate differentiation factor, the expression of PDX1 and HOXA13 increases, and just can identify that candidate's differentiation factor can promote the negative definitive entoderm cell of PDX1-to the positive anterior intestine endoderm cell's of PDX1-differentiation. Similarly, with contact differentiation factor before relatively, if behind contact candidate differentiation factor, the expression of PDX1 and HOXC6 increases, just can identify that candidate's differentiation factor can promote the negative definitive entoderm cell of PDX1-to the positive anterior intestine endoderm cell's of PDX1-differentiation. In preferred embodiments, identify candidate's differentiation factor that can promote that the negative definitive entoderm cell of PDX1-breaks up to the positive anterior intestine endoderm cell of PDX1-by the expression of measuring respectively PDX1, HOXA13 and HOXC6 before and after contacting with candidate's differentiation factor at the cell that makes cell culture or cell mass. In the preferred embodiment, detect the expression of PDX1, HOXA13 and HOXC6 with Q-PCR.

Should be appreciated that in some embodiments, soon detect among PDX1, HOXA13 and the HOXC6 one or more expression when the cell in making cell culture or cell mass contacts with candidate's differentiation factor or behind contact candidate differentiation factor, rather than before cells contacting candidate differentiation factor, detect. In this class embodiment, will be when cell be contacted with candidate's differentiation factor or behind contact candidate differentiation factor the soon expression of one or more among PDX1, HOXA13 and the HOXC6 compare with PDX1, the HOXA13 of one or more time points behind cells contacting candidate differentiation factor and the one or more expression among the HOXC6.

In some embodiments of said method, the scope that detects one or more time points of PDX1 expression after cell contacts with candidate's differentiation factor can be from about 1 hour to about 10 days. For example, can contact rear about 1 hour with candidate's differentiation factor at cell, cell contacts rear about 2 hours with candidate's differentiation factor, cell contacts rear about 4 hours with candidate's differentiation factor, cell contacts rear about 6 hours with candidate's differentiation factor, cell contacts rear about 8 hours with candidate's differentiation factor, cell contacts rear about 10 hours with candidate's differentiation factor, cell contacts rear about 12 hours with candidate's differentiation factor, cell contacts rear about 16 hours with candidate's differentiation factor, cell contacts rear about 24 hours with candidate's differentiation factor, cell contacts rear about 2 days with candidate's differentiation factor, cell contacts rear about 3 days with candidate's differentiation factor, cell contacts rear about 4 days with candidate's differentiation factor, cell contacts rear about 5 days with candidate's differentiation factor, cell contacts rear about 6 days with candidate's differentiation factor, cell contacts rear about 7 days with candidate's differentiation factor, cell contacts rear about 8 days with candidate's differentiation factor, cell contacts rear about 9 days with candidate's differentiation factor, cell contacts rear about 10 days with candidate's differentiation factor or cell contacts the rear expression that detected PDX1 in 10 days that surpasses with candidate's differentiation factor.

The candidate's differentiation factor that is used for method described herein can be selected from compound, for example polypeptide and little molecule. For example, candidate's polypeptide includes but not limited to growth factor, cell factor, chemotactic factor (CF), extracellular matrix protein and synthetic peptide. In preferred embodiments, growth factor is from FGF family, for example FGF-10. The little molecule of candidate includes but not limited to from combinatorial chemistry synthetic compound and natural products, for example steroids, isoprenoid, terpenoid, class phenyl-propane (phenylpropanoid), alkaloids and flavonoids. It will be understood by a person skilled in the art that thousands of kinds of natural and synthetic little molecules can utilize, the little molecule that expection can be used for method described herein is not limited to above kind of giving an example. Typically, micromolecular molecular weight is lower than 10,000 amu. In preferred embodiments, little molecule is retinoids, for example RA.

Can promote the evaluation of the factor of the positive anterior intestine endoderm cell differentiation of PDX1-

Other aspects of the present invention relate to the method for identifying that one or more can promote the differentiation factor of the positive anterior intestine endoderm cell differentiation of PDX1-. In these class methods, obtain to comprise the positive anterior intestine endoderm cell's of PDX1-cell culture or cell mass, and measure the expression of mark in cell culture or the cell mass. After the expression of measuring mark, the cell of cell culture or cell mass is contacted with candidate's differentiation factor. In some embodiments, when cell is contacted with candidate's differentiation factor or behind contact candidate differentiation factor, detect soon the expression of mark. One or more time points after the contact of cell and candidate's differentiation factor detect the expression of identical mark then. With contact differentiation factor before relatively, if behind contact candidate differentiation factor, the expression of mark increases or reduces, and just can identify that candidate's differentiation factor can promote PDX1-positive anterior intestine endoderm cell's differentiation. In preferred embodiments, detect the expression of mark with Q-PCR.

In some embodiments of said method, the scope that detects one or more time points of marker expression after cell is contacted with candidate's differentiation factor can be from about 1 hour to about 10 days. For example, can make cell contact rear about 1 hour with candidate's differentiation factor, make cell contact rear about 2 hours with candidate's differentiation factor, make cell contact rear about 4 hours with candidate's differentiation factor, make cell contact rear about 6 hours with candidate's differentiation factor, make cell contact rear about 8 hours with candidate's differentiation factor, make cell contact rear about 10 hours with candidate's differentiation factor, make cell contact rear about 12 hours with candidate's differentiation factor, make cell contact rear about 16 hours with candidate's differentiation factor, make cell contact rear about 24 hours with candidate's differentiation factor, make cell contact rear about 2 days with candidate's differentiation factor, make cell contact rear about 3 days with candidate's differentiation factor, make cell contact rear about 4 days with candidate's differentiation factor, make cell contact rear about 5 days with candidate's differentiation factor, make cell contact rear about 6 days with candidate's differentiation factor, make cell contact rear about 7 days with candidate's differentiation factor, make cell contact rear about 8 days with candidate's differentiation factor, after cell is contacted with candidate's differentiation factor about 9 days, cell contact with candidate's differentiation factor after about 10 days or make cell contact the expression of the rear mark of the detection above 10 days with candidate's differentiation factor.

As previously described, the candidate's differentiation factor for method described herein can be selected from for example polypeptide or micromolecular compound.

Although every kind of method disclosed herein is all about the positive anterior intestine endoderm cell of PDX1-, should be appreciated that in certain embodiments the method can be used for producing the composition of the PDX1-positive entoderm cell that comprises the positive anterior intestine of PDX1-described herein/midgut endoderm cell and/or anterior intestine described herein rear portion. In addition, disclosed any PDX1-positive entoderm cell type all can be used in the screening technique described herein in this specification.

Above general description the present invention, can be by further understanding the present invention with reference to some specific embodiment provided herein, these embodiment only are in order to illustrate rather than limit the present invention.

Embodiment

Below a lot of embodiment the use of people's multipotential cell has been described.The method of generation people multipotential cell is known in the art, and in many scientific publication things description is arranged all, comprises the 5th, 453,357,5,670,372,5,690,926,6,090,622,6,200,806 and 6,251, No. 671 United States Patent (USP)s and publication number are 2004/0229350 U.S. Patent application.

Embodiment 1

People ES cell

As if grow for the research entoderm, we have adopted polyenergic human embryo stem cell, can infinitely divide in substratum and keep normal caryogram simultaneously.Adopt immunology or mechanical means to separate, obtain the ES cell from embryo's inner cell masses of 5 days.Especially, human embryonic stem cell hESCyt-25 obtains from the unnecessary frozen embryo in cycle in vitro fertilization after the patient agrees.After thawing, the blastocyst of hatching is planted on the mouse embryo fibroblasts (MEF) in the ES substratum (DMEM, 20%FBS, nonessential amino acid, beta-mercaptoethanol, ITS additive).The embryo sticks on the culture dish, and after about two weeks, undifferentiated hESC zone is transferred in the new culture dish that contains MEF.Shift to adopt the simple digestion of mechanical shearing and Dispase, remove cell cluster with mechanical force then, clean and repopulating cell again.After obtaining, hESCyt-25 goes down to posterity through a series of about 100 times.We utilize human embryonic stem cell hESCyt-25 as the parent material that produces definitive entoderm.

Described those skilled in the art should be appreciated that stem cell or other multipotential cells also can be used as the parent material of differentiation step described herein.The parent material that for example, can be used as multipotential cell by the isolating cell that derives from embryo sexual fold of method as known in the art.

Embodiment 2

The feature of hESCyt-25

Human embryonic stem cell hESCyt-25 cultivates in substratum and still keeps normal form, caryogram, growth and self character after 18 months.This clone all shows very strong immunoreactivity (these several antigens all are the characteristic antigen that does not break up hESC), shows alkaline phosphatase activities and be similar form to hESC that other has been set up OCT4, SSEA-4 and TRA-1-60 antigen.In addition, human embryonic stem cell hESCyt-25 forms embryoid (EB) easily when suspension culture.HESCyt-25 can be divided into the different cell types of representing three kinds of main germinal layers, versatility that this has shown hESCyt-25.To the Q-PCR of ZIC1 detect and to immunocytochemistry (ICC) assay certificate of nestin and the ripe neural mark of Geng Duo ectodermic generation.Observed the immunocytochemical stain of β-III tubulin in extending cell cluster, this is early stage neuronic feature.Before, we are with the EB in the retinoic acid treatments suspension, with induced multi-potent stem cells clone differentiation outside internal organ entoderm (VE), embryo.After handling in 54 hours, the mark of treated cell great expression VE: α-fetoprotein (AFP) and SOX7.Immunocytochemical stain shows that cell is expressed in the monolayer of AFP and breaks up in fragmentary piece.To describe below, when no AFP expressed, the real-time quantitative polymerase chain reaction (Q-PCR) of SOX-17 and immunocytochemistry detect proof hESCyt-25 clone can also form definitive entoderm.Different time points detected among the EB of differentiation the Brachyury expression of gene with proof to mesoblastic differentiation.Brachyury expresses gradually and improves in experimentation.In sum, hESCyt-25 clone has the ability that forms the cell of representing three kinds of germinal layers, is polyenergic therefore.

Embodiment 3

The preparation of SOX17 antibody

The major obstacle of identifying definitive entoderm in the hESC culture lacks proper tools exactly, so we have prepared the proteic antibody of anti-people SOX17.

Definitive entoderm is after the gastrula formation phase produces, and all definitive entoderms are all expressed mark SOX17, and being expressed in the intestinal tube of it continues (although expression level changes along the A-P axle) until organogenetic beginning.SOX17 also expresses in the extraembryonic endoderm cell subsets.In mesoderm or ectoderm, do not find this proteic expression.Find now when with the eliminating embryo outside during the mark coupling of clone, SOX17 is the suitable landmarks thing of definitive entoderm clone.

As describe in detail herein, it is to produce the different treatment method of the positive definitive entoderm cell of SOX17 and the effect of differentiation step that SOX17 antibody is used for special testing goal.Be used to get rid of the generation of internal organ and body wall entoderm (extraembryonic endoderm) with other antibody of AFP, SPARC and thrombomodulin reaction.

In order to produce the antibody of anti-SOX17, at the GENOVAC (Fu Laibeige of Antibody Preparation company, Germany) according to the step of its exploitation, the groups of people SOX17 cDNA corresponding with SOX17 protein carboxyl terminal amino acid/11 72-414 (SEQID NO:2) (SEQ ID NO:1) is used to the inherited immunity of rat.The inherited immunity step can be referring to the 5th, 830, and 876,5,817,637,6,165,993 and 6,261, No. 281 United States Patent (USP)s, and publication number is the international patent application of WO 00/29442 and WO 99/13915.

Other usability methods of inherited immunity also have description in non-patent literature.For example, people such as Barry are at Biotechniques 16:616-620, have described with the inherited immunity method producing monoclonal antibody in 1994.Below be the specific examples for preparing differential protein antibody with the inherited immunity method: as people such as Costaglia, (1998) (inherited immunity of anti-thyroid stimulating hormone receptor causes thyroiditis to Genetic immunization against the humanthyrotropin receptor causes thyroiditis and allows production ofmonoclonal antibodies recognizing the native receptor, and can produce the monoclonal antibody of discerning natural receptor), J.Immunol.160:1458-1465; People such as Kilpatrick, (1998) Gene gundelivered DNA-based immunizations mediate rapid production of murinemonoclonal antibodies to the Flt-3 receptor (the quick generation that particle gun is carried) based on the mouse monoclonal antibody of the immunization metering needle antagonism Flt-3 acceptor of DNA, Hybridoma 17:569-576; People such as Schmolke (1998) Identification ofhepatitis G virus particles in human serum by E2-specific monoclonalantibodies generated by DNA immunization (hepatitis G virus particle in the E2-specific monoclonal antibody identifier serum that dna immunization produces), J.Virol.72:4541-4545; People such as Krasemann (1999) Generation of monoclonal antibodies againstproteins with an unconventional nucleic acid-based immunizationstrategy (utilizing unconventional immunization strategy to produce) at proteic monoclonal antibody based on Nucleotide, J.Biotechnol.73:119-129; With people (1996) Generation ofa monoclonal antibody to a defined portion of the Heliobacter pylorivacuolating cytotoxin by DNA immunization (producing the monoclonal antibody of anti-Heliobacter pylorivacuolating intracellular toxin specified portions by dna immunization) J.Biotechnol.51:191-194 such as Ulivieri.

Concern shown in the dendrogram that as Fig. 3 SOX7 and SOX18 are the family members nearest with the SOX17 relation in Sox family.We utilize people SOX7 polypeptide special to SOX17 as the SOX17 antibody that negative control proof inherited immunity produces, and not nearest with its relation family member's reaction.Particularly, SOX7 and other albumen are expressed in the human fibroblasts, pass through the cross reactivity of Western trace and ICC methods analyst and SOX17 antibody then.For example, following method is used for producing SOX17, SOX7 and EGFP expression vector, with carrier transfection human fibroblasts, carries out the Western engram analysis then.The expression vector that is used to produce SOX17, SOX7 and EGFP is respectively pCMV6 (OriGene Technologies, Inc., Rockville, MD), pCMV-SPORT6 (Invitrogen, Carlsbad, CA) and pEGFP-N1 (Clonetech, Palo Alto, CA).Be preparation albumen, super coiled DNA adopts Lipofectamine 2000 (Invitrogen, Carlsbad, CA) the telomerase immortalized MDX human fibroblasts of transient transfection.Transfection after 36 hours at 50mM TRIS-HCl (pH 8), 150mM NaCl, 0.1%SDS, 0.5% Septochol, contain mixture (the Roche Diagnostics Corporation of proteinase inhibitor, Indianapolis, IN) the whole lysates of middle collection.Western engram analysis step is as follows: 100 μ g cell proteins are at NuPAGE (4-12% gradient polyacrylamide, Invitrogen, Carlsbad, CA) going up SDS-PAGE separates, electricity forwards PDVF film (Hercules to, CA), rat SOX17 antiserum(antisera) 10mM TRIS-HCl (pH8), 150mM NaCl, 10%BSA, 0.05%Tween-20 (Sigma, St.Louis, MO) 1/1000 dilution is as probe, use mouse IgG (the Jackson ImmunoResearch Laboratories of the alkaline phosphatase link coupled Chinese People's Anti-Japanese Military and Political College then, West Grove PA) is hatched, and uses Vector Black alkaline phosphatase staining (Vector Laboratories at last, Burlingame CA) manifests.Used molecular weight of albumen standard be wide region color-coded thing (Sigma, St.Louis, MO).

Among Fig. 4, from transient transfection human fibroblasts's the protein extract of SOX17, SOX7 or EGFP cDNA carry out the Western trace with SOX17 antibody and detect.Have only transfection the protein extract of cell of hSOX17 produce a band at～51Kda, very approaching with the proteic molecular weight 46Kda of prediction people SOX17.SOX17 antibody with from transfection the extract of cell of people SOX7 or EGFP do not have reactivity.In addition, SOX17 antibody clearly mark transfection hSOX17 express the human fibroblasts's of member nucleus, but the cell of EGFP that do not had a mark transfection.Equally, ICC detects the specificity that has also shown SOX17 antibody.

Embodiment 4

SOX17 antibody is as the confirmation of definitive entoderm mark

The hESC of part differentiation is specific to SOX17 albumen to show SOX17 antibody, the step mark definitive entoderm of going forward side by side with SOX17 and the same tense marker of AFP antibody.Proved that SOX17, SOX7 (the nearest member of relation of SOX gene family subgroup F) and AFP all have expression in the internal organ entoderm.But, in the definitive entoderm cell, do not find the expression of AFP and SOX7 at the ICC detection level, therefore, they can be used as the negative mark of bonifide definitive entoderm cell.Show that the SOX17 antibody labeling exists as discrete cell cluster form or with AFP positive cell blended cell mass.Particularly, Fig. 5 A shows that minority SOX17 cell is by the common mark of AFP; But, at SOX17 ⁺Also found in the cell scope to be close to and do not had or do not had at all AFP ⁺The zone of cell (Fig. 5 B).Similarly, because it is reported that the body wall entoderm expresses SOX17, can identify with the common mark of SOX-17 antibody with body wall mark SPARC and/or thrombomodulin (TM) to belong to the endoblastic SOX17 of body wall ⁺Cell.Shown in Fig. 6 A-C, thrombomodulin and SOX-17 are total to the at random differentiation generation of the body wall endoderm cell of mark by the hES cell.

To sum up the cell marking experiment is described, by mark feature SOX17 ^Hi/ AFP ^Lo/ [TM ^LoOr SPARC ^Lo] can set up the method for identifying the definitive entoderm cell.In other words, the expression of SOX17 mark is higher than the expression of AFP mark, and this is the endoblastic features of internal organ, and TM or SPARC mark are the endoblastic features of body wall.Therefore, those SOX17 positives, and AFP cell negative and TM or SPARC feminine gender is the definitive entoderm cell.

Confirm mark feature SOX17 in order to obtain further evidence ^Hi/ AFP ^Lo/ TM ^LoSPARC ^LoCan predict definitive entoderm, make the cell of the antibody labeling of SOX17 and AFP expression of gene and respective numbers carry out quantitative comparison.Shown in Fig. 7 A, the hESC through vitamin A acid (internal organ entoderm inductor) or activin A (definitive entoderm inductor) processing has 10 times of differences on the SOX17mRNA expression level.This result has reflected 10 times of differences (Fig. 7 B) of SOX17 antibody labeling cell number.In addition, shown in Fig. 8 A, the activated plain A of hESC handles, and with undressed comparison, can suppress 6.8 times of AFP genetic expressions.Shown in Fig. 8 B-C in the culture the remarkable minimizing of the quantity of AFP labeled cell intuitively reflected this point.For further quantitatively, detect (Fig. 9 A-B) through fluidic cell and confirm that it is that AFP antibody labeling cell number reduces about 7 times result that AFP genetic expression reduces about 7 times.This result is extremely important, because the change that it shows the gene expression amount that Q-PCR shows has reflected the variation of cell type, this can observe by antibody staining.

After hatching, hESC and Nodal family member (Nodal, activin A and activin B-NAA) pass the remarkable increase that causes SOX17 antibody labeling cell in time.Can be labeled as SOX17 (Figure 10 A-F) above 50% cell after passing through the continuous 5 days processing of activin.And activated plain to handle after 5 days almost seldom or do not have cell marking be AFP.

In brief, 242 amino acid whose antibody of anti-people SOX17 protein carboxyl terminal of generation have been identified people SOX17 albumen in the Western trace, but nonrecognition SOX17 immediate member SOX7 in Sox family.Subgroup in the hESC culture that the SOX17 antibody recognition is being broken up, they mainly are SOX17 ⁺/ AFP ^Lo/-(surpassing 95% labeled cell) and small part (＜5%) are total to the cell (internal organ entoderm) of mark SOX17 and AFP.Handle the hESC culture with activin and cause the remarkable rising of SOX17 genetic expression and the obvious increase of SOX17 labeled cell, and significantly suppress the expression of AFP mRNA, reduce the number of AFP antibody labeling cell.

Embodiment 5

The Q-PCR gene expression analysis

In following experiment, real-time quantitative RT-PCR (Q-PCR) is the main analytical procedure of hESC differentiation being carried out detecting after the different treatment effect.Especially, the real-time detection of genetic expression is at the multiple marker gene of different time point analysis with Q-PCR.Characteristic mark to cell type expectation and that do not expect is assessed, so that better understand the overall dynamics of cell mass.The advantage that Q-PCR analyzes is its extreme susceptibility and newly-increased convenient relatively must mark the time, because genome sequence can obtain.In addition, the high susceptibility of Q-PCR can detect fewer relatively purpose gene expression of cells in the very large cell mass.And the ability that detects extremely low-level genetic expression is that " the differentiation deflection " of cell mass provides indication.At cell phenotype significantly before the differentiation, can not detect to the deflection immunocytochemical technique of specific differentiation pathway.For this reason, Q-PCR provides a kind of analytical procedure, and this method is replenishing of immunocytochemical technique concerning being used to screen successful differentiation handling at least, and may more be better than it.In addition, Q-PCR provides a kind of mechanism, and whether this mechanism can utilize half high-throughput quantification assay Analytical Chemical Experiment design successful.

The method of Cai Yonging is for utilizing SYBR Green chemistry and two one step RT-PCR patterns to carry out relative quantification on Rotor Gene 3000 devices (Corbett Research) herein.This method allows to preserve the gene that the cDNA sample is used for analyzing in the future other marks, has avoided the difference of sample room reverse transcription efficient.

According to removing from the experience of the genomic dna amplified production that pollutes, the primer of design should be positioned at exon-exon border, perhaps as being across to the intron of few 800bp.When the marker gene that uses did not contain intron or has pseudogene, RNA sample application DNase I handled.

We detect the genetic expression of the multiple mark of target cell and non-target cell type usually with Q-PCR, thereby produce the gene expression profile in the cell sample.It is right that table 1 has been listed mark and the confirmed available primer relevant with hESC differentiation early stage (particularly ectoderm, mesoderm, definitive entoderm and extraembryonic endoderm).The right human specific of verified these primers, this is extremely important, because hESC grows on the mouse feeder layer usually.The most representative is that every kind of condition is got three samples, independently carries out twice analysis then, with assessment and each relevant biology difference of quantitative assay.

For producing pcr template, separate total RNA and quantitative with RiboGreen (Molecular Probes) with RNeasy (Qiagen).The reverse transcription of the total RNA of 350～500ng uses iScript reversed transcriptive enzyme test kit (BioRad) to carry out, and test kit contains the mixture of oligo-dT and random primer.Per 20 μ L reaction solutions are diluted to cumulative volume 100 μ L, and 3 μ L are used for per 10 μ L Q-PCR reactions (containing 400nM forward and reverse primer and 5 μ L, 2 * SYBR Green mastermix (Qiagen)) then.Two step loop parameters are as follows: 85-94 ℃ of sex change 5 seconds (specifically according to every kind of primer the melting temp of amplicon being selected); 60 ℃ of annealing/extensions 45 seconds.Back 15 seconds collection fluorescence datas at each extended peroid.Each some triplicate produces the typical curve of each runs with 10 times of gradient dilutions series, based on this typical curve cycle threshold (Ct ' s) is converted into quantitative values then.The quantitative values of each sample is proofreaied and correct with the house-keeping gene result, calculates the average and standard deviation of three duplicate samples.When the PCR circulation is finished, carry out the specificity that reaction is determined in the melt curve analysis analysis.Single special product is by the T that is suitable for pcr amplification _mThe simple spike at place is represented.In addition,, do not increase as negative control with the reaction that do not contain reversed transcriptive enzyme.

The first step of setting up the Q-PCR method is the suitable house-keeping gene (HG) of conclusive evidence in experimental system.HG through sample RNA add, the correction of RNA integrity and RT efficient, therefore the expression that importantly HG keeps maintenance level in all samples type is like this proofreaied and correct just meaningful.We have detected the expression of cyclophilin G among the hESC that is breaking up (Cyclophilin G), hypoxanthine phosphoribosyltransferase 1 (HPRT), beta-2-microglobulin, hydroxymethylbianesynthase (HMBS), TATA-conjugated protein (TBP) and glucuronidase β (GUS).Our result shows that the expression level of beta-2-microglobulin increases in atomization, so we get rid of this gene and are used for proofreading and correct.Other genes all keep stable at the expression level of the differentiation phase and the phase of processing.We calculate the correction factor of all samples usually with cyclophilin G and GUS.Use multiple HG to reduce the fluctuation of trimming process self simultaneously, thereby improved the reliability of relative genetic expression numerical value.

After acquisition is used for gauged gene, determine the relative gene expression dose of the marker gene of the sample that the process different experiments is handled with Q-PCR.Selecting these marker genes is that what especially pay close attention to is those gene sets at definitive entoderm and extraembryonic endoderm differential expression because of their content height in the cell mass of the early stage germinal layer of representative.These genes and their relative enrichment condition are pointed out in table 1.

Table 1

Germinal layer	Gene	The expression scope
Entoderm extraembryonic ectoderm mesoderm	SOX17 MIXL1 GATA4 HNF3b GSC SOX7 AFP SPARC TM ZIC1 BRACH	Setting, internal organ and body wall entoderm entoderm and mesoderm setting and primitive endoderm definitive entoderm and primitive endoderm, mesoderm, neural plate entoderm and mesoderm internal organ entoderm internal organ entoderm, liver body wall entoderm body wall entoderm/trophectoderm neurocele, the newborn mesoderm of neural precursor

Because many genes are expressed on a more than germinal layer, so be necessary the expression level of quantitative comparison several genes in identical experiment.SOX17 expresses in definitive entoderm, expresses in internal organ and body wall entoderm and will hang down.SOX7 and AFP express in growing early stage internal organ entoderm.SPARC and TM express at the body wall entoderm, and Brauchyury mesoderm in early days expresses.

Definitive entoderm be it is predicted and expressed high-caliber SOX17 mRNA and low-level AFP and SOX7 (internal organ entoderm), SPARC (body wall entoderm) and Brachyury (mesoderm).In addition, further get rid of early stage ectodermic inducing with ZIC1 herein.At last, GATA4 and HNF3b all express at definitive entoderm and extraembryonic endoderm, therefore with SOX17 at the expression of definitive entoderm be related (table 1).Representational experiment is shown in Figure 11-14, and it has proved how the marker gene that table 1 is described connects each other in different samples, thereby has given prominence to definitive entoderm and extraembryonic endoderm and to the specific differentiation model of mesoderm and neural cell type.

Comprehensive above data can know that the dosage increase that draws activin causes SOX17 genetic expression to improve.And this SOX17 expresses main represent definitive entoderm rather than extraembryonic endoderm.This conclusion is derived from the genetic expression negative correlation of observing SOX17 genetic expression and AFP, SOX7 and SPARC.

Embodiment 6

People ES cell is to the directed differentiation of definitive entoderm

If not cultivating under the condition of effectively keeping the cell undifferentiated state, differentiation at random will take place in people ES cell.This heterogeneous differentiation produces the extraembryonic endoderm cell that comprises body wall and internal organ entoderm (expressing AFP, SPARC and SOX7), and the early stage ectoderm and the mesoderm derivative that are expressed as mark with ZIC1 and Nestin (ectoderm) and Brachyury (mesoderm).Because lack the specific antibody mark in the ES cell culture, the definitive entoderm cellular form does not have detected or describes in detail.Similarly, because the shortage means are not furtherd investigate the generation of early stage definitive entoderm in the ES cell culture.Because there is not the antibody reagent of the suitable anti-definitive entoderm cell of available, the most of evaluation concentrates on ectoderm and extraembryonic endoderm.Generally speaking, in the ES cell culture of differentiation at random, the number of the outer and neuroderm cell type of embryo is much larger than SOX17 ^HiThe number of definitive entoderm cell.

When undifferentiated hESC is cloned in into when increasing on the fiber feeder layer, the cell at clone edge shows the another kind of form different with cloning inner cell.So these okioplasts are easily distinguished because the expression of heterogeneity, bigger form and OCT4 is higher.The existing description, when the ES cell begins differentiation phase, the undifferentiated relatively ES cell of the expression level of OCT4 rises or descends.If the change that the OCT4 level rises or descends may not show from the transformation of multipotency state to the differentiation starting stage above breaking up threshold value.

When detecting undifferentiated clone, can find the little cell cluster that contains 10-15 SOX17 positive cell at undifferentiated hESC clone's the edge and the random site of intersection sometimes with the SOX17 immunocytochemistry.As mentioned above, the cell dispersion group at these peripheral clone edges may be that clone size this moment increases, becomes denser from first cell of classical ES cellular form differentiation.More early stage, littler complete undifferentiated clone (＜1mm; 4-5 days ages) do not contain the SOX17 positive cell in clone inside or edge, and more late period, bigger clone's (diameter 1-2mm,＞5 day age) have the fragmentary SOX17 positive, AFP negative cells to distribute in the peripheral or above-mentioned inside, edge of typical hESC form that do not show of some clones.Because this is to develop effective SOX17 antibody for the first time, produces the definitive entoderm cell in " not differentiation " ES cell culture in early days and never be proved in the past.

Because Q-PCR determines SOX17 and SPARC genetic expression negative correlation, the most SOX17 positive, AFP negative cells will be to the mark reactions that is negative altogether of the antibody of body wall entoderm mark.This body wall endoderm cell in Expression of TM obtains special proof (Figure 15 A-B).Contact the number that can reduce TM expression and TM positive cell with B with Nodal factor activin A.The culture that activin is handled with SOX17, AFP and TM antibody labeling after, shown in Figure 16 A-D, observe the SOX17 positive cell bunch of AFP and TM feminine gender.This is first at the SOX17 positive definitive entoderm cell of proof on the cell levels in differentiation hESC culture.

We have designed some steps and can effectively hESC be divided into SOX17 with above-mentioned SOX17 antibody and Q-PCR method ^Hi/ AFP ^Lo/ SPARC/TM ^LoThe definitive entoderm cell.We adopt number and the multiplication capacity of different differentiation methods to improve these cells, and this is by measuring SOX17 genetic expression in group's level with Q-PCR and detecting with antibody labeling SOX17 in the individual cells level.

We (analyze and describe from the effect that embryonic stem cell produces the application of definitive entoderm cell in the vitro culture thing as Nodal/ activin/BMP) TGF 'beta ' family somatomedin first.In representativeness experiment, the combination of activin A, activin B, BMP or these factors is added into not among the differentiation of human stem cell line hESCyt-25 with initial atomization.

As shown in figure 19, adding 100ng/ml activin A causes undifferentiated relatively hESC to induce 19 times SOX17 genetic expression when breaking up the 4th day.Add activin B (second member of activin family) simultaneously and cause the relative hESC37 of differentiation times inducing in the time of the 4th day in the plain back of handling of admixture activation with activin A.At last, add the 3rd member BMP4 from the TGF 'beta ' family of Nodal/ activin and BMP subtribe, with activin A and activin B coupling, causing not breaking up hESC relatively increases by induce (Figure 19) of 57 times.When inducing SOX17 and no factor substratum to compare with activin and BMP, 5,10 and 15 times of appearance induces in the time of 4 days.When using activin A, B and the triple processing of BMP after 5 days, the derivative level of SOX17 is higher 70 times than not breaking up hESC.These data show that can increase SOX17 with high dosage and long Nodal/ activin TGF 'beta ' family member processing expresses.

In vivo or external Nodal and associated molecule activin A, B and BMP promotes SOX17 to express and definitive entoderm forms.In addition, adding BMP and can promote the SOX17 abduction delivering, may be because further induced Nodal acceptor Cripto.

We have proved that the coupling of BMP4 and activin A and B causes the extra increase of SOX17 and the formation of definitive entoderm.In activin A and B mixture, add the BMP4 long period (＞4 days) and can induce SOX17 at body wall and internal organ entoderm and definitive entoderm.Therefore in the some embodiments of the present invention, handle in 4 days and be necessary to remove BMP4 adding BMP4.

Be the effect of handling in the individual cells level detection TGF β factor, detected the time-histories that the TGF β factor is added with the SOX17 antibody labeling.As shown in Figure 10 A-F, the relative number that prolongs the SOX17 labeled cell in time significantly increases as before.Relative quantification (Figure 20) shows that the SOX17 labeled cell increases above 20 times.This result shows that cell number and SOX17 gene expression dose increased with the TGF β factor treatment time.As shown in figure 21, using Nodal, activin A, activin B and BMP handled after 4 days, and the SOX17 level of inducing reaches 168 times that do not break up hESC.Figure 22 shows that the relative number of SOX17 positive cell is also relevant with dosage.100ng/ml or more dosage can effectively be induced SOX17 genetic expression, increase cell number.

Except TGF 'beta ' family member, the Wnt family molecule works in may and/or keeping in the special differentiation of definitive entoderm.The use of Wnt molecule also helps the differentiation of hESC to definitive entoderm, as shown in figure 23, compares with independent use activin, adds Wnt3a with activin and handles that the SOX17 expression of gene increases in the sample of back.

The tissue culture medium (TCM) of the factor that comprises 10% serum and interpolation is all used in all above-mentioned experiments.Surprisingly, we find that serum-concentration is influential to the expression level of SOX17 under the situation of adding activin.When serum level when 10% drops to 2%, under the condition that contains activin A and B, the expression of SOX17 increases by 3 times.

At last, we prove activin inductive SOX17 shown in Figure 25 A-D ⁺Cell divides in substratum.Arrow shows the cell of the SOX17/PCNA/DAPI mark that is in m period, and this is by the mitotic division plate mode of PCNA/DAPI mark and differs the confirmation of mitotic division feature.

Embodiment 7

Chemokine Receptors 4 (CXCR4) is expressed relevant with the mark of definitive entoderm, and irrelevant with the endoblastic mark of mesoderm, ectoderm or internal organ

As mentioned above, use the cytokine of TGF 'beta ' family, especially activin/nodal subtribe can induce hESC to be divided into definitive entoderm.In addition, we show that foetal calf serum (FBS) the level affects hESC of division culture medium is divided into the efficient of definitive entoderm.This influence is that activin A is under the given concentration in substratum, and high-caliber FBS can suppress the at utmost differentiation to definitive entoderm.As lack the activin A of external source, and hESC is very low to the differentiation efficiency of definitive entoderm system, and the influence of FBS in the hESCs atomization also reduces.

In these experiments, hESC is containing 0.5%, 2.0% or RPMI substratum (Invitrogen, Carlsbad, the CA of 10%FBS; Cat#61870-036) Growth and Differentiation in is wherein added or is not added 100ng/ml activin A and cultivated 6 days.In addition, the FBS gradient and the 100ng/ml activin A couplings with 0.5%-2.0% in preceding 3 days of differentiation.After 6 days, every kind of culture condition is collected the double sample, with the relative genetic expression of real-time quantitative PCR analysis.Remaining cell is fixed for the proteic immunofluorescence of SOX17 and detects.

According to 7 kinds of culture condition that use, the expression level of CXCR4 changes (Figure 26) very greatly.In general, in the culture (A100) that activin A handles CXCR4 express higher, if do not contain external source activin A (NF) then CXCR4 expresses lower.In addition, in the substratum that A100 handles, CXCR4 expresses the highest when FBS concentration is minimum.The CXCR4 level has remarkable reduction under the 10%FBS condition, so the relative expression meets the condition that does not contain activin A (NF) more.

As mentioned above, SOX17, GSC, MIXL1 are consistent with the feature of definitive entoderm cell with HNF3 β expression of gene.Under 7 kinds of differentiation condition the relative expression of these 4 kinds of genes and CXCR4 consistent (Figure 27 A-D).This proof CXCR4 also is the mark of definitive entoderm.

Expression ectoderm and mesoderm system according to the unlike signal thing can be different from definitive entoderm.Early stage mesoderm is expressed Brachyury and MOX1 gene, and newborn neuroderm is expressed SOX1 and ZIC1.Figure 28 A-d proves in the culture that does not contain external source activin A and is more prone to mesoderm and ectoderm expression of gene, and in the culture that activin A handles, the condition of 10%FBS also increases the level of mesoderm and ectoderm marker expression.These expression patterns are opposite with CXCR4's, show to express not high in this etap differentiation from mesoderm or the ectodermic CXCR4 of hESC.

Grow in early days Mammals, the differentiation of clone outside embryo also takes place.Especially relevant is the endoblastic differentiation of internal organ herein, and it is identical with the many expression of gene of definitive entoderm, as SOX17.For distinguishing the outer internal organ entoderm of definitive entoderm and embryo, should detect marks different between them.It is not the mark of expressing at definitive entoderm at the internal organ entoderm that SOX7 has represented a kind of.Therefore, show the SOX17 gene high expression and do not have the culture condition that SOX7 expresses and to comprise definitive entoderm rather than internal organ entoderm.Shown in Figure 28 E, SOX7 high expression level in the culture that does not contain activin A, even contain activin A when FBS concentration is 10%, SOX7 also shows to express to be increased.This expression pattern with CXCR4 is opposite, and it is not high to show that CXCR4 expresses in the internal organ entoderm.

Also measured to be present in and under above-mentioned every kind of differentiation condition SOX17 immunoreactivity (SOX17 arranged ⁺) the relative number of cell.As hESC differentiation phase under high dosage activin A and FBS lower concentration (0.5%-0.2%) condition, SOX17 ⁺Cell distribution is in whole culture.When using high dosage activin A and 10%FBS (v/v), SOX17 ⁺The cell frequency of occurrences reduces, and generally appears in the isolated cell cluster rather than in whole culture and is evenly distributed (Figure 29 A and C and B and E).SOX17 when no external source activin A ⁺Cell number further reduces.SOX17 under these conditions ⁺Cell also occurs in cell cluster, and these cell clusters are littler still less than handling the cell cluster that obtains with high dosage activin A and lower concentration FBS.These result's proof not only corresponding definitive entoderm genetic expressions of CXCR4 expression pattern under every kind of condition, and the number of corresponding definitive entoderm cell.

Embodiment 8

The differentiation condition of enrichment definitive entoderm increases the ratio of CXCR4 positive cell

The dosage of activin A also influences the efficient of hESC to the definitive entoderm differentiation.This embodiment proves that the dosage that increases activin A can improve CXCR4 in the culture ⁺The ratio of cell.

HESC added 0.5%-2%FBS (being increased to 1.0% to 2.0% from 0.5%) preceding 3 days of differentiation and 0,10 or the RPMI substratum of 100ng/ml activin A break up.Break up after 7 days with containing 2%FBS and 2mM (EDTA), not containing Ca ²⁺/ Mg ²⁺PBS room temperature dissociated cell 5 minutes.Cell filters with 35 μ m nylon mesh, counting and precipitation.Precipitation suspends again with the normal donkey serum of small volume 50% human serum/50%, and ice bath 2 minutes is to seal the non-specific antibody binding site.50 μ l cell suspensions (contain and have an appointment 10 ⁵Cell) (Abcam cat#ab10403-100), carried out mark 45 minutes on ice to add 1 μ l mouse anti CXCR4 antibody in.Contain PBS (damping fluid) washed cell of 2% human serum with 5ml, then precipitation.Carry out washing the second time back with per 10 with the 5ml damping fluid ⁵Cell suspends in 50 μ l damping fluids again.Add two anti-(the anti-mouse of FITC link coupled donkey with final concentration 5 μ g/ml; JacksonImmunoResearch cat#715-096-151), was hatched 30 minutes, then with above-mentioned damping fluid washing 2 times.Cell is with 5 * 10 ⁶Cell/ml concentration suspends in damping fluid again, and (The Scripps Research Institute) analyzes and sorting with FACS Vantage (BecktonDickenson) at the flow cytometer center.Cell directly uses RLT lysis buffer (Qiagen) to collect, and separates total RNA then, expresses with the real-time quantitative PCR analyzing gene.

When the dosage of activin A in the division culture medium increased, flow cytometer observed CXCR4 ⁺Cell number significantly increases (Figure 30 A-C).CXCR4 ⁺Cell is those cells that fall into R4 door (gate), and this door only uses two anti-to be provided with in contrast, and for this contrast, 0.2% incident is positioned at the R4 door.When activin A dosage improves, CXCR4 ⁺The enhancing (Figure 31 A-D) of the corresponding definitive entoderm genetic expression of remarkable increase of cell number.

Embodiment 9

The CXCR4 positive cell of separation and concentration definitive entoderm genetic expression is also removed the cell of expressing mesoderm, ectoderm and internal organ entoderm mark

Collect the CXCR4 that identifies among the embodiment 8 ⁺And CXCR4 ^-Cell is analyzed relative genetic expression, determines parental cell group's genetic expression simultaneously.

Level relatively with the increase CXCR4 genetic expression of activin A dosage significantly increases (Figure 32).This and the CXCR4 of the dosage that relies on activin A ⁺The increase of cell conform to very much (Figure 30 A-C).And from cell mass separation of C XCR4 ⁺Cell is equivalent to separate nearly all CXCR4 genetic expression in the cell mass.This has proved the efficient of FACS method collecting cell.

Gene expression analysis shows CXCR4 ⁺Cell not only comprises most of CXCR4 genetic expression, and has comprised the genetic expression of other definitive entoderm marks.Shown in Figure 31 A-D, relative parental generation A100 cell mass CXCR4 ⁺The further enrichment of cell the expression of SOX17, GSC, HNF3B and MIXL1.In addition, CXCR4 ^-Part contains the genetic expression of these definitive entoderm marks hardly.And CXCR4 ⁺And CXCR4 ^-Cell mass has shown opposite gene expression pattern to mesoderm, ectoderm and extraembryonic endoderm.Figure 33 A-D shows relative A100 parental cell group, CXCR4 ⁺Cell has been eliminated the genetic expression of Brachyury, MOX1, ZIC1 and SOX7.Compare with low dosage or the condition of not having an activin A, the expression of these marks is lower among the A100 parental cell group.These results are presented under the high dosage activin A differentiation condition from hESCs separation of C XCR4 ⁺Cell has obtained highly enriched, quite pure definitive entoderm cell mass.

Embodiment 10

With definitive entoderm cell in the quantitative cell mass of CXCR4

For confirm as herein noted earlier and submitted on 21 23rd, 2003 the 60/532nd, No. 004 U.S. Provisional Patent Application (title is " definitive entoderm ") is determined the method for definitive entoderm cell proportion in cell culture or the cell mass, and FACS is used to analyze the cell of expressing CXCR4 and other definitive entoderm marks.

Use the method in the foregoing description, hESC is broken up to produce definitive entoderm.Particularly, for increasing the output and the purity of the cell culture that is breaking up, serum-concentration is as follows in the control substratum: first day 0.2%FBS, second day 1.0%FBS, 3-6 days 2.0%FBS.FACS utilizes three kinds of cell surface epi-position E-cadherins (ECAD), CXCR4 and thrombomodulin that the culture of differentiation is classified.Analyze the cell mass of classification to detect relative expression's level of setting and extraembryonic endoderm and other cell type marks with Q-PCR then.The cell of the CXCR4 sorting that obtains from best differentiation culture thing can make isolating definitive entoderm cell purity greater than 98%.

Table 2 has shown with the result of method differentiation described herein from the analysis of markers of the definitive entoderm culture of hESC

The composition of table 2 definitive entoderm culture

Mark	The per-cent of culture	Definitive entoderm per-cent	Extraembryonic endoderm per-cent	HES cell per-cent
					SOX17 thrombomodulin AFP CXCR4 ECAD other (ECAD feminine genders)	70-80 ＜2 ＜1 70-80 10 10-20	100 0 0 100 0 ?	? 75 25 0 ? ?	? ? ? ? ?100 ?
Add up to	100	100	100	?100

Particularly, table 2 shows that CXCR4 and SOX17 positive cell (entoderm) account for 70% to 80% of cell culture.Express in the cell of SOX17 at these, be less than 2% Expression of TM (body wall entoderm), be less than 1% and express AFP (internal organ entoderm).Partly remove TM positive and AFP positive cell (chamber wall and internal organ entoderm mixture from the SOX17/CXCR4 positive cell; Amount to 3%) after, the cell culture that can find out about 67%-77% is a definitive entoderm.About 10% cell is the ECAD positive, and this is the mark of hESC, and approximately the cell of 10-20% is other cell types.

We find to compare with above-mentioned low serum step, can improve the purity of definitive entoderm in the noble cells culture that obtains by keep FBS concentration≤0.5% in whole 5-6 days differentiation step before FACS separates.But the definitive entoderm cell number that keeps FBS concentration≤0.5% also to cause producing in whole 5-6 days differentiation step reduces.

The definitive entoderm cell that produces with method described herein was containing maintenance and amplification under the condition of activin, did not also observe differentiation significantly above 50 days.In these examples, SOX17, CXCR4, MIXL1, GATA4 and HNF3 β keep expression in the training period.In addition, do not detect TM, SPARC, OCT4, AFP in these cultures, SOX7, ZIC1 and BRACH.Possible this class cell can be kept in substratum and increase and surpass 50 days and do not observe obvious differentiation.

Embodiment 11

Other marks of definitive entoderm cell

In following experiment, isolation of RNA from the definitive entoderm of purifying and human embryo stem cell group.In the cell mass of every kind of purifying, use the gene chip analyzing and testing genetic expression of RNA then.Further analyze at definitive entoderm and the possible gene of not expressing with Q-PCR again, with mark as definitive entoderm at embryonic stem cell.

Human embryo stem cell (hESC) is maintained in the DMEM/F12 substratum that adds 20%KnockOut serum substitute, the basic fibroblast growth factor of 4ng/ml recombinant human (bFGF), 0.1mM beta-mercaptoethanol, L-glutaminate, non-essential amino acid and penicillin/streptomycin.Be divided into definitive entoderm by hESC after in the RPMI substratum that adds 100ng/ml recombinant human activin A, FBS and penicillin/streptomycin, cultivating 5 days.FBS concentration every day is according to following variation: 0.1% (first day), 0.2% (second day), 2% (3-5 days).

With the hESC and the definitive entoderm cell mass of FACS isolated cell acquisition purifying, to be used for gene expression analysis.With SSEA4 antigen (R﹠amp; D Systems, cat#FAB1435P) immune purifying hESC is with CXCR4 (R﹠amp; D Systems, cat#FAB170P) purifying definitive entoderm.Cell with trypsinase/EDTA (Invitrogen cat#25300-054) dissociates, with phosphate-buffered saline (PBS) washing that contains 2% human serum, then in 100% human serum resuspended 10 minutes of ice bath with the sealing non-specific binding.To containing 5 * 10 ⁶Adding 200 μ l phycoerythrin coupling antibody in the 800 μ l human serums of cell dyeed on ice 30 minutes.Cell is with 8ml PBS washed twice, and is resuspended in 1ml PBS.It is to carry out with FACS Vantage (BD Bioscience) in the core laboratory of The Scripps Research Institute that FACS separates.Cell directly uses RLT lysis buffer (Qiagen) to collect, and uses the RNeasy isolation of RNA according to working instructions (Qiagen) then.

Submit to the double purified RNA give Expression Analysis (Durham, NC) so that adopt Affymetrix platform and U133 Plus 2.0 high-density polynucleotide arrays to produce express spectra.The data that obtain are one group and compare that it identifies the gene of differential expression in hESC and definitive entoderm cell mass.Be chosen on the expression level with hESC in compare and gene that obvious rise changes arranged as the distinctive new candidate markers of tool of definitive entoderm.The gene of selecting detects the changes in gene expression of finding with the confirmation gene chip with above-mentioned Q-PCR, and these expression of gene patterns in the research hESC atomization.

Figure 34 A-M shows the gene expression results of some marks.Shown the cell culture that adds 100ng/ml activin A1,3 and 5 days post analysis, 5 days the differentiation step (CXDE) finish result among the hESC of the definitive entoderm cell of expression CXCR4 of back purifying and purifying.Six marker genes of the relatively proof of Figure 34 C and GM, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 have shown expression pattern much at one, and also also similar to the ratio of the expression pattern of CXCR4 and SOX17/SOX7.As previously mentioned, SOX17 all has expression in the extraembryonic endoderm of definitive entoderm and expression SOX7.Because SOX7 does not express at definitive entoderm, can reliably estimate the contribution of definitive entoderm to the expression of observed SOX17 in the whole cell mass according to the ratio of SOX17/SOX7.Figure G-L and figure M show that with the similarity of figure C FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 may be the marks of definitive entoderm, and they express not high in the extraembryonic endoderm cell.

Will be appreciated that Q-PCR result described herein can further be confirmed by ICC.

Embodiment 12

Vitamin A acid and FGF-10 induce PDX1 specifically in the definitive entoderm culture

Experimental results show that below RA and FGF-10 induce the expression of PDX1 in the definitive entoderm cell.

Human embryo stem cell was cultivated 4 days under the condition that contains or do not contain activin.At the 4th day, in substratum, add 1 μ M RA and 50ng/ml FGF-10.Adding RA/FGF-10 after 48 hours, with the quantitative PDX1 marker gene of Q-PCR with to the expression of non-specific other marker genes of preceding gut entoderm.

Cause the remarkable increase (referring to Figure 35) of PDX1 genetic expression with RA processing definitive entoderm cell, but do not increase the expression (Figure 36 A-F) of (SOX1, ZIC1) or neuronic (NFM) gene expression markers of internal organ entoderm (SOX7, AFP), nerve.With observed comparing in the definitive entoderm, be exposed to 1 μ M RA and after 50ng/ml FGF-1048 hour the guarantor, the PDX1 gene expression dose is induced to approximately increases about 500 times.In addition, these results' demonstrations do not use the cell culture of activin to compare with those before RA handles, PDX1 expresses and improves 160 times in the cell culture that activin is handled, and shows that higher PDX1 induces only to be present in the cell culture that is divided into definitive entoderm (SOX17).

Embodiment 13

Compare more high expression level of FGF-10 promotion PDX1 with single with RA

This embodiment demonstration is compared with independent use RA, and RA and FGF-10 coupling induce higher PDX1 to express.

As among the embodiment in front, hESC was cultivated 4 days under the condition that contains or do not contain activin.At the 4th day, cell was handled with one of following substances: 1 independent μ M RA; 1 μ M RA and FGF-4 or FGF-10 coupling; Or 1 μ M RA and FGF-4 and FGF-10 coupling.RA or RA/FGF handle after 96 hours, with the expression of the quantitative PDX1 of Q-PCR, SOX7 and NFM.

Can induce PDX1 genetic expression to increase by 60 times with retinoic acid treatments hESC culture then with activin earlier.RA adds FGF-4 and can slightly increase PDX1 and express (be approximately use separately RA 3 times) when handling.But, add FGF-10 and vitamin A acid simultaneously, use RA to strengthen 60 times (seeing Figure 37 A) separately to the induction ratio of PDX1.This very strong PDX1 induction ratio is not handled high about 1400 times with activin or RA/FGF.What is interesting is, add the beneficial effect that FGF-4 and FGF-10 have offset FGF-10 simultaneously, the limited PDX1 that has only produced the interpolation that ascribes FGF-4 to strengthens.

Compare with the cell of handling without RA/FGF, coupling RA/FGF-4 or RA/FGF-10 do not increase the marker gene expression (Figure 37 B-C) irrelevant with preceding gut entoderm.

Embodiment 14

(Anterior-Posterior, A-P) position before and after external vitamin A acid dosage influence

Carry out following experiment to determine whether vitamin A acid dosage influences A-P position in the vitro cell culture.

Human embryo stem cell was cultivated 4 days under the condition that contains or do not contain activin.At the 4th day, add 50ng/ml FGF-10 and 0.04 μ M, 0.2 μ M or 1.0 μ M RA in the substratum.Expression by quantitative PDX1 marker gene of Q-PCR and the non-special mark of other anterior intestine entoderms.

Add the vitamin A acid and the 50ng/ml FGF-10 coupling of various dose, induce the differential gene expression pattern relevant with special front and back position pattern.The preferential expression of inducing anterior entoderm mark (HOXA3) of the RA of maximum dose level (1 μ M), and the strongest increase (Figure 38 A-B) that produces PDX1.The expression of gut entoderm mark (CDX1, HOXC6) (seeing Figure 38 C and 41E) during median dose RA (0.2 μ M) induces, and the preferential expression of inducing back gut entoderm mark (HOXA13) (seeing Figure 38 D) of lowest dose level RA (0.04 μ M).RA dosage does not have materially affect (seeing Figure 38 F-G) to the relative expression of neural (SOX1) or neurone (NFM) mark.This embodiment has given prominence to the purposes as morphogen at external use RA, and especially conduct is derived from the morphogen of the entoderm derivative of hESC.

Embodiment 15

Add the expression that B27 strengthens PDX1

By using some factors and cell growth/differentiation condition can influence PDX1 expression in the definitive entoderm.In following experiment, we show that adding B27 can strengthen PDX1 expression in the definitive entoderm cell.

Handled the not differentiation hES cell on the mouse embryo fibroblasts feeder layer, cultivated 4 days with the activin A (100-200ng/ml is dissolved among the 0.5-2%FBS/DMEM/F12) of high dosage, be divided into definitive entoderm with inducing human embryo stem cell.No activin A cultivates impinging upon among the 0.5-2%FBS/DMEM/F12 that does not have activin A.The 4th day, cell culture is handled with following substratum: no activin A (nothing) among the 2%FBS, no activin A (SR) in 2% serum substitute, or the 2%FBS/DMEM/F12 that contains 50ng/ml activin A and 2 μ M RA and 50ng/ml FGF-10 (does not have, + FBS ,+B27) with similar in 2% serum substitute (SR).B27 (Gibco/BRL) through 1/50 the dilution after be added directly to 2%FBS/DMEM/F12 (+B27).Get the double cell sample at every, as above separate total RNA and carry out the Q-PCR analysis.

Figure 39 A-E demonstration is compared with the cell of serum-free culture, and serum-free additive B27 can further induce PDX1 genetic expression, but does not induce the increase of the non-special marker expression of anterior intestine.

Embodiment 16

Using activin B to strengthen PDX1 induces

Present embodiment be presented at cell in vitro cultivate in activin B strengthen of the differentiation of PDX1 negative cells to the PDX1-positive cell.

The not differentiation hES cell of cultivating on the mouse embryo fibroblasts feeder layer with high dosage activin A (50ng/ml) processing in low serum/RPMI 6 days is divided into definitive entoderm with inducing human embryo stem cell.FBS dosage first day was 0%, the second day 0.2%, and 3-6 day 2%.The negative control (NF) that is used for definitive entoderm is cultivated at the 2%FBS/RMPI that does not add activin A.For inducing PDX1 to express, contain the 2%FBS/RPMI of 2 μ M vitamin A acids every kind of culture acceptance in the 6th day.Add the activin A and the activin B of various dose combination in the culture of using activin A to handle in 1-5 days or still keep the independent 50ng/ml of use activin A.Do not add activin A and activin B in the control cultures of no activin A (NF).This RA/ activin is handled and was carried out 3 days, detects the PDX1 genetic expression of double cell sample then with Q-PCR.

Figure 40 A is presented under the condition of 25ng/ml (A25) or 50ng/ml (A50) activin A existence, and the activin B of additive capacity scope 10-50ng/ml (a10, a25 and a50) improves at least 2 times of PDX1 expression than the culture that uses 50ng/ml activin A separately.Increase the increase (Figure 40 B) of not following HNF6 as the PDX1 that adds activin B result, it is this liver and the mark of pancreas that is growing time period.This result shows that with respect to liver the cell proportion that is divided into pancreas increases.

Embodiment 17

Using serum dosage to strengthen PDX1 induces

In whole atomization, the PDX1 that the serum amount of cell culture influences the definitive entoderm cell expresses.Below experiment is presented at hESC serum level in the culture in the atomization of the negative definitive entoderm of PDX1-influences the PDX1 expression of these cells in the further atomization of PDX1-positive entoderm.

The not differentiation hESC that cultivates on the mouse embryo fibroblasts feeder layer with high dosage activin A (100ng/ml) processing in low serum/RPMI 5 days is divided into definitive entoderm with inducing human embryo stem cell.FBS dosage first day was 0.1%, second day 0.5%, 3-5 day 0.5%, 2% or 10%.No activin A contrast (NF) accepts not contain the same FBS/RMPI dosage of activin A.Induced PDX1 to express at the 6th day by adding RA.At 6-7 days, culture was cultivated in the 0.5%FBS/RPMI that contains 2 μ M vitamin A acids, the 8th day 1 μ M, 9-11 days 0.2 μ M.Activin A is reduced to 50ng/ml during retinoic acid treatments, and no activin A contrast (NF) does not still add activin A.

Figure 41 A is presented at the definitive entoderm inductive during 3 days (the 3rd, 4 and 5 day), and FBS dosage has the ability of inducing PDX1 genetic expression during the retinoic acid treatments that continue to change.This does not follow the obvious change of ZIC1 (Figure 41 B) or SOX7 (Figure 41 C) gene expression pattern.

Embodiment 18

The working conditions substratum strengthens PDX1 and induces

We have also studied influences other factors and the growth conditions that definitive entoderm cell PDX1 expresses.Below experiment display condition substratum is to the influence of the negative definitive entoderm cell of PDX1-to the differentiation of PDX1-positive entoderm cell.

The not differentiation hESC that cultivates on the mouse embryo fibroblasts feeder layer with high dosage activin A (100ng/ml) processing in low serum/RPMI 5 days is divided into definitive entoderm with inducing human embryo stem cell.FBS dosage first day was 0.1%, the second day 0.5%, the 3-5 day 2%.

Add RA (being dissolved in the 2%FBS/RPMI that contains 25ng/ml activin A) 4 days in the definitive entoderm culture that after activin A handled 5 days, produces and express endoblastic differentiation to induce to PDX1.A few days ago add 2 μ M RA, the 3rd day 1 μ M, the 4th day 0.5 μ M.Be used for PDX1 inductive minimum medium and be fresh culture (2A25R) or by the substratum of one of four kinds of different cell masses conditioning after 24 hours.The substratum of conditioning (CM) produces by mouse embryo fibroblasts (MEFCM) or with 5 days hESC of one of following three kinds of conditions differentiation: i) 3%FBS/RPMI (CM2), ii) activin A (CM3) or iii) bone morphogenic protein 4 (BMP4) is (CM4).The concentration of adding activin A or BMP4 down at above-mentioned identical FBS dosage (0.2%, 0.5%, 2%) is 100ng/ml.These three kinds different differentiation examples produce three kinds of very different people's cell masses, can carry out the conditioning of PDX1 inducing culture by them.The 3%FBS (NF) that does not add somatomedin produces the foreign cell group who mainly is made up of extraembryonic endoderm, ectoderm and mesoblastema.The culture (A100) that activin A handles produces a high proportion of definitive entoderm, and the culture (B100) that BMP4 handles mainly produces trophectoderm and some extraembryonic endoderms.

Figure 42 A be presented in a few days ago fresh and conditioned medium that RA handles the PDX1 induction phase with.But, to PDX1 expression beginning reduction in fresh culture and the processing of MEF conditioned medium in the 3rd day.The conditioned medium that the hESC of differentiation produces can be kept or further improve PDX1 genetic expression, compares with fresh culture and can improve 3-4 doubly.The hESC-conditioned medium is kept the effect of PDX1 high expression level and is handled further reinforcement in the 4th day at RA, than the high about 6-7 of fresh culture doubly.Figure 42 B display condition substratum is handled and to be caused the CDX1 genetic expression that reduces greatly, and this gene is not partly expressed at the entoderm of expressing PDX1.This shows the conditioned medium processing definitive entoderm of using from the hESC culture of differentiation, can significantly improve PDX1 and express the total purity of entoderm.

Figure 43 shows that PDX1 genetic expression is positive dose response to the amount of the conditioned medium that is used for the definitive entoderm cell.The substratum cumulative volume that adds each culture dish is 5ml, and the conditioned medium of designated volume (as shown in figure 43) is diluted to (A25R) in the fresh culture.It should be noted that only adding the 1ml conditioned medium in the 4ml fresh culture just can induce and keep than single with the higher PDX1 expression level of 5ml fresh culture.This shows that conditioned medium induces PDX1 to express the release of material in conditioned medium that endoblastic beneficial effect depends on certain or some cell, and this or these dosages of substance rely on ground and strengthen PDX1 and express endoblastic generation.

Embodiment 19

Confirmation in conjunction with PDX1 antibody

Help monitoring inducing that PDX1 expresses in the cell mass with PDX1 bonded antibody.This embodiment shows that the rabbit polyclonal IgY antibody of PDX1 can be used for detecting this proteic existence.

In first experiment, the Western engram analysis confirm IgY anti--(antibody of IgY α-PDX1) combines with PDX1 in the cell lysate PDX1.In this analysis, compared the combining of total lysate of IgY α-PDX1 antibody and 50 μ g MDX12 cell after 24 hours from MDX12 human fibroblasts or transfection PDX1 expression vector.Cell lysate separates with SDS-PAGE, and electricity forwards on the film, resists-IgY (Rb α-IgY) two anti-detections with IgY α-PDX1 one anti-antiserum(antisera) and alkaline phosphatase link coupled rabbit subsequently.Different dilution one is anti-and two anti-ly be used for different strip films: A (anti-a 500 * dilution, two anti-10,000 * as to dilute) by following combination, B (2,000 *, 10,000 *), C (500 *, 40,000 *), D (2,000 *, 40,000 *), E (8,000 *, 40,000 *).

In all antibody combinations, the cell of transfection PDX1 expression vector all detects combination.Just detect combination when in the inoblast of untransfected (PDX1-feminine gender), only using one anti-, two anti-(the combination A) of maximum concentration at the same time.This is non-specific combination, because all detect the extra band that a molecular weight is slightly larger than PDX1 in the inoblast of transfection and untransfected.

In second experiment, with immunocytochemistry detect rabbit anti--(polyclonal antibody of Rb α-PDX1) combines with PDX1's PDX1.For producing a kind of PDX1 express cell to this experiment, MS1-V cell (ATCC#CRL-2460) (is made up with pEGFP-N1, Clontech) by transient transfection PDX1-EGFP expression vector.Cells transfected is used Rb α-PDX1 and α-EGFP antiserum(antisera) mark then.Can be observed cells transfected with EGFP fluorescence and use Cy5 link coupled two anti-α-EGFP immunocytochemistries.Use α-Rb Cy3-link coupled two to resist and to see the PDX1 immunofluorescence.

The combination of Rb α-PDX1 and α-EGFP antibody is expressed location altogether to GPF.

Embodiment 20

The immunocytochemistry of people's pancreas tissue

This embodiment shows that the PDX1 specific antibody can be used for immunocytochemistry identifier PDX1-positive cell.

In first experiment, section is dyeed detecting Regular Insulin to paraffin-embedded people's pancreas, and (Gp α-Ins) one is anti-, then the anti-cavy of Cy2 link coupled dog of 1/100 dilution (two anti-the carrying out of D α-Gp) for the cavy synalbumin by 1/200 dilution earlier.In second experiment,, anti-to identical paraffin-embedded people's pancreas section statining by the IgY α-PDX1 one of 1/4000 dilution earlier to detect PDX1, AF555 link coupled Rb α-IgY two anti-carrying out of 1/300 dilution then.The picture that to collect from first and second experiment is integrated.In the 3rd experiment, also dye with DAPI through the painted cell of IgY α-PDX1.

Analysis to the section of people's pancreas shows that pancreas islet dyeing is darker.Although pancreas islet (the Regular Insulin positive) shows the strongest PDX1 signal, acinar tissue (Regular Insulin feminine gender) also shows faint dyeing.DAPI and PDX1 dye altogether and show that PDX1 mainly but be not to be positioned nuclear fully.

Embodiment 21

PDX1 immunoprecipitation through the cell of retinoic acid treatments

For further confirming in the presence of RA, to exist PDX1 to express in the definitive entoderm cell of differentiation, and the definitive entoderm cell that does not break up under the RA condition do not have PDX1 and expresses, with rabbit anti--PDX1 (the antibody mediated immunity precipitation of Rb α-PDX1) from RA differentiation with PDX1 undifferentiated definitive entoderm cell.Use IgY α-PDX1 antibody to detect the RA of immunoprecipitation by the Western trace.

For obtaining to be used for the definitive entoderm cell that does not break up and break up of immunoprecipitation, hESC handled 5 days with the low serum that contains 100ng/ml activin A, handled 2 days 1 μ M 1 day, 0.2 μ M 1 day (gut entoderm before the PDX1-positive) then with 50ng/ml activin A and 2 μ M alltrans RA.The preparation transfection cell lysate of MS1-V cell (ATCC#CRL-2460) of PDX1 expression vector as positive control.Interpolation Rb α-PDX1 and rabbit specificity two are anti-with immunoprecipitation PDX1 in each lysate.Centrifugal collecting precipitation.Immunoprecipitate is dissolved in the damping fluid that contains SDS, separates through polyacrylamide gel, and the albumen electricity is forwarded on the film, resists with IgY α-PDX1 one link coupled Rb α-IgY two anti-and subsequently and detects.

The collect immunoprecipitate PDX1 albumen of from MS1-V positive control cell and the 8th day cell of (d8 swimming lane, beginning RA handled back 3 days) and the 9th day (the d9 swimming lane begins RA and handled back 4 days) all shows the positive (Figure 44).Throw out from undifferentiated definitive entoderm cell (activin A handles among cell-Figure 44 of the 5th day and is designated as (A)) and undifferentiated hESC (being designated as (NF) among undressed the 5th day cell-Figure 44) shows negative to PDX1.

Embodiment 22

The generation of PDX1 promotor-EGFP transgenosis hESC system

Be to use PDX1 mark isolated cell, we are with effable reporter gene genetic marker PDX1-positive anterior intestine endoderm cell.Present embodiment is described to make up and is comprised the carrier of reporting box, and this report box comprises the reporter gene that is subjected to the control of PDX1 regulatory region.Present embodiment has also been described the preparation transfection, and the cell of this carrier (for example human embryo stem cell) and this report box are incorporated into genomic cell.

By the GFP reporter gene is placed under the control in PDX1 generegulation district (promotor), made up definitive entoderm clone with the expression PDX1 of reporter gene genetic marker.At first, personnel selection PDX1 control region (Genebank registration number AF192496) is replaced the CMV promotor of pEGFP-N1 (Clontech) carrier, make up EGFP and express by the initial carrier of people PDX1 gene promoter, wherein people PDX1 control region comprises the nucleotide sequence of 85 base pairs (bp) to the downstream from the about 4.4kb in PDX1 transcription initiation site upstream (kilobase to).This zone comprises the characteristic controlling element of PDX1 gene, is enough to show in transgenic mice normal PDX1 expression pattern.In the carrier that makes up, the initial EFGP of PDX1 promotor expresses.In some experiments, this carrier is transfected to advance hESC.

From above carrier excision PDX1 promotor/EGFP box, subclone is gone into one and is selected carrier, and this carrier comprises the neomycin phosphotransferase gene that is subjected to the control of phosphoglyceric kinase-1 promotor.Selecting the box both sides is flp recombinase recognition sites, to remove this box.Make this select the carrier linearizing, dye the back and import hESC with the standard liposomal body method.The G418 screening separated the undifferentiated transgenosis hESC clone of amplification after 10-14 days.

Embodiment 23

The endoblastic separation of the positive anterior intestine of PDX1-

The hESC that following examples proof comprises PDX1 promotor/EGFP box can be divided into the PDX1-positive entoderm cell, uses fluorescence amplifying cell separator (FACS) to separate then.

PDX1 promotor/EGFP transgenosis hESC broke up 5 days in the substratum that contains activin A, broke up 2 days in the substratum that contains activin A and RA then.The cell of differentiation is collected with trysinization, directly is added among RNA lysis buffer or the PBS after Becton Dickinson FACS Diva sorting.Take out single viable cell sample, and not with EGFP (Live) circle choosing (gating), and single viable cell circle selects into the EGFP positive (GFP) and GFP feminine gender (Neg) group.In an experiment, the positive part of EGFP is assigned among the group of two identical sizes according to fluorescence intensity (Hi and Lo).

After the sorting, with Q-PCR and immunocytochemical assay cell mass.Preparing RNA with Qiagen RNeasy post transfers cDNA then to and is used for Q-PCR and analyzes.Q-PCR is undertaken by preceding method.For carrying out immunocytochemical assay, be added to behind the cell sorting among the PBS, in 4% Paraformaldehyde 96, fix 10 minutes, with the centrifugal cell adhesion that makes of cytospin in slide glass.One of cytokeratin 19 (KRT19) resists from Chemicon; One of hepatocyte neclear factor 3 β (HNF3 β) resist from Santa Cruz; Glucose transporter 2 (GLUT2) is from R﹠amp; D system.Use two of suitable coupling FITC (green) or rhodamine (redness) to resist and detect an anti-combination.

The representative FACS sorting of noble cells as shown in figure 45.Isolating PDX1-positive cell ratio is about 7% among this embodiment, changes between 1-20% according to differentiation efficiency.

The cell of sorting is further used for Q-PCR and analyzes.The cell of differentiation shows that EGFP fluorescence is relevant with endogenous PDX1 genetic expression.Compare with no fluorocyte, the PDX1 expression level of EGFP positive cell increases about more than 20 times (Figure 46).Separating of high and low EGFP intensity cell shows EGFP expression level relevant with the PDX1 expression level (Figure 47).Except the PDX1 analysis of markers, in the cell of sorting, utilize Q-PCR to analyze several genes of expressing at the pancreas entoderm.The product of these marker genes (NKX2.2, GLUT2, KRT19, HNF4 α and HNF3 β) obtains enrichment (Figure 48 A-E) at EGFP male portion branch.On the contrary, neural mark ZIC1 and GFAP not enrichment in the EGFP of sorting express cell (Figure 49 A and B).

Use immunocytochemistry to find out, in fact all expressing K RT19 and GLUT2 of the separative PDX1-positive cell of institute.This result indicates that the pancreas entoderm is a cell.Through the many this cells of antibody staining also is HNF3 β male.

Method described herein, composition and device are the representatives of present preferred embodiment, play an exemplary role, and are not in order to limit the scope of the invention.In being included in the scope of spirit of the present invention, those skilled in the art can make it and changing or as other purposes, these change and other purposes also in open scope of the present invention.Correspondingly, it will be apparent for a person skilled in the art that do not depart from the scope of the present invention with spirit under, can make different substitutions and modifications to invention disclosed herein.

Be used in phrase in appended claims and the whole disclosure " mainly by ... form " implication be to comprise any key element behind the phrase and be limited to other to disclose the key element that special activity of listed key element or function do not play interference or facilitate effect.Therefore, phrase " mainly by ... form " show that listed key element is essential or compulsory, but other key elements are that optionally being present in of they do not depend on its activity that whether influences listed key element or function.

Reference

Present patent application has been quoted lot of documents and patent.

Provided the complete of some documents in the literary composition and quoted, only listed author and age during other reference citations, it is complete quote as follows:

Alexander, J., Rothenberg, M., Henry, G.L., and Stainier, D.Y. (1999) .Casanova plays an early and essential role in endoderm formationin zebrafish (Casanova plays a part early stage and important in the zebra fish entoderm forms) .Dev Biol 215,343-357.

Alexander, J., and Stainier, D.Y. (1999) .A molecular pathwayleading to endoderm formation in zebrafish (molecular pathways that causes entoderm to form in the zebra fish) .Curr Biol 9,1147-1157.

Aoki, T.O., Mathieu, J., Saint-Etienne, L., Rebagliati, M.R., Peyrieras, N., and Rosa, F.M. (2002) .Regulation of nodal signalling andmesendoderm formation by TARAM-A, a TGFbeta-related type Ireceptor (regulating the nodal signal and mesendoderm forms) .Dev Biol 241,273-288. with TGF β-correlation type I acceptor TARAM-A

Beck, S., Le Good, J.A., Guzman, M., Ben Haim, N., Roy, K., Beermann, F., and Constam, D.B. (2002) .Extra-embryonic proteasesregulate Nodal signalling during gastrulation (the embryo exoproteinase is regulated the Nodal signal in the gastrula formation phase) .Nat Cell Biol 4,981-985.

Beddington, R.S., Rashbass, P., and Wilson, V. (1992) .Brachyury-agene affecting mouse gastrulation and early organogenesis (one kind of Brachyury-influences the mouse gastrula and forms and organogenetic gene) .Dev Suppl, 157-165.

Bongso, A., Fong, C.Y., Ng, S.C, and Ratnam, S. (1994) .Isolationand culture of inner cell mass cells from human blastocysts (separating and cultivation inner cell mass cell) .Hum Reprod 9,2110-2117. from people's blastocyst

Chang, H., Brown, C.W., and Matzuk, M.M. (2002) .Geneticanalysis of the mammalian transforming growth factor-betasuperfamily (genetic analysis of Mammals transforming growth factor-beta superfamily) .Endocr Rev23,787-823.

Conlon, F.L., Lyons, K.M., Takaesu, N., Barth, K.S., Kispert, A., Herrmann, B., and Robertson, E.J. (1994) .A primary requirement fornodal in the formation and maintenance of the primitive streak in themouse (former of mouse form and keep in mainly need nodal) .Development 120,1919-1928.

Dougan, S.T., Warga, R.M., Kane, D.A., Schier, A.F., and Talbot, W.S. (2003) .The role of the zebrafish nodal-related genes squint andcyclops in patterning of mesendoderm (zebra fish nodal-genes involved squint and the effect of cyclops in mesendoderm forms) .Development 130,1837-1851.

Feldman, B., Gates, M.A., Egan, E.S., Dougan, S.T., Rennebeck, G., Sirotkin, H.L, Schier, A.F., and Talbot, W.S. (1998) .Zebrafish organizerdevelopment and germ-layer formation require nodal-related signals (zebra fish tissue development and germinal layer form needs the relevant signal of nodal-) .Nature 395,181-185.

Feng, Y., Broder, C.C, Kennedy, P.E., and Berger, E.A. (1996) .HIV-I entry cofactor:functional cDNA cloning of a seven-transmembrane, (HIV-1 enters cofactor to G protein-coupled receptor: stride membrane structure seven times, the functional cDNA clone of g protein coupled receptor) .Science 272,872-877.

Futaki, S., Hayashi, Y., Yamashita, M., Yagi, K., Bono, H., Hayashizaki, Y., Okazaki, Y., and Sekiguchi, (basement membrane components continues the molecular basis of formation to K. (2003) .Molecular basisof constitutive production of basement membrane components:Geneexpression profiles of engelbreth-holm-swarm tumor and F9 embryonalcarcinoma cells: the .J BiolChem. gene expression pattern of engelbreth-holm-swarm tumour and F9 embryo cells)

Grapin-Botton, A., and Melton, D.A. (2000) .Endodermdevelopment:from patterning to organogenesis (entoderm is grown: be formed into tissue from pattern and take place) .Trends Genet 16,124-130.

Harris, T.M., and Childs, G. (2002) .Global gene expression patternsduring differentiation of F9 embryonal careinoma cells into parietalendoderm (F9 embryo cells in body wall entoderm atomization overall gene expression pattern) .Funct Integr Genomics 2,105-119.

Hogan, B.L. (1996) .Bone morphogenetic proteins indevelopment (developmental bone morphogenetic protein) .Curr Opin Genet Dev 6,432-438.

Hogan, B.L. (1997) .Pluripotent embryonic cells and methods ofmaking same (pluripotent embryonic cell and the method for preparing the pluripotent embryonic cell) (U.S.A., Vanderbilt University).

Howe, C.C, Overton, G.C, Sawicki, J., Solter, D., Stein, P., andStrickland, S. (1988) .Expression of SPARC/osteonectin transcript inmurine embryos and gonads (expression of SPARC/ osteonectin transcript in mice embryonic and the sexual gland) .Differentiation 37,20-25.

Hudson, C, Clements, D., Friday, R.V., Stott, D., and Woodland, H.R. (1997) .Xsox17alpha and-beta mediate endoderm formation inXenopus (in the African toad Xsox17 α and-β regulates entoderm and form) .Cell 91,397-405.

Imada, M., Imada, S., Iwasaki, H., Kume, A., Yamaguchi, H., andMoore, E.E. (1987) .Fetomodulin:marker surface protein of fetaldevelopment which is modulatable by cyclic AMP (fetomodulin: the mark surface protein of fetation is regulated by ring-type AMP) .Dev Biol 122,483-491.

Kanai-Azuma, M., Kanai, Y., Gad, J.M., Tajima, Y., Taya, C, Kurohmaru, M., Sanai, Y., Yonekawa, H., Yazaki, K., Tam, P.P., andHayashi, Y. (2002) .Depletion of definitive gut endoderm in Sox 17-nullmutant mice (disappearance of setting gut entoderm in the no Sox17 mutant mice) .Development129,2367-2379.

Katoh, M. (2002) .Expression of human SOX7 in normal tissues andtumors (expression of people SOX7 in healthy tissues and the tumour) .Int J MoI Med 9,363-368.

Kikuchi, Y., Agathon, A., Alexander, J., Thisse, C, Waldron, S., Yelon, D., Thisse, B., and Stainier, D.Y. (2001) .casanova encodes a novelSox-related protein necessary and sufficient for early endoderm formationin zebrafish (early stage entoderm forms required albumen in the relevant zebra fish of new Sox of casanova coding) .Genes Dev 15,1493-1505.

Kim, C.H., and Broxmeyer, H.E. (1999) .Chemokines:signal lampsfor trafficking of T and B cells for development and effector function (chemokine: grow and the T of effector function and the signal lamp that the B cell transports) .J Leukoc Biol 65,6-15.

Kimelman, D., and Griffin, K.J. (2000) .Vertebrate mesendoderminduction and patterning (inducing and forming of vertebrates mesendoderm) .Curr OpinGenet Dev 10,350-356.

Kubo A, Shinozaki K, Shannon JM, Kouskoff V, Kennedy M, Woo S, Fehling HJ, Keller G. (2004) the Development of definitive endoderm fromembryonic stem cells in culture growth of the definitive entoderm of embryonic stem cell (in the culture from) .Development.131,1651-62.

Kumar, A., Novoselov, V., Celeste, A.J., Wolfman, N.M., ten Dijke, P., and Kuehn, M.R. (2001) .Nodal signaling uses activin andtransforming growth factor-beta receptor-regulated Smads (Smad that the Nodal signal uses activin and transforming growth factor to regulate) .J Biol Chem 276,656-661.

Labosky, P.A., Barlow, D.P., and Hogan, B.L. (1994a) .Embryonicgerm cell lines and their derivation from mouse primordial germ cells (embryonic genital cell line and be derived from deriving of mouse archeocyte) .Ciba Found Symp 182,157-168; Discussion 168-178.

Labosky, P.A., Barlow, D.P., and Hogan, B.L. (1994b) .Mouseembryonic germ (EG) cell lines:transmission through the germline anddifferences in the methylation imprint of insulin-like growth factor 2receptor (Igf2r) gene compared with embryonic stem (ES) cell lines (mice embryonic reproduction (EG) clone: by kind be to transmit and rhIGF-1 2 acceptors (Igf2r) gene and embryonic stem cell (ES) are the difference of mark of methylating) .Development 120,3197-3204.

Lickert, H., Kutsch, S., Kanzler, B., Tamai, Y., Taketo, M.M., andKemler, R. (2002) .Formation of multiple hearts in mice followingdeletion of beta-catenin in the embryonic endoderm (the β catenin is removed the formation of the multiple heart in back in the mice embryonic entoderm) .Dev Cell 3,171-181.

Lu, C.C, Brennan, J., and Robertson, E.J. (gastrula forms (2001) .From fertilizationto gastrulation:axis formation in the mouse embryo from being fertilized: the formation of mice embryonic axis) .Curr Opin Genet Dev 11,384-392.

Ma, Q., Jones, D., and Springer, T.A. (1999) .The chemokinereceptor CXCR4 is required for the retention of B lineage andgranulocytic precursors within the bone marrow microenvironment (B system and keeping of granulocyte precursor need Chemokine Receptors CXCR4 in the bone marrow microenvironment) .Immunity 10,463-471.

McGrath KE, Koniski AD, Maltby KM, McGann JK, Palis J. (1999) Embryonic expression and function of the chemokine SDF-1 and itsreceptor, CXCR4 (embryo of chemokine SDF-1 and its receptor CXCR 4 expresses and function) .Dev Biol.213,442-56.

Miyazono, K., Kusanagi, K., and Inoue, H. (2001) .Divergence andconvergence of TGF-beta/BMP signaling (diversity and the convergence of TGF-β/BMP signal) .J Cell Physiol 187,265-276.

Nagasawa, T., Hirota, S., Tachibana, K., Takakura, N., Nishikawa, S., Kitamura, Y., Yoshida, N., Kikutani, H., and Kishimoto, T. (1996) .Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in micelacking the CXC chemokine PBSF/SDF-1 (the B-cell lymphocyte generates and the Myelopoietic defective of marrow in the mouse of CXC chemokine PBSF/SDF-1 disappearance) .Nature382,635-638.

Niwa, H. (2001) .Molecular mechanism to maintain stem cellrenewal of ES cells (molecule mechanism that keeps the stem cell renewal of ES cell) .Cen StructFunct 26,137-148.

Ogura, H., Aruga, J., and Mikoshiba, K. (2001) .Behavioralabnormalities of Zicl and Zic2 mutant mice:implications as models forhuman neurological disorders (dystropy of Zic1 and Zic2 mutant mice :) .Behav Genet 31 as the model of people's nervous dysfunction, 317-324.

Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A., and Bongso, A. (2000) .Embryonic stem cell lines from human blastocysts:somaticdifferentiation in vitro is (from the embryonic stem cell line of people's blastocyst: external somatocyte differentiation) .Nat Biotechnol 18,399-404.

Rodaway, A., and Patient, R. (2001) .Mesendoderm.an ancient germlayer? (mesendoderm, an ancient germinal layer?) Cell 105,169-172.

Rodaway, A., Takeda, H., Koshida, S., Broadbent, J., Price, B., Smith, J.C, Patient, R., and Holder, N. (1999) .Induction of the mesendoderm inthe zebrafish germ ring by yolk cell-derived TGF-beta family signals anddiserimination of mesoderm and endoderm by FGF (yolk cell deutero-TGF-beta family signal to zebra fish germ ring mesendoderm induce and with FGF to mesoderm and endoblastic discriminating) .Development 126,3067-3078.

Rohr, K.B., Schulte-Merker, S., and Tautz, D. (1999) .Zebrafish zic1expression in brain and somites is affected by BMP and hedgehogsignalling (zic1 in zebra fish brain and the body segment expresses and is subjected to BMP and hedgehog effect of signals) .Mech Dev 85,147-159.

Schier, A.F. (2003) .Nodal signaling in vertebrate development (the developmental Nodal signal of vertebrates) .Annu Rev Cell Dev Biol 19,589-621.

Schoenwolf, G.C, and Smith, J.L. (2000) .Gastrulation and earlymesodermal patterning in vertebrates (gastrula forms and early stage mesoderm formation pattern in the vertebrates) .Methods MoI Biol 135,113-125.

Shamblott, M.J., Axelman, J., Wang, S., Bugg, E.M., Littlefield, J.W., Donovan, P.J., Blumenthal, P.D., Huggins, G.R., and Gearhart, J.D. (1998) .Derivation of pluripotent stem cells from cultured humanprimordial germ cells (from the derivation of the multipotential stem cell of people's archeocyte of cultivating) .Proc Natl Acad Sci USA 95,13726-13731.

Shapiro, A.M., Lakey, J.R., Ryan, E.A., Korbutt, G.S., Toth, E., Wamock, G.L., Kneteman, N.M., and Rajotte, R.V. (2000) .Islettransplantation in seven patients with type 1 diabetes mellitus using aglucocorticoid-free immunosuppressive regimen (using the pancreatic islets transplantation do not contain the glucocorticosteroid immunosuppressive therapy among the 7 routine type 1 diabetes patients) .N Engl J Med 343,230-238.

Shapiro, A.M., Ryan, E.A., and Lakey, J.R. (2001a) .Pancreaticislet transplantation in the treatment ofdiabetes mellitus (pancreatic islets transplantation in treating diabetes) .Best Pract Res Clin Endocrinol Metab 15,241-264.

Shapiro, J., Ryan, E., Warnock, G.L., Kneteman, N.M., Lakey, J., Korbutt, G.S., and Rajotte, R.V. (2001b) .Could fewer islet cells betransplanted in type 1 diabetes? can Insulin independence should be dominantforce in islet transplantation (transplant in the type 1 diabetes by a small amount of islet cells? the Regular Insulin dependent/non-dependent is the principal element in the pancreatic islets transplantation) .Bmj 322,861.

Shiozawa, M., Hiraoka, Y., Komatsu, N., Ogawa, M., Sakai, Y., andAiso, S. (1996) .Cloning and characterization of Xenopus laevis xSox7cDNA (clone and the evaluation of African toad laevis xSox7 cDNA) .Biochim BiophysActa 1309,13-76.

Smith, J. (1997) .Brachyury and the T-box genes (Brachyury and T-box gene) .Curr Opin Genet Dev 7,474-480.

Smith, J.C, Armes, N.A., Conlon, F.L., Tada, M., Umbhauer, M., and Weston, K.M. (1997) .Upstream and downstream from Brachyury, the agene required for vertebrate mesoderm formation (upstream and downstream of Brachyury, a kind of vertebrates mesoderm forms the gene that needs) .Cold Spring Harb SympQuant Biol 62,337-346.

Takash, W., Canizares, J., Bonneaud, N., Poulat, F., Mattei, M.G., Jay, P., and Berta, P. (2001) .SOX7 transcription factor:sequence, chromosomal localisation, expression, transactivation and interferencewith Wnt signalling (SOX7 transcription factor: sequence, chromosomal localization, express, the commentaries on classics of Wnt signal is lived and is disturbed) .Nucleic Acids Res 29,4274-4283.

Taniguchi, K., Hiraoka, Y., Ogawa, M., Sakai, Y., Kido, S., and Aiso, S. (1999) .Isolation and characterization of a mouse SRY-related cDNA, the mSox7 (cDNA that mouse SRY-is relevant, the separation of mSox7 and evaluation) .BiochimBiophys Acta 1445,225-231.

Technau, U. (2001) .Brachyury, the blastopore and the evolution ofthe mesoderm (Brachyury, blastopore and mesoblastic evolution) .Bioessays 23,788-794.

Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. (1998) .Embryonic stemcell lines derived from human blastocysts (embryonic stem cell line of derived from human blastocyst) .Science 282,1145-1147.

Tremblay, K.D., Hoodless, P.A., Bikoff, E.K., and Robertson, E.J. (2000) .Formation of the definitive endoderm in mouse is a Smad2-dependent process (formation of mouse definitive entoderm is the process that Smad2 relies on) .Development 127,3079-3090.

Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., and Speleman, F. (2002) .Accurate normalization ofreal-time quantitative RT-PCR data by geometric averaging of multipleinternal control genes (geometric mean by multiple inherent crt gene is accurately proofreaied and correct real-time quantitative RT-PCR) .Genome Biol 3, RESEARCH0034.

Varlet, I., Collignon, J., and Robertson, E.J. (1997) .nodal expressionin the primitive endoderm is required for specification of the anterior axisduring mouse gastrulation (mouse gastrula form phase front axle specificityization need the expression of nodal in primitive endoderm) .Development 124,1033-1044.

Vincent, S.D., Dunn, N.R., Hayashi, S., Norris, D.P., and Robertson, E.J. (2003) .Cell fate decisions within the mouse organizer are governedby graded Nodal signals (in the mouse organizer cell fate decision be subjected to gradient Nodal signal control) .Genes Dev 17,1646-1662.

Weiler-Guettler, H., Aird, W.C, Rayburn, H., Husain, M., andRosenberg, R.D. (1996) .Developmentally regulated gene expression ofthrombomodulin in postimplantation mouse embryos (transplanting the genetic expression of growing the thrombomodulin of regulating in the mice embryonic of back) .Development 122,2271-2281.

Weiler-Guettler, H., Yu, K., Soff, G., Gudas, L.J., and Rosenberg, R.D. (1992) .Thrombomodulin gene regulation by cAMP and retinoic acid inF9 embryonal carcinoma cells (cAMP and vitamin A acid are regulated the thrombomodulin gene in the F9 embryo cells) .Proceedings Of The National Academy Of SciencesOf The United States Of America 89,2155-2159.

Wells, J.M., and Melton, D.A. (1999) .Vertebrate endodermdevelopment (growth of vertebrates entoderm) .Annu Rev Cell Dev Biol 15,393-410.

Wells, J.M., and Melton, D.A. (2000) .Early mouse endoderm ispatterned by soluble factors from adjacent germ layers (early stage mouse entoderm is by the solvable factor modeization from contiguous germinal layer) .Development 127,1563-1572.

Willison, K. (1990) .The mouse Brachyury gene and mesodermformation (mouse Brachyury gene and mesoderm form) .Trends Genet 6,104-105.

Zhao, G.Q. (2003) .Consequences of knocking out BMP signaling inthe mouse (knocking out the result of BMP signal in the mouse) .Genesis 35,43-56.

Zhou, X., Sasaki, H., Lowe, L., Hogan, B.L., and Kuehn, M.R. (1993) .Nodal is a novel TGF-beta-like gene expressed in the mouse nodeduring gastrulation (nodal is that gastrula forms the new TGF β sample gene that the phase expresses at the mouse joint) .Nature 361,543-547.

Sequence table

＜110〉Novocell Inc. (Novocell, Inc.)

Kevin Alan Da Muer (D ' Amour, Kevin Allen)

Alan D A Gunike (Agulnick, Alan D.)

Susan Ai Lize (Eliazer, Susan)

Rahm Emanuel E Bethe lattice (Baetge, Emmanuel E.)

＜120〉entoderm of expression PDX1

<130>CYTHERA.043VPC

<150>US?11/021618

<151>2004-12-23

<150>US?60/587942

<151>2004-07-14

<150>US?60/586566

<151>2004-07-09

<150>US?60/566293

<151>2004-04-24

<160>2

<170>FastSEQ?for?Windows?Version?4.0

<210>1

<211>1245

<212>DNA

<213>Homo?sapiens

<400>1

atgagcagcc?cggatgcggg?atacgccagt?gacgaccaga?gccagaccca?gagcgcgctg?60

cccgcggtga?tggccgggct?gggcccctgc?ccctgggccg?agtcgctgag?ccccatcggg?120

gacatgaagg?tgaagggcga?ggcgccggcg?aacagcggag?caccggccgg?ggccgcgggc?180

cgagccaagg?gcgagtcccg?tatccggcgg?ccgatgaacg?ctttcatggt?gtgggctaag?240

gacgagcgca?agcggctggc?gcagcagaat?ccagacctgc?acaacgccga?gttgagcaag?300

atgctgggca?agtcgtggaa?ggcgctgacg?ctggcggaga?agcggccctt?cgtggaggag?360

gcagagcggc?tgcgcgtgca?gcacatgcag?gaccacccca?actacaagta?ccggccgcgg?420

cggcgcaagc?aggtgaagcg?gctgaagcgg?gtggagggcg?gcttcctgca?cggcctggct?480

gagccgcagg?cggccgcgct?gggccccgag?ggcggccgcg?tggccatgga?cggcctgggc?540

ctccagttcc?ccgagcaggg?cttccccgcc?ggcccgccgc?tgctgcctcc?gcacatgggc?600

ggccactacc?gcgactgcca?gagtctgggc?gcgcctccgc?tcgacggcta?cccgttgccc?660

acgcccgaca?cgtccccgct?ggacggcgtg?gaccccgacc?cggctttctt?cgccgccccg?720

atgcccgggg?actgcccggc?ggccggcacc?tacagctacg?cgcaggtctc?ggactacgct?780

ggccccccgg?agcctcccgc?cggtcccatg?cacccccgac?tcggcccaga?gcccgcgggt?840

ccctcgattc?cgggcctcct?ggcgccaccc?agcgcccttc?acgtgtacta?cggcgcgatg?900

ggctcgcccg?gggcgggcgg?cgggcgcggc?ttccagatgc?agccgcaaca?ccagcaccag?960

caccagcacc?agcaccaccc?cccgggcccc?ggacagccgt?cgccccctcc?ggaggcactg?1020

ccctgccggg?acggcacgga?ccccagtcag?cccgccgagc?tcctcgggga?ggtggaccgc?1080

acggaatttg?aacagtatct?gcacttcgtg?tgcaagcctg?agatgggcct?cccctaccag?1140

gggcatgact?ccggtgtgaa?tctccccgac?agccacgggg?ccatttcctc?ggtggtgtcc?1200

gacgccagct?ccgcggtata?ttactgcaac?tatcctgacg?tgtga 1245

<210>2

<211>414

<212>PRT

<213>Homo?sapiens

<400>2

Met?Ser?Ser?Pro?Asp?Ala?Gly?Tyr?Ala?Ser?Asp?Asp?Gln?Ser?Gln?Thr

1 5 10 15

Gln?Ser?Ala?Leu?Pro?Ala?Val?Met?Ala?Gly?Leu?Gly?Pro?Cys?Pro?Trp

20 25 30

Ala?Glu?Ser?Leu?Ser?Pro?Ile?Gly?Asp?Met?Lys?Val?Lys?Gly?Glu?Ala

35 40 45

Pro?Ala?Asn?Ser?Gly?Ala?Pro?Ala?Gly?Ala?Ala?Gly?Arg?Ala?Lys?Gly

50 55 60

Glu?Ser?Arg?Ile?Arg?Arg?Pro?Met?Asn?Ala?Phe?Met?Val?Trp?Ala?Lys

65 70 75 80

Asp?Glu?Arg?Lys?Arg?Leu?Ala?Gln?Gln?Asn?Pro?Asp?Leu?His?Asn?Ala

85 90 95

Glu?Leu?Ser?Lys?Met?Leu?Gly?Lys?Ser?Trp?Lys?Ala?Leu?Thr?Leu?Ala

100 105 110

Glu?Lys?Arg?Pro?Phe?Val?Glu?Glu?Ala?Glu?Arg?Leu?Arg?Val?Gln?His

115 120 125

Met?Gln?Asp?His?Pro?Asn?Tyr?Lys?Tyr?Arg?Pro?Arg?Arg?Arg?Lys?Gln

130 135 140

Val?Lys?Arg?Leu?Lys?Arg?Val?Glu?Gly?Gly?Phe?Leu?His?Gly?Leu?Ala

145 150 155 160

Glu?Pro?Gln?Ala?Ala?Ala?Leu?Gly?Pro?Glu?Gly?Gly?Arg?Val?Ala?Met

165 170 175

Asp?Gly?Leu?Gly?Leu?Gln?Phe?Pro?Glu?Gln?Gly?Phe?Pro?Ala?Gly?Pro

180 185 190

Pro?Leu?Leu?Pro?Pro?His?Met?Gly?Gly?His?Tyr?Arg?Asp?Cys?Gln?Ser

195 200 205

Leu?Gly?Ala?Pro?Pro?Leu?Asp?Gly?Tyr?Pro?Leu?Pro?Thr?Pro?Asp?Thr

210 215 220

Ser?Pro?Leu?Asp?Gly?Val?Asp?Pro?Asp?Pro?Ala?Phe?Phe?Ala?Ala?Pro

225 230 235 240

Met?Pro?Gly?Asp?Cys?Pro?Ala?Ala?Gly?Thr?Tyr?Ser?Tyr?Ala?Gln?Val

245 250 255

Ser?Asp?Tyr?Ala?Gly?Pro?Pro?Glu?Pro?Pro?Ala?Gly?Pro?Met?His?Pro

260 265 270

Arg?Leu?Gly?Pro?Glu?Pro?Ala?Gly?Pro?Ser?Ile?Pro?Gly?Leu?Leu?Ala

275 280 285

Pro?Pro?Ser?Ala?Leu?His?Val?Tyr?Tyr?Gly?Ala?Met?Gly?Ser?Pro?Gly

290 295 300

Ala?Gly?Gly?Gly?Arg?Gly?Phe?Gln?Met?Gln?Pro?Gln?His?Gln?His?Gln

305 310 315 320

His?Gln?His?Gln?His?His?Pro?Pro?Gly?Pro?Gly?Gln?Pro?Ser?Pro?Pro

325 330 335

Pro?Glu?Ala?Leu?Pro?Cys?Arg?Asp?Gly?Thr?Asp?Pro?Ser?Gln?Pro?Ala

340 345 350

Glu?Leu?Leu?Gly?Glu?Val?Asp?Arg?Thr?Glu?Phe?Glu?Gln?Tyr?Leu?His

355 360 365

Phe?Val?Cys?Lys?Pro?Glu?Met?Gly?Leu?Pro?Tyr?Gln?Gly?His?Asp?Ser

370 375 380

Gly?Val?Asn?Leu?Pro?Asp?Ser?His?GlV?Ala?Ile?Ser?Ser?Val?Val?Ser

385 390 395 400

Asp?Ala?Ser?Ser?Ala?Val?Tyr?Tyr?Cys?Asn?Tyr?Pro?Asp?Val

405 410

Claims (109)

1. the cell culture that comprises people's cell, be pancreas-positive anterior intestine endoderm cell of the duodenum homology frame factor-1 (PDX1) at least about described people's cell of 2% wherein, described PDX1-is positive, and the anterior intestine endoderm cell is the multipotential cell that can be divided into derived from intestinal tube forward cell, tissue or organ.

2. cell culture as claimed in claim 1 is the positive anterior intestine endoderm cell of PDX1-at least about described people's cell of 5% wherein.

3. cell culture as claimed in claim 1 is the positive anterior intestine endoderm cell of PDX1-at least about described people's cell of 10% wherein.

4. cell culture as claimed in claim 1 is the positive anterior intestine endoderm cell of PDX1-at least about described people's cell of 25% wherein.

5. wherein there are people's feeder cell in cell culture as claimed in claim 1 in described culture, wherein except described people's feeder cell, the people's cell at least about 2% is the positive anterior intestine endoderm cell of PDX1-.

6. cell culture as claimed in claim 1, wherein said PDX1-is positive, and the anterior intestine endoderm cell expresses homology frame A13 (HOXA13) gene.

7. cell culture as claimed in claim 1, wherein said PDX1-is positive, and the anterior intestine endoderm cell expresses homology frame C6 (HOXC6) gene.

8. cell culture as claimed in claim 1, wherein said PDX1-is positive, and the anterior intestine endoderm cell expresses SOX17.

9. cell culture as claimed in claim 1, wherein the expression of PDX1 is higher than the expression of the mark that is selected from α-fetoprotein (AFP), SOX7, SOX1, ZIC1 and NFM in the positive anterior intestine endoderm cell of described PDX1-.

10. cell culture as claimed in claim 1, wherein said cell culture is substantially free of the cell that is selected from internal organ endoderm cell, body wall endoderm cell and neurocyte.

11. cell culture as claimed in claim 1, the negative definitive entoderm cell of per approximately 10 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of a PDX1-in the wherein said cell culture.

12. cell culture as claimed in claim 1, the negative definitive entoderm cell of per approximately 5 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of a PDX1-in the wherein said cell culture.

13. cell culture as claimed in claim 1, the negative definitive entoderm cell of per approximately 4 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of a PDX1-in the wherein said cell culture.

14. cell culture as claimed in claim 1 also comprises embryonic stem cell.

15. cell culture as claimed in claim 14, wherein said embryonic stem cell is derived from the tissue that is selected from morula, embryo's inner cell mass (ICM) and embryo sexual fold.

16. cell culture as claimed in claim 1 also comprises retinoid.

17. cell culture as claimed in claim 16, wherein said retinoid are vitamin A acid (RA).

18. cell culture as claimed in claim 1 also comprises FGF-10.

19. cell culture as claimed in claim 1 also comprises B27.

20. cell culture as claimed in claim 1 also comprises RA and FGF-10.

21. cell culture as claimed in claim 20 also comprises B27.

22. comprise the cell mass of cell, be the positive anterior intestine endoderm cell of people PDX1-wherein at least about 90% described cell, described PDX1-is positive, and the anterior intestine endoderm cell is the multipotential cell that can be divided into derived from intestinal tube forward cell, tissue or organ.

23. cell mass as claimed in claim 22 is the positive anterior intestine endoderm cell of PDX1-at least about 95% described cell wherein.

24. cell mass as claimed in claim 22 is the positive anterior intestine endoderm cell of PDX1-at least about 98% described cell wherein.

25. cell mass as claimed in claim 22, wherein said PDX1-is positive, and the anterior intestine endoderm cell expresses the HOXA13 gene.

26. cell mass as claimed in claim 22, wherein said PDX1-is positive, and the anterior intestine endoderm cell expresses the HOXC6 gene.

27. cell mass as claimed in claim 22, wherein said PDX1-is positive, and the anterior intestine endoderm cell expresses SOX17.

28. cell mass as claimed in claim 22, wherein the expression of PDX1 is higher than the expression of the mark that is selected from AFP, SOX7, SOX1, ZIC1 and NFM in the positive anterior intestine endoderm cell of described PDX1-.

29. produce the positive anterior intestine endoderm cell's of PDX1-method, described method comprises following steps:

Acquisition comprises the cell mass of the negative definitive entoderm cell of PDX1-; And

For providing, described cell mass is enough to promote that wherein said PDX1-is positive, and the anterior intestine endoderm cell is the multipotential cell that can be divided into derived from intestinal tube forward cell, tissue or organ to the retinoid of the negative definitive entoderm cell of the described PDX1-of small part to the amount of the positive anterior intestine endoderm cell differentiation of PDX1-.

30. method as claimed in claim 29, also comprise giving the step that the enough time forms for the positive anterior intestine endoderm cell of PDX1-, the enough time that the positive anterior intestine endoderm cell of the wherein said PDX1-of confession forms is determined by the existence that detects the positive anterior intestine endoderm cell of PDX1-in the described cell mass.

31. method as claimed in claim 29 is the positive anterior intestine endoderm cell of PDX1-at least about the negative definitive entoderm cytodifferentiation of 2% described PDX1-wherein.

32. method as claimed in claim 29 is the positive anterior intestine endoderm cell of PDX1-at least about the negative definitive entoderm cytodifferentiation of 5% described PDX1-wherein.

33. method as claimed in claim 29 is the positive anterior intestine endoderm cell of PDX1-at least about the negative definitive entoderm cytodifferentiation of 10% described PDX1-wherein.

34. method as claimed in claim 29 is the positive anterior intestine endoderm cell of PDX1-at least about the negative definitive entoderm cytodifferentiation of 25% described PDX1-wherein.

35. method as claimed in claim 29, the existence that wherein detects the positive anterior intestine endoderm cell of PDX1-in described cell mass comprises the expression that detects PDX1.

36. method as claimed in claim 35, wherein the expression of PDX1 is higher than the expression of the mark that is selected from α-fetoprotein (AFP), SOX7, SOX1, ZIC1 and NFM in the positive anterior intestine endoderm cell of described PDX1-.

37. method as claimed in claim 35, the expression of wherein said PDX1 is measured by quantitative polyase chain reaction (Q-PCR).

38. method as claimed in claim 35, the expression of wherein said PDX1 is measured by immunocytochemistry.

39. method as claimed in claim 29, wherein said retinoid is RA.

40. method as claimed in claim 39, wherein RA provides to the concentration range of about 50 μ M with about 0.01 μ M.

41. method as claimed in claim 39, wherein RA provides to the concentration range of about 20 μ M with about 0.04 μ M.

42. method as claimed in claim 39, wherein RA provides to the concentration range of about 10 μ M with about 0.1 μ M.

43. method as claimed in claim 39, wherein RA provides to the concentration range of about 2.5 μ M with about 0.2 μ M.

44. method as claimed in claim 39, wherein RA provides to the concentration range of about 1.5 μ M to be about 0.5 μ M.

45. method as claimed in claim 39, wherein RA provides with the concentration of about 1 μ M.

46. method as claimed in claim 39, wherein RA provides when described cultivation is carried out about 4 days.

47. method as claimed in claim 29, also comprise the factor of the amount that the generation that is enough to strengthen the positive anterior intestine endoderm cell of PDX1-is provided in described culture, the described factor is selected from the combination of FGF-10, FGF-4, activin A, activin B, B27, conditioned medium and the described factor.

48. method as claimed in claim 47, the wherein said factor are selected from FGF-10, FGF-4, activin A and activin B.

49. method as claimed in claim 48, the wherein said factor provides to the concentration range of about 500ng/ml with about 10ng/ml.

50. method as claimed in claim 48, the wherein said factor provides to the concentration range of about 200ng/ml with about 20ng/ml.

51. method as claimed in claim 48, the wherein said factor provides to the concentration range of about 75ng/ml with about 25ng/ml.

52. method as claimed in claim 48, the concentration of the about 50ng/ml of the wherein said factor provides.

53. method as claimed in claim 48, the wherein said factor and described retinoid roughly provide simultaneously.

54. method as claimed in claim 47, the wherein said factor is B27.

55. method as claimed in claim 54, wherein B27 provides with about 0.1% to about 20% concentration range of whole substratum.

56. method as claimed in claim 54, wherein B27 provides with about 0.2% to about 5% concentration range of whole substratum.

57. method as claimed in claim 54, wherein B27 provides with about 0.5% to about 2% concentration range of whole substratum.

58. method as claimed in claim 54, wherein B27 provides with about 1% concentration of whole substratum.

59. method as claimed in claim 54, wherein B27 and described retinoid roughly provide simultaneously.

60. method as claimed in claim 47, the wherein said factor is a conditioned medium.

61. method as claimed in claim 60, wherein conditioned medium provides with about 10% to about 100% concentration range of whole substratum.

62. method as claimed in claim 60, wherein conditioned medium provides with about 20% to about 80% concentration range of whole substratum.

63. method as claimed in claim 60, wherein conditioned medium provides with about 40% to about 60% concentration range of whole substratum.

64. method as claimed in claim 60, wherein conditioned medium provides with about 50% concentration of whole substratum.

65. method as claimed in claim 60, wherein conditioned medium and described retinoid roughly provide simultaneously.

66. method as claimed in claim 60, wherein conditioned medium contacts about 24 hours by the human embryo stem cell (hESC) that makes differentiation and prepares with cell culture medium.

67. as the described method of claim 66, wherein said hESC is being selected from the RPMI that adds 3% serum, is adding the low serum RPMI of activin A and was adding in the substratum of low serum RPMI of BMP4 differentiation about 5 days.

68. the positive anterior intestine endoderm cell of the PDX1-that the described method of claim 29 produces.

69. produce the method for the positive anterior intestine endoderm cell's of enrichment PDX1-cell mass, said method comprising the steps of:

Obtain the multipotential cell group, at least one cell comprises the nucleic acid that is subjected to the control of PDX1 promotor of at least one copy among the wherein said multipotential cell group, and described nucleic acid comprises the sequence of encoding green fluorescent protein (GFP) or its biological active fragment;

Break up described multipotential cell so that produce the positive anterior intestine endoderm cell of PDX1-, described PDX1-is positive, and the anterior intestine endoderm cell is the multipotential cell that can be divided into derived from intestinal tube forward cell, tissue or organ; And

The positive anterior intestine endoderm cell of described PDX1-is separated with the PDX1-negative cells.

70. as the described method of claim 69, the cell mass of wherein said enrichment comprises the positive anterior intestine endoderm cell of PDX1-at least about 95%.

71. as the described method of claim 69, the cell mass of wherein said enrichment comprises the positive anterior intestine endoderm cell of PDX1-at least about 98%.

72. as the described method of claim 69, wherein said differentiation step also is included as to provide among the described multipotential cell group is enough to promote the somatomedin of described multipotential cell at least a TGF beta superfamily of the amount of the negative definitive entoderm cytodifferentiation of PDX1-, and is enough to promote the retinoid of the negative definitive entoderm cell of described PDX1-to the amount of the positive anterior intestine endoderm cell differentiation of PDX1-for the negative definitive entoderm cell of described PDX1-provides.

73. as the described method of claim 72, wherein said retinoid is RA.

74. the positive anterior intestine endoderm cell's of PDX1-that the described method of claim 69 produces enriched populations.

75. increase the method for the expression of PDX1 gene product in expressing the definitive entoderm cell of SOX17, described method comprises makes described definitive entoderm cell contact with the differentiation factor of the amount that is enough to increase described PDX1 gene product expression.

76. as the described method of claim 75, wherein said differentiation factor is a retinoid.

77. as the described method of claim 76, wherein said differentiation factor is RA.

78. as the described method of claim 75, wherein said differentiation factor is selected from the combination of FGF-10, FGF-4, activin A, activin B, B27, conditioned medium and the described factor.

79. identify to promote the method for the negative definitive entoderm cell of PDX1-, said method comprising the steps of to the differentiation factor of the positive anterior intestine endoderm cell differentiation of PDX1-:

Acquisition comprises the group of the negative definitive entoderm cell of PDX1-;

The group of the negative definitive entoderm cell of the described PDX1-of comprising is contacted with candidate's differentiation factor; And

With with described cell mass before described candidate's differentiation factor contacts in PDX1 expression ratio, determine with described cell mass after described candidate's differentiation factor contacts in the expression of PDX1 whether increase, wherein the increase of expressing at PDX1 described in the described cell mass shows that described candidate's differentiation factor can promote the negative definitive entoderm cell of described PDX1-to the positive anterior intestine endoderm cell differentiation of PDX1-, and described PDX1-is positive, and the anterior intestine endoderm cell is the multipotential cell that can be divided into derived from intestinal tube forward cell, tissue or organ.

80. as the described method of claim 79, wherein said PDX1 expresses and detects by Q-PCR.

81., also be included in the step that contacts HOXA13 expression of gene in the described cell mass of front and back mensuration with described candidate's differentiation factor as the described method of claim 79.

82. as the described method of claim 79 also be included in detect before and after candidate's differentiation factor as described in adding as described in the method for HOXC6 expression of gene in the cell mass.

83. as the described method of claim 79, wherein said candidate's differentiation factor is a small molecules.

84. as the described method of claim 83, wherein said small molecules is a retinoid.

85. as the described method of claim 84, wherein said retinoid is RA.

86. as the described method of claim 79, wherein said candidate's differentiation factor is a polypeptide.

87. as the described method of claim 79, wherein said candidate's differentiation factor is a somatomedin.

88. as the described method of claim 79, wherein said candidate's differentiation factor is FGF-10.

89. identify the method for the differentiation factor that can promote the positive anterior intestine endoderm cell differentiation of PDX1-, said method comprising the steps of:

Acquisition comprises the positive anterior intestine endoderm cell's of PDX1-group;

The positive anterior intestine endoderm cell's of the described PDX1-of comprising group is contacted with candidate's differentiation factor; And

With with described cell mass before described candidate's differentiation factor contacts in mark expression ratio, determine to be after described candidate's differentiation factor contact that the expression of identical mark is to increase or reduce in the described cell mass, wherein increase or the reduction in marker expression described in the described cell mass shows that described candidate's differentiation factor can promote the positive anterior intestine endoderm cell's of PDX1-differentiation.

90. as the described method of claim 81, wherein said marker expression is measured by Q-PCR.

91. as the described method of claim 81, wherein said candidate's differentiation factor is a small molecules.

92. as the described method of claim 81, wherein said candidate's differentiation factor is a polypeptide.

93. as the described method of claim 81, wherein said candidate's differentiation factor is a somatomedin.

94. comprise the carrier of the reporter gene that is operably connected to the PDX1 control region.

95. as the described carrier of claim 94, wherein said reporter gene is EGFP.

96. comprise the cell of the described carrier of claim 94.

97. comprise the cell of the reporter gene that is operably connected to the PDX1 control region.

98. as the described cell of claim 97, the wherein said reporter gene that is operably connected to described PDX1 control region is integrated into karyomit(e).

99. as the described cell of claim 97, wherein said reporter gene is EGFP.

100. as the described cell of claim 97, wherein said cell is a versatility.

101. as the described cell of claim 100, wherein said cell is hESC.

102. as the described cell of claim 97, wherein said cell is the definitive entoderm cell.

103. as the described cell of claim 97, wherein said cell is the positive anterior intestine endoderm cell of PDX1-.

104. conditioned medium by the following steps preparation:

Make fresh cell culture medium contact about 24 hours with the hESC group of differentiation, wherein said hESC is being selected from the RPMI that adds 3% serum, is adding the low serum RPMI of activin A and was adding in the cell culture medium of low serum RPMI of BMP4 differentiation about 5 days; And

From described substratum, remove the hESC group of described differentiation.

105. as the described conditioned medium of claim 104, wherein said fresh cell culture medium is RPMI.

106. as the described conditioned medium of claim 105, wherein said RPMI is low serum RPMI.

107. the method for conditioning substratum said method comprising the steps of:

Make fresh cell culture medium contact about 24 hours with the hESC group of differentiation, wherein said hESC is being selected from the RPMI that adds 3% serum, is adding the low serum RPMI of activin A and was adding in the cell culture medium of low serum RPMI of BMP4 differentiation about 5 days; And

From described substratum, remove the hESC group of described differentiation.

108. as the described method of claim 107, wherein said fresh cell culture medium is RPMI.

109. as the described conditioned medium of claim 108, wherein said RPMI is low serum RPMI.

Images