CN109628371A - Definitive entoderm - Google Patents

Abstract

The invention discloses the cell cultures and preparation method thereof including definitive endodenn cells.The method for being separated invention also discloses the cell mass of the definitive endodenn cells purified substantially and from other cell types, being enriched with and purifying definitive endodenn cells.

Description

Definitive entoderm

The application be the applying date be on December 23rd, 2004, application No. is the divisions of 201410120990.2 application of the same name Application.

Related application

The application is non-provisional application, enjoys in and mentions under the regulation of 35U.S.C. § 119 (e) item on December 23rd, 2003 U.S. Provisional Patent Application No. 60/532,004, the priority of entitled " definitive entoderm " of friendship, in 35U.S.C. § 119 (e) The entitled of U.S. Provisional Patent Application No. 60/586,566 that on July 9th, 2004 submits also is enjoyed under the regulation of item " to be used for Separate the chemokines cell surface receptor of definitive entoderm " priority of item, and the regulation in 35U.S.C. § 119 (e) item Under enjoy in the U.S. Provisional Patent Application No. 60/587,942 submitted on July 14th, 2004, it is entitled " for separating in setting The chemokines cell surface receptor of germinal layer " priority.The open of each priority application listed above is all drawn herein Enter, it is for reference.

Invention field

The present invention relates to medicine and cell biologies.In particular, the present invention relates to composition, including mammal is fixed Shape endoderm cell and preparation, separation and the method using these cells.

Background of invention

In 1994, human pluripotent stem cells, example were separated in the culture for not having fibroblast nutrient for the first time If embryo does (ES) cell and embryonic germ (EG) cell (Bongso et al., 1994), then grown with fibroblast It supports in the culture of object and has separated these stem cells (Hogan, 1997).Later, Thomson, Reubinoff and Shamblott Continuous culture (the Reubinoff et of people's ES and EG cell is established using the Mouse trophoblast for inactivating mitosis al.,2000；Shamblott et al.,1998；Thomson et al.,1998).

People ES and EG cell (hESCs) are to study the early development of people and to some such as diabetes and Parkinson's disease Disease treatment intervention provides new chance.For example, will be to current utilization using the cell of the generation insulin derived from hESCs The cell therapeutic approach of donor pancreatic cell provides huge improvement.However, being generated at present it is not understood that how to be generated from hESCs The β cell of insulin.Similarly, the cell therapy of islet cells of the utilization from donor pancreas of diabetes is received at present The limitation of high quality islet cells shortage needed for transplanting.The cell therapy of one type-1 diabetes mellitus patient is needed to transplant about 8x 10⁸Pancreatic islet cell (Shapiro et al., 2000；Shapiro et al.,2001a；Shapiro et al., 2001b).Similarly, it is necessary to which at least two healthy donors organs are successfully transplanted with obtaining enough islet cells. HESCs provides a kind of source of starting material, and the noble cells for going out the high quality of fundamental quantity from the Materials is thin for people Born of the same parents' treatment.

Two kinds of characteristics for making hESCs be very suitable for cell therapy application are versatility and keep these in long-term culture The accumulation of heredity variation will not occur for the ability of cell.Versatility refers to that hESCs is divided into all 3 kinds of primary germ layers The ability of the derivative of (entoderm, mesoderm, ectoderm), all bodies that these subsequent primary germ layers form adult are thin Born of the same parents' type and embryo outside organization's (e.g., placenta) and reproduction cell.Although versatility imparts the particularly useful property of hESCs, this Kind of characteristic also gives research and operates these cells and its derivative brings special challenge.Due to the hESC culture in differentiation In there may be a large amount of cell types, therefore, the efficiency that most cell types generate is very low.In addition, success evaluation is any The generation of given cell type, which depends critically on, determines suitable marker.Realize efficient directed differentiation for hESCs's Treatment use is extremely important.

In order to which hESCs to be used to generate the cell that can be used for cell therapy application as starting material, foregoing problems are overcome It is beneficial.For example, it is horizontal in order to reach the cell material that islet cell transplantation treatment needs, it, will in the earliest period of differentiation Expeditiously directed differentiation is that pancreas islet/β cell line is advantageous to hESCs.

In addition to the differentiation of the efficiently and directionally of atomization, intermediate cell from differentiation pathway to pancreas islet/β cell line that break up along The separation of type and qualitative, and using this cell as suitable lineage precursors be used to break up in other steps, and have Benefit.

Summary of the invention

Some embodiments of the invention be related to include definitive endodenn cells cell culture, wherein the setting Endoderm cell is pluripotent cell, can be divided into intestinal tube cell or the organ derived from intestinal tube.According to some embodiments, setting Endoderm cell is mammalian cell, and in preferred embodiments, and definitive endodenn cells are people's cell.In the present invention Some embodiments in, definitive endodenn cells express or do not express specific marker significantly.In some embodiments, Definitive endodenn cells express one or more markers, the marker be selected from SOX17, CXCR4, MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.In other embodiments, definitive endodenn cells are not significant Express one or more markers, the marker be selected from OCT4, α-fetoprotein (AFP), thrombomodulin (TM), SPARC and SOX7。

According to other embodiments of the present invention, the method for generating definitive entoderm from pluripotent cell is described.Some In embodiment, pluripotent cell is derived from mulberry body.In some embodiments, multipotential stem cell is stem cell.For these The stem cell of method may include, but be not limited to embryonic stem cell.Embryonic stem cell can be derived from embryo inner cell mass or embryo Genital ridge.Embryonic stem cell can originate from various animal species, these types include, but are not limited to various mammal kinds Class, including people.In preferred embodiments, human embryo stem cell is used to generate definitive entoderm.

In some embodiments of the present invention, one or more growth factors are used for from pluripotent cell to the interior embryo that shapes In the atomization of confluent monolayer cells.These one or more growth factors for being used for atomization may include from TGF beta superfamily Growth factor.In these embodiments, one or more growth factors include Nodal/ activin and/or growth factor The BMP subgroup of TGF beta superfamily.In some embodiments, one or more growth factors are selected from Nodal, activin A, The combination of activin B, BMP4, Wnt3a or any of these growth factors.

Embodiment of the present invention further relates to be enriched in the cell mass in definitive endodenn cells.In certain embodiments, The definitive endodenn cells are separated or are purified substantially.In some embodiments, the separation or the setting purified substantially Endoderm cell expresses SOX17 and/or CXRC4 marker, and expression degree is higher than OCT4, AFP, TM, SPARC and/or SOX7 Marker.

Additionally providing enrichment has the method for cell mass of definitive entoderm.It in some embodiments, can will be in setting Endoderm cell is separated from mixed cell population or basic purifying, by the way that the cell is contacted with a kind of reagent, the reagent and A kind of molecule combination, which is located at definitive endodenn cells surface, rather than other cell surfaces in mixed cell population, Then cell of the separation in conjunction with reagent.In certain embodiments, the molecule positioned at the definitive endodenn cells surface is CXCR4。

Other embodiments of the present invention further relates to CXCR4 antibody, and other ligands of SDF-1 ligand or CXCR4 are used for The definitive endodenn cells of obtaining enrichment, separation or basic purified form.For example, can be by CXCR4 antibody, SDF-1 ligand Another ligand of CXCR4 be used as such as it is affine separation or Magneto separate method in reagent, with enrichment, separation or it is substantially pure Change the definitive endodenn cells in conjunction with the reagent.

Other embodiments of invention as described herein are related to the composition of such as cell culture comprising multipotency is thin Born of the same parents and definitive endodenn cells.In certain embodiments, cell culture includes simultaneously stem cell and definitive endodenn cells. Stem cell population in these cultures can be greater than, equal to or less than definitive endodenn cells number in the culture.One In a little embodiments, stem cell is human embryo stem cell.In certain embodiments, hESCs is maintained on trophoderm.At this In a little embodiments, the trophocyte can be such as fibroblastic to obtain from people, mouse or other suitable biologicals Cell.

In some embodiments of the present invention, the composition including definitive endodenn cells and hESCs further includes One or more growth factors.These growth factors may include the growth factor from TGF beta superfamily.In these embodiments In, one or more growth factors include the BMP subgroup of Nodal/ activin and/or growth factor TGF beta superfamily.? In some embodiments, one or more growth factors are selected from Nodal, activin A, activin B, BMP4, Wnt3a or appoint The combination of what these growth factor.

Other embodiments of the present invention is described with reference to following number paragraphs:

1. including the cell culture of people's cell, wherein at least about 10% people's cell is definitive endodenn cells, The definitive endodenn cells are the pluripotent cell that can be divided into intestinal tube cell or the organ derived from intestinal tube.

2. cell culture described in paragraph 1, wherein at least about 50% people's cell is definitive endodenn cells.

3. cell culture described in paragraph 1, wherein at least about 80% people's cell is definitive endodenn cells.

4. cell culture described in paragraph 1, wherein the definitive endodenn cells expression is selected from SOX17 and CXCR4 Marker.

5. cell culture described in paragraph 4, wherein in the definitive endodenn cells, selected from SOX17's and CXCR4 The expression of the marker is higher than the mark for being selected from OCT4, α-fetoprotein (AFP), thrombomodulin (TM), SPARC and SOX7 The expression of object.

6. cell culture described in paragraph 4, wherein the definitive endodenn cells do not express selected from OCT4, AFP, TM, The marker of SPARC and SOX7.

7. cell culture described in paragraph 4, wherein definitive endodenn cells expression selected from MIXL1, GATA4 and The marker of HNF3b.

8. cell culture described in paragraph 4, wherein definitive endodenn cells expression selected from FGF17, VWF, The marker of CALCR, FOXQ1, CMKOR1 and CRIP1.

9. cell culture described in paragraph 1, wherein the definitive endodenn cells express SOX17 and CXCR4.

10. cell culture described in paragraph 9, wherein in the definitive endodenn cells, the SOX17 and The expression of CXCR4 is higher than the expression of OCT4, AFP, TM, SPARC and SOX7.

11. cell culture described in paragraph 9, wherein the definitive endodenn cells do not express OCT4, AFP, TM, SPARC and SOX7.

12. cell culture described in paragraph 9, wherein definitive endodenn cells expression MIXL1, GATA4 and HNF3b。

13. cell culture described in paragraph 9, wherein definitive endodenn cells expression selected from FGF17, VWF, The marker of CALCR, FOXQ1, CMKOR1 and CRIP1.

14. cell culture described in paragraph 1, wherein in the cell culture, each corresponding pluripotent cell In the presence of at least about 2 definitive endodenn cells.

15. cell culture described in paragraph 14, wherein the pluripotent cell includes embryonic stem cell.

16. cell culture described in paragraph 15 is selected from wherein the embryonic stem cell is derived from from mulberry body, embryo Inner cell mass (ICM) and embryo genital ridge tissue.

17. cell culture described in paragraph 1 further comprises culture medium, which includes less than about 10% blood Clearly.

18. the growth that cell culture described in paragraph 1 further comprises the Nodal/ Activin subgroup of TGF beta superfamily The factor.

19. cell culture described in paragraph 1 further comprises selected from Nodal, activin A, activin B and combinations thereof Growth factor.

20. including the cell mass of cell, wherein at least about 90% cell is people's definitive endodenn cells, described People's definitive endodenn cells are pluripotent cell, which can be divided into intestinal tube cell or the organ derived from intestinal tube.

21. cell mass described in paragraph 20, wherein at least about 95% cell is people's definitive endodenn cells.

22. cell mass described in paragraph 20, wherein at least about 98% cell is people's definitive endodenn cells.

23. cell mass described in paragraph 20, wherein people's definitive endodenn cells expression is selected from SOX17's and CXCR4 Marker.

24. cell mass described in paragraph 23, wherein in people's definitive endodenn cells, selected from SOX17's and CXCR4 The expression of the marker is higher than the expression of the marker selected from OCT4, AFP, TM, SPARC and SOX7.

25. cell mass described in paragraph 23, wherein people's definitive endodenn cells do not express selected from OCT4, AFP, TM, The marker of SPARC and SOX7.

26. cell mass described in paragraph 23, wherein people's definitive endodenn cells expression selected from MIXL1, GATA4 and The marker of HNF3b.

27. cell mass described in paragraph 23, wherein definitive endodenn cells expression selected from FGF17, VWF, CALCR, The marker of FOXQ1, CMKOR1 and CRIP1.

28. cell mass described in paragraph 20, wherein people's definitive endodenn cells express SOX17 and CXCR4.

29. cell mass described in paragraph 28, wherein in people's definitive endodenn cells, the table of SOX17 and CXCR4 Up to the expression for being higher than OCT4, AFP, TM, SPARC and SOX7.

30. cell mass described in paragraph 28, wherein people's definitive endodenn cells do not express OCT4, AFP, TM, SPARC And SOX7.

31. cell mass described in paragraph 28, wherein described people's definitive endodenn cells expression MIXL1, GATA4 and HNF3b。

32. cell mass described in paragraph 28, wherein definitive endodenn cells expression selected from FGF17, VWF, CALCR, The marker of FOXQ1, CMKOR1 and CRIP1.

33. cell mass described in paragraph 20, wherein each corresponding pluripotent cell exists extremely in the cell colony Few about 2 definitive endodenn cells.

34. cell mass described in paragraph 33, wherein the pluripotent cell includes embryonic stem cell.

35. cell mass described in paragraph 34, wherein the embryonic stem cell is derived from the ICM selected from mulberry body, embryo With the tissue of the genital ridge of embryo.

36. the method for generating definitive endodenn cells, the method include the following steps:

Obtain the cell mass including people's pluripotent cell；

At least one TGF beta superfamily growth factor is provided for the cell mass, the amount of the growth factor is enough to promote The pluripotent cell is divided into definitive endodenn cells, and the definitive endodenn cells are that can be divided into intestinal tube cell or derivative In the pluripotent cell of the organ of intestinal tube；And

It gives sufficient time to form definitive endodenn cells, wherein the formation definitive endodenn cells is enough Time is determined with detecting the presence of definitive endodenn cells in the cell colony.

37. method described in paragraph 36, wherein at least about 10% pluripotent cell is divided into definitive endodenn cells.

38. method described in paragraph 36, wherein at least about 50% pluripotent cell is divided into definitive endodenn cells.

39. method described in paragraph 36, wherein at least about 70% pluripotent cell is divided into definitive endodenn cells.

40. method described in paragraph 36, wherein at least about 80% pluripotent cell is divided into definitive endodenn cells.

41. method described in paragraph 36, wherein presence of the detection definitive endodenn cells in the cell colony includes The expression and at least one that at least one marker selected from SOX17 and CXCR4 is detected in the cell population are selected from The expression of the marker of OCT4, AFP, TM, SPARC and SOX7, wherein in the definitive endodenn cells, selected from SOX17 and The expression of the marker of CXCR4 is higher than the expression of the marker selected from OCT4, AFP, TM, SPARC and SOX7.

42. method described in paragraph 36, wherein presence of the detection definitive endodenn cells in the cell mass includes The expression and at least one that at least one marker selected from SOX17 and CXCR4 is detected in the cell population are selected from The expression of the marker of AFP, TM and SOX7, wherein in the definitive endodenn cells, described in SOX17 and CXCR4 The expression of marker is higher than the expression of the marker selected from AFP, TM and SOX7.

43. method described in paragraph 42, the expression of marker described in wherein at least one is measured with Q-PCR.

44. method described in paragraph 42, the expression of marker described in wherein at least one is surveyed with immunocytochemistry It is fixed.

45. method described in paragraph 36, wherein detect in cell colony definitive endodenn cells there are packets It includes and detects at least one marker selected from VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 in the cell colony cell Expression and the expression of at least one marker selected from OCT4, AFP, TM, SPARC and SOX7, wherein in the setting In endoderm cell, the expression of the marker selected from FGF17, VWF, CALCR, FOXQ1 and CRIP1 be higher than selected from OCT4, AFP, TM, The expression of the marker of SPARC and SOX7.

46. method described in paragraph 36, wherein the Nodal/ that at least one growth factor is TGF beta superfamily lives Change plain subgroup.

47. method described in paragraph 46, wherein at least one growth factor is selected from Nodal, activin A, activation Plain B and combinations thereof.

48. method described in paragraph 47, wherein at least one growth factor is Nodal.

49. method described in paragraph 47, wherein at least one growth factor is activin A.

50. method described in paragraph 47, wherein at least one growth factor is activin B.

51. method described in paragraph 36, which provide a variety of growth factors of TGF beta superfamily.

52. method described in paragraph 51, wherein a variety of growth factors include Nodal, activin A and activin B.

53. method described in paragraph 36, wherein the concentration of at least one growth factor is at least about 10ng/ml.

54. method described in paragraph 36, wherein the concentration of at least one growth factor is at least about 100ng/ml.

55. method described in paragraph 36, wherein the concentration of at least one growth factor is at least about 500ng/ml.

56. method described in paragraph 36, wherein the concentration of at least one growth factor is at least about 1000ng/ml.

57. method described in paragraph 36, wherein the concentration of at least one growth factor is at least about 5000ng/ml.

58. method described in paragraph 36, wherein the cell colony is grown on the culture medium including less than about 10% serum In.

59. method described in paragraph 36, wherein the pluripotent cell includes stem cell.

60. method described in paragraph 59, wherein the pluripotent cell includes embryonic stem cell.

61. method described in paragraph 60, wherein the embryonic stem cell be derived from selected from mulberry body, embryo ICM and The tissue of the genital ridge of embryo.

62. the definitive endodenn cells generated in the method for paragraph 36.

63. generating the cell mass method of the definitive endodenn cells of enrichment, include the following steps:

Break up the cell in pluripotent human cell colony, to generate definitive endodenn cells, the definitive endodenn cells For pluripotent cell, which can be divided into intestinal tube cell or the organ derived from intestinal tube；

Reagent is provided to the cell mass, the reagent in conjunction with marker, determine in described by the marker expression It is not expressed in substantially in other cell types in the cell mass in shape endoderm cell；And

By the definitive endodenn cells in conjunction with the reagent and it is located at other cell classes described in the cell mass Type separation, to generate the cell mass of the definitive endodenn cells of enrichment.

64. method described in paragraph 63, wherein differentiation step further comprises,

The cell mass including multipotency people's cell is obtained,

At least one TGF beta superfamily growth factor is provided to the cell colony, the amount of the growth factor is enough to promote Definitive endodenn cells are divided by the pluripotent cell, the definitive endodenn cells are pluripotent cell, and the multipotency is thin Born of the same parents can be divided into intestinal tube cell or the organ derived from intestinal tube, and

Give time enough and form definitive endodenn cells, wherein the formation definitive endodenn cells it is enough when Between determined with detecting the presence of definitive endodenn cells in the cell colony.

65. method described in paragraph 63, wherein detection includes,

The expression of at least one marker selected from SOX17 and CXCR4 is detected in the cell of the cell colony, and The expression of at least one marker selected from OCT4, AFP, TM, SPARC and SOX7, wherein in the definitive endodenn cells In, the expression of the marker selected from SOX17 and CXCR4 is higher than the table of the marker selected from OCT4, AFP, TM, SPARC and SOX7 It reaches.

66. method described in paragraph 63 detects at least one choosing wherein detection includes in the cell of the cell colony From the expression of the marker of SOX17 and CXCR4 and the expression of at least one marker selected from AFP, TM and SOX7, wherein In the definitive endodenn cells, the expression of the marker selected from SOX17 and CXCR4 is higher than selected from AFP, TM and SOX7 The expression of marker.

67. method described in paragraph 63 detects at least one choosing wherein detection includes in the cell of the cell colony From the expression of the marker of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1, and it is at least one selected from OCT4, AFP, The expression of the marker of TM, SPARC and SOX7, wherein in the definitive endodenn cells, selected from FGF17, VWF, The expression of the marker of CALCR, FOXQ1, CMKOR1 and CRIP1 is higher than the mark selected from OCT4, AFP, TM, SPARC and SOX7 The expression of object.

68. method described in paragraph 63, wherein at least about 95% cell is definitive endodenn cells.

69. method described in paragraph 63, wherein at least about 98% cell is definitive endodenn cells.

70. method described in paragraph 63, wherein the marker is CXCR4.

71. method described in paragraph 63, wherein the reagent is antibody.

72. 71 the method for paragraph, wherein the antibody is affinity to CXCR4.

73. the cell mass for the enrichment definitive endodenn cells that method described in paragraph 63 generates.

74. described in any item cell cultures of paragraph 4 or 9, wherein the not significant table of definitive endodenn cells Up to the marker for being selected from OCT4, AFP, TM, SPARC and SOX7.

75. the cell mass of any one of paragraph 23 or 28, wherein the definitive endodenn cells not significantly expression choosing From the marker of OCT4, AFP, TM, SPARC and SOX7.

It is to be appreciated that above-mentioned method and composition is related to Cell culture invitro.However, above-mentioned vitro differentiation groups of cells Closing object can be in vivo applications.

Other embodiments of the present invention can be also found that in following U.S. Provisional Patent Applications, i.e. on December 23rd, 2003 The U.S. Provisional Patent Application of submission the 60/532nd, 004, it is entitled " definitive entoderm "；On July 9th, 2004 U.S. submitted It is Provisional Patent Application No. 60/586,566, entitled " for separating the chemotactic factor (CF) cell surface receptor of definitive entoderm "；With And U.S. Provisional Patent Application the 60/587th, 942 that on July 14th, 2004 submits, it is entitled " for separating definitive entoderm Cell surface receptor ", these disclosures are fully incorporated herein, it is for reference.

Brief description

Fig. 1 is the schematic diagram that the slave hESCs proposed generates the differentiation pathway of beta cell.The first step in the approach is by ES Cells switch is definitive endodenn cells system, is represented further by ES cell differentiation as embryo in endoderm, endocrine gland Layer or earliest one of the known steps of pancreas islet/beta cell.The some factors that can be used for that this is mediated to change are the member of TGF 'beta ' family, These members include, but are not limited to activin, nodals and BMPs.Determine the representative marker of definitive entoderm target cell For SOX17, GATA4, HNF3b, MIX1 and CXCR4.

Fig. 2 behaviour SOX17 cDNA diagram which show the position of conserved motifs and is highlighted for the immune of GENOVAC The region of method.

Fig. 3 is a kind of correlation dendrogram, shows that the correlation of SOX17 and SOX7 is most strong, slightly with the correlation of SOX18 It is weak.SOX17 protein in allied species is more much better than than the correlation of other members of SOX group's F subtribe in same species.

Fig. 4 is using the anti-SOX17 antibody of rat as the western hybridization (Western blot) of probe.The hybridization demonstrates this Antibody to the specificity (swimming lane 1) of people's SOX17 protein of overexpression in fibroblast, and to EGFP (swimming lane 2) or Maximally related SOX family member SOX7 (swimming lane 3) lacks immunoreactivity.

Fig. 5 A-B is display SOX17⁺The microphoto of cell cluster, the AFP that display largely marks altogether⁺Cell (A).This with Other SOX17⁺Cell cluster (B) observes a small amount of AFP⁺Cell does not observe AFP⁺Cell forms sharp contrast.

Fig. 6 A-C is the microphoto for showing parietal endoderm and SOX17.Embedding figure A is shown to people's thrombomodulin (TM) Immunocytochemistry, which is located at the cell table of the parietal endoderm cells in the hES cell culture of random differentiation Face.Embedding figure B is the region identical with embedding figure A that TM and SOX17 double-marking is shown.Embedding figure C is to mark the identical of core with DAPI The phase difference image in region.It is perfectly correlated to notice that DAPI label core is marked with SOX17.

Fig. 7 A-B is the anti-SOX17 sun for the SOX17 gene expression and SOX17 specific antibody for showing quantitative PCR (Q-PCR) The histogram of property cell.For the control medium (SR20) of undifferentiated, embedding figure A shows that activin A increases SOX17 base Because of expression, and retinoic acid (RA) strong inhibition SOX17 is expressed.Embedding figure B shows that the reacting condition of model identical and similarity degree exists SOX17⁺On cell number, show that the Q-PCR test of SOX17 gene expression is very sensitive to the variation of individual cell level.

Fig. 8 A is histogram, is shown in the hESCs culture broken up in the presence of activin A and keeps low-level AFP gene Expression, and in 10% fetal calf serum (FBS), cell random differentiation shows AFP strong upregulation.Difference on expression About 7 times.

Fig. 8 B-C is two microphoto images, it is shown that activin A is also very bright in individual cell level to AFP expression inhibiting It is aobvious, for the FBS (top) only with 10%, (bottom) is observed in the condition that activin A is handled AFP⁺Cell Cluster seldom and also very little.

Fig. 9 A-B is comparative diagram, and display quantifies AFP using flow cytometer⁺Cell number.The chart is bright, (right embedding existing Figure) or there is no AFP changes in gene expression amplitude (Fig. 8 A) and AFP when (left embedding figure) activin A⁺Cell number is very consistent, into One step shows Q-PCR analysis to the practicability for being shown in the variation occurred on individual cell level.

Figure 10 A-F is microphoto, and hESCs was exposed to nodal, activin A and activin B (NAA) by display, at 5 days What the time produced cell number dramatically increases (A-C).By comparing SOX17⁺The phase of cell and cell total amount existing for the region To amount, as shown in DAPI colour developing core (D-F), it can be seen that all cells of about 30-50% are after with NAA processing 5 days to SOX17 With immunoreactivity.

Figure 11 is histogram, and display activin A (0,10,30 or 100ng/mL) dose-dependently increases differentiation The SOX17 gene expression of hESCs.After handling 3 days adherent cultures, continue settling flux culture 3~5 days, expression increases Through apparent.

Figure 12 A-C is histogram, it was confirmed that activin A is to MIXL1 (embedding figure A), GATA4 (embedding figure B) and HNF3b (embedding figure C) the effect expressed.It also observed in other three definitive endoderm markers MIXL1, GATA4 and HNF3b to activin In dose-dependent increase.It is extremely similar to SOX17's to the increased expression amplitude of activin dose dependence, it is strong to show Activin A specific action is in coexpression all four genes (SOX17⁺,MIXL1⁺,GATA4⁺andHNF3b⁺) cell mass.

Figure 13 A-C is histogram, it was confirmed that activin A is to AFP (embedding figure A), SOX7 (embedding figure B) and SPARC (embedding figure C) table Effect in reaching.Reduce to activin A dose dependence the expression of visceral endoderm marker AFP.Primitive endoderm (SOX7) And parietal endoderm (SPARC) marker is still constant or only inhibits in certain point displays, shows activin A not specific action In these extra-embryonic endoderm cells types.This further support the expression increase of SOX17, MIXL1, GATA4 and HNF3b be due to Activin A causes the increase of definitive endodenn cells quantity.

Figure 14 A-B is histogram, shows the work that activin A expresses ZIC1 (embedding figure A) and Brachyury (embedding figure B) With.The continuous expression of neural marker ZIC1 shows that Activin A on neural breaks up without dose-dependent effect.From Brachyury expression reduces as can be seen that the processing of 100ng/mL activin A has significant inhibition to mesoblastic differentiation.This can It can be the increased result of definitive entoderm specificity from mesendoderm precursors.With non-treated control cultures phase Than time point phase maintains brachyury expression to low-level activin A processing (10 and 30ng/mL) after differentiation.

Figure 15 A-B is microphoto, and activin processing causes parietal endoderm differentiation to reduce.Only with the training of serum differentiation It supports and finds TM in object (A)^hiParietal endoderm region, however TM is seldom divided into when including activin (B)⁺Cell and total immune Reactive intensity is lower.

Figure 16 A-D is microphoto, shows that activin A and activin B handle caused marker expression.Continuously with activation Plain A and activin B are handled hESCs4 days, carry out three labels with SOX17, AFP and TM antibody.Embedding figure A-SOX17；Embedding figure B-AFP； Embedding figure C-TM；And embedding figure D-Phase/DAPI.Pay attention to visible a large amount of when entirely without AFP (B) and TM (C) immunoreactivity SOX17 positive cell (A).

Figure 17 is displaing micro picture, shows definitive entoderm and visceral endoderm one occur from hESCs in vitro.Visceral endoderm one Region is with AFP^hi/SOX17^lo/-Identification, however definitive entoderm shows antipodal feature, SOX17^hi/AFP^lo/-.Selection The domain is since the two regions are closer to each other, however, having repeatedly in the AFP being kept completely separate^hiCell compartment is observed SOX17^hi/AFP^lo/-Region shows part definitive endodenn cells derived from visceral endoderm cells.

Figure 18 is the chart for describing TGF 'beta ' family ligand and receptor.The factor of activation AR-Smads and BR-Smads is conducive to Generate definitive entoderm from human embryo stem cell (referring to J Cell Physiol.187:265-76).

Figure 19 is histogram, shows to express at any time with the SOX17 of single one or more TGF-β factor treatments induction.

Figure 20 is histogram, shows to cause SOX17 with a variety of TGF-β factor treatments⁺The increase of cell quantity at any time.

Figure 21 is histogram, shows that the SOX17 induced with a variety of TGF-β factor treatments is expressed at any time.

Figure 22 is histogram, increases SOX17 with showing activin A dose dependence⁺Cell quantity.

Figure 23 is histogram, and display Wnt3a, which is added, increases SOX17 table into activin A and the culture of activin B processing It reaches, higher than the level that induction is used alone in activin A and activin B.

Figure 24 A-C is histogram, is shown under the conditions of low FBS, and definitive entoderm increase is divided into.It is including 2%FBS It is handled under similarity condition with activin A and B processing hESCs in 10%FBS (10AA) (embedding figure A) in the culture medium of (2AA) Than SOX17 expression is 2-3 times high.Definitive endoderm markers MIXL1 (embedding figure B) is also affected in an identical manner, and And 2%FBS is higher than under the conditions of 10%FBS to the inhibition of AFP (visceral endoderm one) (embedding figure C).

Figure 25 A-D is microphoto, the SOX17 in display culture⁺Cell division.SOX17 immunoreactive cell is in HESC clones dividing edge (C, D), with proliferating cell nuclear antigen (PCNA) (embedding figure B) label, without with OCT4 (embedding figure C) Mark altogether.In addition, core is marked with DAPI, in SOX17⁺Cell (arrow) and OCT4⁺, can in undifferentiated hESCs (arrow) (D) Clearly see mitosis picture.

Figure 26 is histogram, is shown in various culture mediums, relative expression water of the CXCR4 in the hESCs broken up It is flat.

Figure 27 A-D be histogram, by with Figure 26 same treatment, show how a series of definitive endoderm markers have Closely similar CXCR4 expression pattern.

Figure 28 A-E is histogram, in display mesoderm (BRACHYURY, MOX1), ectoderm (SOX1, ZIC1) and internal organ How germinal layer (SOX7) marker is by with Figure 26 same treatment there is opposite CXCR4 to express.

Figure 29 A-F is microphoto, is shown in SOX17 immunoreactive cell under three kinds of culture medium conditions of Figure 26-28 Relative different.

Figure 30 A-C is flow cytometry dot plots, it was demonstrated that as the concentration for the activin A being added into differential medium increases Add, CXCR4⁺Cell quantity increases.

Figure 31 A-D is histogram, the CXCR4 that display high dose activin A processing (A100-CX+) separates afterwards⁺Cell is determined Shape entoderm marker quantity is compared with its mother cell group (A100) more horn of plenty.

Figure 32 is histogram, the CXCR4 that display is separated using fluorescence activated cell sorts device (FACS)⁺And CXCR4^-Cell And the gene expression of mother cell group.This confirms CXCR4⁺Cell includes all present on each mother cell group substantially CXCR4 gene expression, CXCR4^-Then include seldom or do not include CXCR4 gene expression.

Figure 33 A-D is histogram, it was demonstrated that the CXCR4 of high dose activin A processing⁺Cell mesoderm (BRACHYURY, MOX1), ectoderm (SOX1, ZIC1) and visceral endoderm one (SOX7) gene expression missing, these non-definitive endoderm markers Expression be suppressed.

Figure 34 A-M is histogram, shows marker gene expression pattern, can be used for identifying definitive endodenn cells.It is fixed The expression analysis of shape entoderm marker FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 are respectively as shown in embedding figure G-L. Aforementioned pedigree marker gene SOX17, SOX7, SOX17/SOX7, TM, ZIC1 and MOX1 are respectively as shown in embedding figure A-F.Embedding figure M is aobvious Show the expression analysis of CXCR4.About embedding figure A-M, the human embryo stem cell gene expression of mono- column hESC display purifying is marked；2NF It shows the cell handled with 2%FBS, activin is not added；0.1A100 is shown with the processing of 0.1%FBS, 100ng/ml activin A Cell；1A100 shows the cell handled with 1%FBS, 100ng/ml activin A；2A100 shows living with 2%FBS, 100ng/ml Change the cell of element A processing.

Detailed description of the invention

One critical stage of people's early development, the i.e. embryogenesis of term primitive gut occur after fertilization 2-3 weeks.Primitive gut embryogenesis It is extremely important because three original embryos of this phase Focus and ordering first (Lu et al., 2001；Schoenwolf and Smith,2000).Ectoderm is responsible for the formation of body covering and whole nervous systems, however, heart, blood, bone, skeletal muscle And other connective tissues are derived from mesoderm.Definitive entoderm is defined as being responsible for the germinal layer that whole intestinal tubes are formed, the whole Intestinal tube includes esophagus, stomach, small intestine and large intestine, and the organ derived from enteron aisle, such as lung, liver, thymus gland, parathyroid gland and first shape Gland, gall-bladder and pancreas (Grapin-Botton and Melton, 2000；Kimelman and Griffin,2000；Tremblay et al.,2000；Wells and Melton,1999；Wells and Melton,2000).Definitive entoderm with it is referred to as original Have between the cell line of entoderm being kept completely separate obviously different.Primitive endoderm primarily forms embryo outside organization, main If the body wall and visceral endoderm portions of yolk sac placenta and the cell epimatrix material of Reichert's film.

In primitive gut forming process, definitive entoderm forming process starts from cellular migration event, wherein mesendoderm is thin Born of the same parents' (cellular component for being capable of forming mesoderm or entoderm) pass through the structure for being referred to as former line.Definitive entoderm is derived from and wears Cross the cell of former line front and knot (one is located at the specific structure of former line foremost part).When migrating generation, shape interior embryo Layer, which is initially formed the foremost part of intestinal tube when until forming intestinal tube rear end, to be terminated.

The internal analysis that definitive entoderm is formed, such as Conlon et al., 1994；Feldman et al.,1998； Zhou et al.,1993；Aoki et al.,2002；Dougan et al.,2003；Tremblay et al.,2000； Vincent et al.,2003；Alexander et al.,1999；Alexander and Stainier,1999；Kikuchi et al.,2001；Hudson et al., 1997 and mouse Kanai-Azuma et al., 2002 zebra fish carried out and Africa The research of toad, these researchs are how to complete the development of specific endoderm cell type using human embryo stem cell in culture dish It lays a good foundation.The main bottleneck that development is restarted in culture dish is constituted in terms of cultivating relevant two to external ESC.It is first First, orderly germinal layer or organ structure will not be generated.In the hESC culture systems broken up, most of germinal layer and organ Specific genetic markers object can be expressed in heterologous form.Therefore, because lacking organ specific boundaries, it is difficult estimation specificity The formation of tissue or cell type.The nearly all gene expressed in the cell type of specific germinal layer or organization type It is expressed in the cell type of other germinal layers or organization type.Not special boundary is difficult to assign base with 1-3 cdna sample Because of the specificity of expression.It is, therefore, necessary to carefully detect considerable gene, some genes should can also lose interest in organ or It is expressed on the specific cell type of tissue.Secondly, the opportunity of gene expression pattern is to the activity developed along special modality to Guan Chong It wants.

As for more complicated event, it is noted that stem cell differentiation in vitro is quite nonsynchronous and may be than in vivo Significantly much.In this way, one group of cell may be when expression forms related gene with primitive gut, and another group may start Final differentiation.Moreover, handling hESC single layer or embryoid (EBs) may lead when participating in or without extrinsic factor Cause the significant difference of full gene expression pattern or differentiation state.Thus, in order to effectively advance along specific differentiation channel, root According to the gene expression pattern of heterogeneous cell mixture, the use of exogenous factor must carry out time control.It is examined in culture vessel It is also beneficial for considering the morphological association of these cells.With grow up in medium container or be divided into single layer and/or hESC grams Ability is compared when grand, and the ability of hESCs when uniform influence forms so-called embryoid is ideal far from most.

As an above-mentioned heterogeneous and nonsynchronous effective ways are handled, some embodiments of the invention consider will differentiation The method of cell is combined with enrichment, the method for separating and/or purifying the intermediate cell types in differentiation channel.

Embodiment of the present invention is related to generating the new determination method of definitive endodenn cells in culture, pass through by The pluripotent cell of such as stem cell is divided into multipotency definitive endodenn cells." multipotency " used herein or " pluripotent cell " refer to energy Generate the cell type of other specific cell types of limited quantity.As described above, definitive endodenn cells are not divided into source In ectoderm or mesoblastic tissue, however, intestinal tube and the organ derived from intestinal tube can be divided into.In some preferred embodiments In, definitive endodenn cells are derived from hESCs.These methods, which provide, efficiently generates tissue derived from people's entoderm, as pancreas, Liver, lung, stomach, intestines and thyroid basis.For example, it is functional production that the generation first step of definitive entoderm, which can break up stem cell, The β cell of raw insulin.In order to obtain the β cell for generating insulin of dosage, before reaching pancreas islet/β cell, it is expected that each Differentiation step is all efficient differentiation step.Because perhaps stem cell is divided into definitive endodenn cells represents generating functionality Pancreas islet/β cell initial step (as shown in Figure 1), it is accordingly required in particular to which this step differentiation is efficient.

In view of the needs of differentiation pluripotent cell to definitive endodenn cells, some aspects of the present invention are related to in-vitro method, The pluripotent cell of about 50-80% is changed into definitive endodenn cells.Typically, these methods include defined and temporarily specified Mode use culture and growth factor.Using can with the reagent of definitive endodenn cells specific bond, from cell mass will Definitive endodenn cells are separated and/or are purified with other cells, can get more enrichments of definitive endodenn cells group.In this way, The present invention relates to definitive endodenn cells and preparative separation and/or the methods for purifying these cells.

In order to determine the quantity of definitive endodenn cells in cell culture or cell mass, need from culture or cell mass The middle method for distinguishing this kind of cell and other cells.Therefore, embodiment of the present invention is related to cell sign object, exists, missing And/or relative expression levels are special to definitive entoderm, and detect the method for determining these marker expressions.Make herein " expression " refers to the level or content that the generation of material or substance or material or substance generate.Accordingly it is determined that specific marker The expression of object refers to the opposite or absolute content of the marker of detection expression, or only detection marker whether there is.It is used herein " marker " refers to any molecule that is observed or detecting.For example, marker may include but be not limited to nucleic acid, it is such as special The transcript of gene, the polypeptide product of gene, non-genomic polypeptide product, glycoprotein, sugar, glycolipid, lipid, lipoprotein or small point Son.

In some embodiments of the invention, the presence or absence of marker expression and/or its level are by quantitative PCR (Q- PCR it) determines.For example, the transcript amount that some genetic markers generate, for example, SOX17, CXCR4, OCT4, AFP, TM, SPARC, SOX7, MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1, CRIP1 and as described herein other Marker is determined by quantitative Q-PCR.In other embodiments, Q-PCR and immunohistochemistry technique for identification and determine these The amount or relative scale of marker.

By using the method for one or more suitable marker expressions of such as above-mentioned determination, in cell culture or carefully The ratio of identification definitive endodenn cells and determining definitive endodenn cells is possible in born of the same parents group.For example, of the invention In some embodiments, the level of definitive endodenn cells or cell mass the expression SOX17 and/or CXCR4 gene of generation is than non- Definitive endodenn cells type or high about 2 orders of magnitude of cell mass.In other embodiments, the definitive endodenn cells of generation Or the level of cell mass expression SOX17 and/or CXCR4 gene is than non-definitive endodenn cells type or cell mass high 2 or more The order of magnitude.In other other embodiments, definitive endodenn cells or the cell mass expression of generation are one or more to be selected from The marker of SOX17, CXCR4, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 are thinner than non-definitive entoderm The order of magnitude of high about 2 or 2 of the expression of born of the same parents' type or cell mass or more.

The present invention relates to cell culture, including with a large amount of definitive entoderms and including the definitive endodenn cells of enrichment Cell mass.As a result, some embodiments be related to include definitive endodenn cells cell culture, wherein in the culture At least about the cell of 50-80% is definitive endodenn cells.One preferred embodiment be related to include people's cell cell culture Object, wherein at least about 50-80% people's cell is definitive endodenn cells in culture.Since the effect of differentiation method can pass through Change some parameter regulations, the including but not limited to time of cell growth conditions, growth factor concentration and incubation step, the present invention The differentiation method can make about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, 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 it is greater than 95% pluripotent cell is converted into definitive entoderm.In the other embodiments of the present invention, by the multipotency of such as stem cell population Cell mass is converted into substantially pure definitive endodenn cells.

Composition of the present invention and method have several useful features.E.g., including the cell of definitive entoderm Culture and cell mass, and the method for preparing these cell cultures and cell mass can be used for mould and build the early stage rank that human hair is educated Section.In addition, composition of the present invention and method can also be used for the therapeutic intervention of the disease such as diabetes.For example, due to fixed Shape entoderm is only the tissue-derived of limited quantity, thus it can be used for developing pure tissue or cell type.

Definitive entoderm is prepared from pluripotent cell

Definitive endodenn cells culture and composition including definitive endodenn cells as described herein can embryos freely It is prepared in the pluripotent cell of stem cell." embryo " used herein refers to organism stage of development range, from single fertilized eggs Start to the multi-cellular structure in addition to the gametid of development no longer comprising multipotency or totipotent cell to terminate.Except derived from spouse The embryo of son fusion, term " embryo " refer to the embryo derived from somatic cell nuclear transfer.Preferred derivative definitive endodenn cells Method is using human embryo stem cell as the starting material for preparing definitive entoderm.The embryonic stem cell that this method uses can be Derived from mulberry body, embryo inner cell mass or the cell obtained from embryo's sexual gland ridge.Using art methods, human stem cell can In the medium keep multipotency state and it is substantially undifferentiated.The method, for example, U.S. Patent No. 5,670,5,690, Method disclosed in 9265,843,6,200,806 and 6,251, No. 671, is fully incorporated herein, for reference.

In some embodiments of the present invention, hESCs is maintained at trophoderm.In these embodiments, Any trophoderm that hESCs can be made to be maintained at multipotency state can be used in method of the present invention.One common culture The trophoderm of human embryo stem cell is one layer of l cell.Recently, human fibroblasts trophoderm also developed It (referring to method disclosed in U.S. Patent Application No. 2002/0072117, is fully incorporated herein, for ginseng for cultivating hESCs It examines).Other embodiments of the method for the invention allow to keep multipotency hESCs without using trophoderm.These methods such as U.S. Described in number of patent application 2003/0175956, it is published here and is fully incorporated, it is for reference.

Human embryo stem cell of the present invention may remain in the culture medium with or without serum.In some realities It applies in scheme, uses serum replacement.In other embodiments, the culture medium technology of serum-free, such as U.S. Patent Application No. Described in 2003/0190748, it is published here and is fully incorporated, it is for reference.

Keep the stem cell of multipotency state according to routine passage in the medium, until being divided into definitive entoderm.One In a little embodiments, break up to the completion of definitive entoderm be by be added into stem cell media the growth of TGF beta superfamily because Son, the amount being added are enough to promote differentiation to definitive entoderm.It is used to prepare the TGF beta superfamily growth factor of definitive entoderm Selected from Nodal/ activin or BMP subgroup.In some embodiments of differentiation method of the present invention, growth factor is selected from Nodal, activin A, activin B and BMP4.In addition, growth factor Wnt3a and other Wnt family members can be used for preparing setting Endoderm cell.In some embodiments of the present invention, the above-mentioned any growth factor referred to can be used.

In some embodiments of differentiation method of the present invention, the above-mentioned growth factor being added into cell, Concentration in culture medium is enough to promote at least partly stem cell to break up the embryo in shaping.In some embodiments of the present invention, The concentration of above-mentioned growth factor at least about 5ng/ml, at least about 10ng/ml, at least about 25ng/ml in culture medium, 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 1000ng/ml, at least about 2000ng/ml, at least about 3000ng/ml, at least About 4000ng/ml, at least about 5000ng/ml are greater than 5000ng/ml.

In some embodiments of the invention, above-mentioned growth factor needs to remove from cell culture after culture medium is added. For example, the about after the addition 1 day removing time of growth factor, about 2 days, about 3 days, about 4, about 5 days, about 6 days, about 7 days, about 8 days, About 9 days or about 10 days.In a preferred embodiment, the removing time of growth factor about after the addition 4 days.

The culture of definitive endodenn cells can be in the culture medium comprising a small amount of serum or serum-free.Of the invention some In embodiment, the concentration range of serum is about 0.05%v/v to about 20%v/v.For example, in some embodiments, culture The concentration of serum can be below about 0.05% (v/v), be below about 0.1% (v/v), be below about 0.2% (v/v), be below about in base 0.3% (v/v), it is below about 0.4% (v/v), is below about 0.5% (v/v), is below about 0.6% (v/v), is below about 0.7% (v/ V), below about 0.8% (v/v), below about 0.9% (v/v), below about 1% (v/v), below about 2% (v/v), below about 3% (v/v), below about 4% (v/v), below about 5% (v/v), below about 6% (v/v), below about 7% (v/v), below about 8% (v/v), below about 9% (v/v), below about 10% (v/v), below about 15% (v/v) or below about 20% (v/v).Some In embodiment, definitive endodenn cells are grown under serum-free.In other embodiments, definitive endodenn cells are in serum It is grown in the presence of substitute.In other embodiments, definitive endodenn cells are grown in the presence of B27.In these embodiment party In case, the concentration range of B27 additive is about 0.2% to about 20%v/v.

HESC is cultivated to definitive entoderm and can be monitored by determining the marker feature expression of definitive entoderm.Some In embodiment, the expression of some markers can be by whether there are markers to determine.Selectively, the table of some markers Up to can be determined by level of the measurement marker in cell culture or cell population.In these embodiments, may be used To qualitatively or quantitatively determine the expression of marker.The method for the expression marker that quantitative marker gene generates can be used quantitative PCR(Q-PCR).The method for carrying out Q-PCR is the prior art.Other methods of the prior art can also be used for quantitative marker gene Expression.For example, the expression of marker gene product can be by using for interested specific markers gene product Antibody detects.In some embodiments of the present invention, it is determined that the table of the marker genes characteristic with definitive entoderm It reaches, and lacks the expression of the marker genes characteristic of the hESCs and other cell types that significantly express.

As the following examples it is further described that a reliable markers object of definitive entoderm be SOX17 gene.In this way, The definitive endodenn cells of the method for the invention preparation express SOX17 marker gene, thus generate SOX17 gene product. The marker of other definitive entoderms be MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.In some embodiments of the present invention, the level of definitive endodenn cells expression SOX17 marker gene is higher than The level of SOX7 marker gene, with original and visceral endoderm one feature (referring to table 1).In addition, in some embodiments In, SOX17 marker gene expression is higher than the expression of OCT4 marker gene, the feature with hESCs.It is other in the present invention In embodiment, the level that definitive endodenn cells express SOX17 marker gene is higher than AFP, SPARC or thrombomodulin (TM) level of marker gene.In some embodiments of the present invention, the method expresses determining for SOX17 according to the present invention Shape endoderm cell does not express the PDX1 of the level of signifiance or amount (PDX1- is negative).

Other markers of definitive entoderm are CXCR4 gene.CXCR4 gene Codocyte surface chemokine receptor, Its ligand is chemoattractant SDF-1.In adult comprising CXCR4 receptor cell main function believed as migration hematopoietic cell extremely Marrow, delivery lymphocyte, the various B cells of differentiation and macrophage blood cell line [Kim, C., and Broxmeyer, H.J.Leukocyte Biol.65,6-15(1999)].CXCR4 receptor also plays the co-receptor effect that HIV-1 enters in T cell [Feng,Y.,et al.Science,272,872-877(1996)].It is a series of [McGrath, K.E.etal.Dev.Biology213,442-456 (1999)] carry out research in, describe adult mice and its early stage send out Educate middle Chemokine receptor CXCR4 and its unique ligand SDF-1 expression [Kim, C., andBroxmyer, H., J.Leukocyte Biol.65,6-15(1999)].CXCR4/SDF1 is clear in developmental interaction, in transgenosis In mouse, when any gene disruption [Nagasawa et al.Nature, 382,635-638 (1996)], Ma, Q., et Al Immunity, 10,463-471 (1999)] result in late embryonic lethality.McGrath etc. using ribonuclease protecting and In-situ hybridization method confirms, early stage forming primitive gut (E7.5), CXCR4 is the most abundant chemokine receptors mRNA. In gastrula stage, CXCR4/SDF-1 signal seems the migration for mainly inducing former line cell, and expression is existing at this moment fixed On shape entoderm, mesoderm and ectoderm.In E7.2-7.8 mice embryonic, CXCR4 and alpha fetal protein are mutually exclusive, are shown in Visceral endoderm one lacks expression [McGrath, K.E.et al.Dev.Biology 213,442-456 (1999)].

In some embodiments of the present invention, the definitive endodenn cells expression of the method preparation according to the present invention CXCR4 marker gene.In other embodiments, the definitive endodenn cells expression of the method preparation according to the present invention CXCR4 marker gene and other definitive endoderm markers, including but not limited to SOX17, MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.In some embodiments of the invention, definitive endodenn cells The level for expressing CXCR4 marker gene is higher than SOX7 marker gene.In addition, in some embodiments, CXCR4 marker Gene expression is higher than the level of OCT4 marker gene expression.In the other embodiments of the present invention, definitive endodenn cells table It is higher than the level of AFP, SPARC or thrombomodulin (TM) marker gene up to the level of CXCR4 marker gene.In this hair In bright some embodiments, the definitive endodenn cells of method preparation expression CXCR4 according to the present invention are not expressed significantly Horizontal or amount PDX1 (PDX1- is negative).

It should be appreciated that the expression of SOX17 is not repelled in the expression of CXCR4 in endoderm cell.Correspondingly, in the present invention In some embodiments, the level of definitive endodenn cells expression SOX17 and CXCR4 marker gene is above SOX7 marker The level of gene.In addition, in some embodiments, the expression of SOX17 and CXCR4 marker gene is above OCT4 marker The expression of gene.In other embodiments of the present invention, definitive endodenn cells express SOX17 and CXCR4 marker gene Level be higher than the level of AFP, SPARC or thrombomodulin (TM) marker gene.In some embodiments of the invention, The definitive endodenn cells of method according to the present invention preparation expression SOX17/CXCR4 do not express the level of signifiance or amount PDX1 (PDX1- is negative).

It should be appreciated that according to differentiation condition, induced in definitive endodenn cells different level range SOX17 and/or CXCR4 marker expression.In this way, SOX17 marker and/or CXCR4 marker are shaping in some embodiments of the invention Non- setting of the expression ratio SOX17 marker and/or CXCR4 marker of endoderm cell or cell mass in such as multipotential stem cell Expression in endoderm cell or cell mass is at least about 2 times at least about 10,000 times high.In the other embodiments of the present invention, SOX17 marker and/or CXCR4 marker definitive endodenn cells or cell mass expression ratio SOX17 marker and/or CXCR4 marker expresses up at least about 4 times, at least about 6 in the non-definitive endodenn cells or cell mass of such as multipotential stem cell Again, at least about 8 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 40 times, at least about 80 times, at least about 100 Again, at least about 150 times, at least about 200 times, at least about 500 times, at least about 750 times, at least about 1000 times, at least about 2500 times, At least about 5000 times, at least about 7500 times or at least about 10,000 times.In some embodiments, SOX17 marker and/or CXCR4 marker is unlimitedly higher than SOX17 marker and/or CXCR4 mark in the expression of definitive endodenn cells or cell mass Will object is expressed in the non-definitive endodenn cells or cell mass of such as multipotential stem cell.

It should be appreciated that in some embodiments of the invention, and in non-definitive endodenn cells or cell mass The expression of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 marker is compared, GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1 in definitive endodenn cells or cell colony, The expression of CMKOR1 and CRIP1 marker increases.

It will also be understood that in definitive endodenn cells, the expression and OCT4, SPARC of SOX17 marker, AFP, TM and/or SOX7 marker expression level existence difference range.Similarly, in definitive endodenn cells, CXCR4 marker Expression and OCT4, SPARC, AFP, TM and/or SOX7 marker expression level existence difference range.Similarly, in this hair In bright some embodiments, expression ratio OCT4, SPARC, AFP, TM and/or SOX7 of SOX17 marker or CXCR4 marker are marked Up at least about 2 times at least about 10,000 times of the expression of will object.In the other embodiments of the present invention, SOX17 marker or Up at least about 4 times of the expression of expression ratio OCT4, SPARC, AFP, TM and/or SOX7 marker of CXCR4 marker, at least about 6 Again, at least about 8 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 40 times, at least about 80 times, at least about 100 Again, at least about 150 times, at least about 200 times, at least about 500 times, at least about 750 times, at least about 1000 times, at least about 2500 times, At least about 5000 times, at least about 7500 times or at least about 10,000 times.In some embodiments, OCT4, SPARC, AFP, TM And/or SOX7 marker is not significant in definitive endodenn cells expression.

It should be appreciated that in some embodiments of the invention, in definitive endodenn cells, with OCT4, SPARC, AFP, TM and/or SOX7 are compared, and are selected from GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 Marker expression increase.

Composition including definitive entoderm

Some aspects of the present invention are related to the composition of such as cell colony and cell culture comprising such as stem cell Pluripotent cell and definitive endodenn cells.For example, using method of the present invention, can prepare including hESCs mixture and The composition of definitive endodenn cells.It in some embodiments, often include that 95 pluripotent cells are just wrapped in the composition of preparation Include at least about 5 definitive endodenn cells.In other embodiments, often include 5 pluripotent cells just include at least about 95 Definitive endodenn cells.In addition, composition includes the definitive endodenn cells and pluripotent cell of other ratios.For example, combining It often include that 1,000,000 pluripotent cell just includes at least about 1 definitive endodenn cells, often includes more than 100,000 in object Can cell just include at least about 1 definitive endodenn cells, often include 100,000 pluripotent cells just include at least about 1 fixed Shape endoderm cell often includes that 10,000 pluripotent cells just include at least about 1 definitive endodenn cells, often include 1,000 A pluripotent cell just includes at least about 1 definitive endodenn cells, often include 500 pluripotent cells just includes at least about 1 fixed Shape endoderm cell often includes that 100 pluripotent cells just include at least about 1 definitive endodenn cells, often include 10 multipotencys Cell just include at least about 1 definitive endodenn cells, often include 5 pluripotent cells just include at least about 1 definitive entoderm Cell, often include 2 pluripotent cells just include at least about 1 definitive endodenn cells, often include 1 pluripotent cell just include to Few about 2 definitive endodenn cells often include that 1 pluripotent cell just includes at least about 5 definitive endodenn cells, often includes 1 A pluripotent cell just includes at least about 10 definitive endodenn cells, often include 1 pluripotent cell just includes at least about 20 fixed Shape endoderm cell often includes that 1 pluripotent cell just includes at least about 50 definitive endodenn cells, often includes that 1 multipotency is thin Born of the same parents just include at least about 100 definitive endodenn cells, often include 1 pluripotent cell just include at least about 1,000 setting in Endoderm cell often includes that 1 pluripotent cell just includes at least about 10,000 definitive endodenn cells, often includes that 1 multipotency is thin Born of the same parents just include at least about 100,000 definitive endodenn cells, often include 1 pluripotent cell just include at least about 1,000,000 A definitive endodenn cells.In some embodiments of the invention, pluripotent cell is human pluripotent stem cells.In some embodiments In, it is stem cell-derived in mulberry body, embryo inner cell mass or the genital ridge of embryo.In some of the other embodiments, multipotency is thin Born of the same parents are derived from the sexual gland or germinal tissue for having been subjected to the multi-cellular structure of embryo stage development.

Some aspects of the present invention are related to cell culture or cell mass, including at least about 5% definitive endodenn cells to extremely Few about 95% definitive endodenn cells.In some embodiments, cell culture or cell mass include mammalian cell.? In preferred embodiment, cell culture or cell mass include people's cell.For example, cell involved in some specific embodiments is trained Object, including people's cell are supported, wherein at least about 5% at least about 95% people's cell is definitive endodenn cells.The other realities of the present invention The scheme of applying is related to cell culture, including people's cell, wherein at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or the people's cell greater than 90% be definitive endodenn cells.

The other embodiments of the present invention are related to the composition of such as cell culture or cell colony comprising such as people is fixed The people's cell of shape endoderm cell, wherein at least the expression of SOX17 or CXCR4 marker is big in about 5% people's cell In the expression of OCT4, SPARC, alpha fetal protein (AFP), thrombomodulin (TM) and/or SOX7 marker.In other embodiment party In case, wherein at least in about 10% people's cell, at least about 15% people's cell, at least about 20% people's cell, In at least about 25% people's cell, at least about 30% people's cell, at least about 35% people's cell, at least about 40% people In cell, at least about 45% people's cell, at least about 50% people's cell, at least about 55% people's cell, at least about In 60% people's cell, at least about 65% people's cell, at least about 70% people's cell, at least about 75% people's cell In, at least about 80% people's cell, at least about 85% people's cell, at least about 90% people's cell, at least about 95% In people's cell or be greater than 95% people's cell in, the expression of SOX17 or CXCR4 marker be all larger than OCT4, SPARC, AFP, TM and/ Or the expression of SOX7 marker.

It should be appreciated that some embodiments of the invention is related to the composition of such as cell culture or cell mass comprising Such as the people's cell of people's definitive endodenn cells, wherein at least about 5% to more than at least about 95% people's cell, wherein being selected from One or more markers of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 Expression is higher than the expression of OCT4, SPARC, AFP, TM and/or SOX7 marker.

The other embodiments of the present invention are related to the composition such as cell culture or cell mass comprising such as embryo in people's setting The people's cell of confluent monolayer cells, wherein at least about 5% people's cell the expression of SOX17 and CXCR4 marker be above OCT4, SPARC, AFP, TM and/or SOX7 marker expression.In other embodiments, at least about 10% people's cell, at least about In 15% people's cell, at least about 20% people's cell, at least about 25% people's cell, at least about 30% people's cell In, at least about 40% people's cell, at least about 50% people's cell, at least about 55% people's cell, at least about In 60% people's cell, at least about 65% people's cell, at least about 70% people's cell, at least about 75% people's cell, In at least about 80% people's cell, at least about 85% people's cell, at least about 90% people's cell, at least about 95% people In cell or be greater than 95% people's cell in, the expression of SOX17 and CXCR4 marker be above OCT4, SPARC, AFP, TM and/or The expression of SOX7 marker.

It should be appreciated that some embodiments of the invention is related to the composition such as cell culture or cell mass comprising such as The people's cell of people's definitive endodenn cells, at least about 5% to more than at least about 95% people's cell, wherein GATA4, MIXL1, The expression of the marker of HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 be higher than OCT4, SPARC, The expression of AFP, TM and/or SOX7 marker.

The other embodiments of the present invention are related in the composition of such as cell culture or cell mass, including such as people's setting The mammalian endodermal cells of endoderm cell, wherein indicating in the SOX17 at least in about 5% endoderm cell or CXCR4 The expression of object is all larger than the expression of OCT4, SPARC, alpha fetal protein (AFP), thrombomodulin (TM) and/or SOX7 marker. In other embodiments, wherein at least in about 10% endoderm cell, at least about 15% endoderm cell, extremely In few about 20% endoderm cell, at least about 25% endoderm cell, at least about 30% endoderm cell, at least In about 35% endoderm cell, at least about 40% endoderm cell, at least about 45% endoderm cell, at least about In 50% endoderm cell, at least about 55% endoderm cell, at least about 60% endoderm cell, at least about In 65% endoderm cell, at least about 70% endoderm cell, at least about 75% endoderm cell, at least about In 80% endoderm cell, at least about 85% endoderm cell, at least about 90% endoderm cell, at least about In endoderm cell in 95% endoderm cell or greater than 95%, the expression of SOX17 or CXCR4 marker is all larger than The expression of OCT4, SPARC, AFP, TM and/or SOX7 marker.

It should be understood that some embodiments of the invention is related to the composition such as cell culture or cell mass comprising feed Newborn animal endoderm cell, wherein in the endoderm cell of at least about 5% to more than at least about 95%, one or more choosings It is higher than from the expression of the marker of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 The expression of OCT4, SPARC, AFP, TM and/or SOX7 marker.

The other further embodiments of the present invention are related to the composition such as cell culture or cell mass, including such as people is fixed The mammalian endodermal cells of shape endoderm cell, wherein in the SOX17 at least in about 5% endoderm cell and The expression of CXCR4 marker is all larger than OCT4, SPARC, alpha fetal protein (AFP), thrombomodulin (TM) and/or SOX7 mark The expression of object.In other embodiments, wherein at least about 10% endoderm cell, at least about 15% entoderm it is thin In born of the same parents, at least about 20% endoderm cell, at least about 25% endoderm cell, at least about 30% endoderm cell In, at least about 35% endoderm cell, at least about 40% endoderm cell, at least about 45% endoderm cell In, at least about 50% endoderm cell, at least about 55% endoderm cell, at least about 60% endoderm cell In, at least about 65% endoderm cell, at least about 70% endoderm cell, at least about 75% endoderm cell In, at least about 80% endoderm cell, at least about 85% endoderm cell, at least about 90% endoderm cell In, at least about 95% endoderm cell or in the endoderm cell greater than 95%, the expression of SOX17 and CXCR4 marker It is all larger than the expression of OCT4, SPARC, AFP, TM and/or SOX7 marker.

It should be appreciated that some embodiments of the invention is related to the composition of such as cell culture or cell mass, including feed Newborn animal endoderm cell, wherein at least about 5% to more than at least about 95% endoderm cell, GATA4, MIXL1, The expression of the marker of HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 be higher than OCT4, SPARC, The expression of AFP, TM and/or SOX7 marker.

Using method of the present invention, can prepare be substantially free of other cell types include definitive endodenn cells Composition.About the cell in cell culture or cell mass, term " being substantially free of " refers in cell culture or cell mass In specific cell be not present or less than about 5% of total cell number in cell culture or cell mass.Of the invention some The definitive endodenn cells group or cell culture prepared in embodiment using method of the present invention is substantially free of specifically The cell of significant expression OCT4, SOX7, AFP, SPARC, TM, ZIC1 or BRACH marker gene.

In one embodiment of the invention, based on the expression of marker gene, definitive endodenn cells are described as SOX17 high, MIXL1 high, AFP are low, SPARC is low, thrombomodulin is low, SOX7 is low, CXCR4 high.

Enrichment, separation and/or the purifying of definitive entoderm

About other aspects of the invention, definitive endodenn cells can be used specifically for the affinity labeling of these cells come Enrichment, separation and/or purifying.The example of the affinity labeling of specific for definitive endoderm cell is antibody, ligand or other spies The different binding reagents for the marker molecules such as polypeptide, the marker molecules are present in the table of cell shape endoderm cell Face, but be substantially not present in the other cell types found in the cell culture medium of method according to the present invention preparation. In some embodiments, the antibody in conjunction with CXCR4 is used for the enrichment of definitive entoderm, the affine mark for separating and/or purifying Note.In other embodiments, chemotactic factor (CF) SDF-1 or other can also be used for affinity labeling based on the molecule of SDF-1.These points Son includes, but are not limited to SDF-1 segment, SDF-1 fusions or SDF-1 analogies.

Antibody and its method for cell separation are prepared known in the art, these methods can utilize institute of the present invention The antibody and cell stated is implemented.In one embodiment, it will combine CXCR4 antibody in conjunction with magnetic bead, then in cell In culture medium in conjunction with definitive endodenn cells, iuntercellular and the adherency with substrate are reduced after enzyme treated.By cell/anti- Body/bead complexes are exposed in shifting magnetic field definitive endodenn cells and unbonded cell to combine magnetic bead and separate. Once definitive endodenn cells are physically separate with other cells in the medium, the antibody of combination is destroyed, cell is replaced in In suitable tissue culture medium (TCM).

Embodiment of the present invention consider other definitive endodenn cells cultures or cell mass enrichment, separation and/or Purification process.For example, in some embodiments, CXCR4 antibody is incubated in the cell culture comprising definitive entoderm, warp Iuntercellular and the adherency with substrate are reduced after processing.Then by cell washing, centrifugation and settling flux.Cell suspension then again with Secondary antibody, such as can be with the conjugated antibody incubation of the FITC in conjunction with first antibody.Then again by cell washing, centrifugation and settling flux In buffer.With post analysis cell suspension, sorted using fluorescence activated cell sorts device (FACS).By CXCR4^-Negative cells CXCR4 is collected in separation^-Thus positive cell has separated the type cell.When needing, isolated cell composition can be further For several times using the affine method of substitution or increase sorting, the identical or different marker of specific for definitive entoderm is used.

In the other embodiments of the present invention, using in conjunction with CXCR4 ligand or the enrichment of other molecules, separate and/or Purify definitive entoderm.In some embodiments, the molecule is SDF-1 or its segment, fusions or its analogies.

In preferred embodiments, after stem cell culture is induced to break up to definitive entoderm system, from other non-fixed Definitive endodenn cells are enriched with, separate and/or purified in shape endoderm cell.It should be understood that above-mentioned enrichment, separation and/or pure Change method can utilize this culture of any differential period.

Except above-mentioned method, definitive endodenn cells can also be separated with other cell separation technologies.In addition, definitive entoderm Cell can also be enriched under growth conditions by a series of methods being further cultured for or separation, the growth conditions are conducive to described Definitive endodenn cells selectively survival or selective amplification.

It, can be in vitro certainly at least through some differentiation such as stem cell culture or cell mass using method of the present invention Pluripotent cell culture or cell mass in enrichment, separation and/or purifying definitive endodenn cells.In some embodiments, The cell carries out random differentiation.However, the cell primary differentiation is definitive entoderm in certain preferred embodiments.One A little preferred enrichments, separation and/or purification process are related to preparing definitive entoderm from human embryo stem cell in vitro.Use the present invention The method, compared with untreated cell mass or cell culture, the cell mass or cell being enriched in definitive entoderm are trained Supporting object is at least about 2 times to about 1000 times.In some embodiments, compared with untreated cell mass or cell culture, At least about 5 times to about 500 times of the definitive entoderm of enrichment.In other embodiments, it is trained with untreated cell mass or cell Feeding object is compared, and the definitive entoderm of enrichment is at least about 10 times to about 200 times.In other embodiments, with it is untreated thin Born of the same parents group or cell culture are compared, and the definitive entoderm of enrichment is at least about 20 times to about 100 times.In other embodiments, Compared with untreated cell colony or cell culture, the definitive entoderm of enrichment is at least about 40 times to about 80 times.At certain In a little embodiments, compared with untreated cell colony or cell culture, the definitive entoderm of enrichment be at least about 2 times extremely About 20 times.

General description has been carried out to the present invention, has been further appreciated that the present invention with reference to some specific embodiments, this The purpose that these embodiments of invention are merely illustrative, and it is not intended to the limitation present invention.

Embodiment

Following many embodiments describe the use of multipotency people's cell.The method for preparing multipotency people's cell is that this field is general Technical staff is known, a large amount of technical presses, including U.S. Patent number 5,453,357,5,670,372,5, and 690,926,6, 090,622,6,200,806 and 6,251,671 and U.S. Patent Application Publication No. 2004/0229350 be described, herein by it It is fully incorporated, as reference.

Embodiment 1

People's ES cell

To study endoderm development, it is multipotential stem cell, seeming in the medium can that we, which utilize human embryo stem cell, Infinitely to divide, while keeping normal caryotype.The cell-derived embryo inner cell mass in 5 days sizes of ES, using exempting from Epidemiology or mechanical means separation.In particular, human embryonic stem cell hESCyt-25 is from the IVF cycles agreed to through patient Supernumerary frozen embryo.After thawing, the blastaea of hatching is inoculated on mouse embryonic fibroblasts (MEF), is cultivated using ES Base (DMEM, 20%FBS, nonessential amino acid, beta -mercaptoethanol, ITS regulator).About after two weeks, embryo is adhered to culture In ware, by undifferentiated hESCs zone-transfer to MEFs.Transfer is completed with machine cuts, after simply being digested using neutral proteinase, Cell cluster is removed with machinery, washs, inoculate.After self-derivedization, continuous passage hESCyt-25 100 times.We utilize HESCyt-25 human embryonic stem cell prepares definitive entoderm as starting material.

Persons skilled in the art should be understood that stem cell or other pluripotent cells also are used as of the present invention point The starting material of change method.For example, embryo's sexual gland crest cell can be separated according to methods known in the art, it is used as pluripotent cell Starting material.

Embodiment 2

The characterization of hESCyt-25

Human embryonic stem cell cultivate 18 middle of the month be always maintained at normal morphology, caryotype, growth and oneself My renewing speciality.The cell line shows strong immunoreactivity, above-mentioned antigen to OCT4, SSEA-4 and TRA-1-60 antigen All it is the feature of undifferentiated hESCs, and shows the identical alkaline phosphatase activity of other established hESC systems and morphology.This Outside, it when human stem cell system hESCyt-25, which suspends, to be cultivated, is also easy to form class embryoid body (EBs).Due to the card of its multipotency essence Bright, hESCyT-25 is divided into the different cell types for representing three kinds of principal germ layers.ZIC1 and Tri-labeling method are detected with Q-PCR (ICC) detection nestin and more mature neural markers are learned to confirm ectodermic generation.The immunocyte of β-III micro-pipe Chemical staining can observe there is the feature of early stage neuron in the cell cluster of elongation.Before this, we are including retinoic acid EBs induced multipotent stem cells are handled in suspension is divided into the visceral endoderm one (VE) outside an embryo being.By 54 hours Processing, two VE markers of the alpha fetal proteins (AFP) of the cells express high levels of processing, SOX7.Immunocytochemical stain is aobvious Show with the cell expression AFP of single layer differentiation for fragmentary sheet.As described below, hESCyT-25 cell line can also be formed in setting Germinal layer is confirmed by realtime quantitative inspection (Q-PCR) and detection SOX17, the immunocytochemistry without AFP expression. In order to confirm to be divided into mesoderm, in the EB that several time point analysis are breaking up to detect short-tail (Brachyury) gene table It reaches.During the test, Brachyury expression progressive increases.As previously mentioned, hESCyT-25 system is multipotency, it is capable of forming Represent the cell of three germinal layers.

Embodiment 3

The preparation of SOX17 antibody

The main bottleneck that definitive entoderm is identified in hESC culture is to lack proper implements.We thus prepare anti- The antibody of SOX17 albumen.

When being formed during primitive gut embryogenesis, marker SOX17 is expressed in entire definitive entoderm, and its expression is maintained at Intestinal tube (axis is variant although expression prolongs A-P) is until organ initially forms.SOX17 is also expressed in a series of extraembryonic endoderms On cell.This protein is in mesoderm or ectoderm without expression.When excluding pedigree outside embryo in conjunction with other markers, It was found that SOX17 is the suitable marker of definitive entoderm pedigree.

As detailed herein, in order to prepare the definitive endodenn cells of the SOX17 positive, SOX17 antibody is used for specific detection The effect of various processing and differentiation method.Other antibody reacted with AFP, SPARC and thrombomodulin are also used in exclusion The generation of dirty and parietal endoderm (extraembryonic endoderm).

In order to prepare the antibody of anti-SOX17, according to Antibody preparation company GENOVAC (Freiberg, Germany) exploitation Method, groups of people SOX17 cDNA corresponding with carboxylic terminal amino acid 172-414 (SEQNO:2) of SOX17 albumen (attached drawing 2) (SEQIDNO:1) it is used for the inherent immunity of rat.The method of inherent immunity is found in U.S. Patent number 5,830,876,5,817, 637,6,165,993 and 6,261,281 and International Patent Application Publication No. WO 00/29442 and WO99/13915, is disclosed in This is introduced, for reference.

Other methods of inherent immunity are also seen in non-patent literature.For example, according to inherent immunity system described in Barry etc. Standby monoclonal antibody, Biotechniques 16:616-620,1994, it is published here and is fully incorporated, it is for reference.According to something lost The immune specific method for preparing anti-differential protein antibody is passed, for example, the something lost of Costaglia etc. (1998) human thyrotropin receptor Pass the immune monoclonal antibody for causing thyroiditis and preparation identification autoreceptor, J.Immunol.160:1458-1465； The immune-mediated mouse monoclonal antibody of Flt-3 receptor based on DNA of Kilpatrick et al. (1998) particle gun transmission Quickly preparation, Hybridoma 17:569-576；Schmolke et al., (1998) are generated in human serum by DNA immunization E2- monoclonal antibody specific identifies hepatitis G virus particle J, Virol.72:4541-4545；Krasemann et al., (1999) The monoclonal antibody for being directed to albumen is generated using unconventional nucleic acid immunization strategy, J.73:119-129；And Ulivieri et al. (1996) monoclonal antibody that helicobacter pylori cavitating toxin determines part, 51:191-194, above-mentioned public affairs are generated by DNA immunization This is opened in be fully incorporated, it is for reference.

As shown in the relational tree of Fig. 3, in Sox family, SOX7 and SOX18 and SOX17 are most close.We are more using people SOX7 Peptide confirmed as negative control SOX17 antibody be to SOX17 it is special, do not reacted with most similar family member.In particular, In order to prove inherent immunity generate antibody be to SOX17 it is special, by SOX7 and other protein expressions in human fibroblasts On, then pass through the overall reaction activity of Western blot and ICC analysis and SOX17 antibody.For example, being prepared using following methods The expression vector of SOX17, SOX7 and EGFP are transfected into human fibroblasts and are analyzed with Western blot.For Prepare SOX17, SOX7 and EGFP expression vector be respectively pCMV6 (OriGene Technologies, Inc., Rockville, MD), pCMV-SPORT6 (Invitrogen, Carlsbad, CA) and pEGFP-N1 (Clonetech, Palo Alto,CA).To prepare albumen, transiently transfected using Lipofectamine 2000 with super coiled DNA telomerase immortalized MDX human fibroblasts (Invitrogen, Carlsbad, CA).After transfection 36 hours, total cell lysate is collected in 50mM TRIS-HCl (pH 8), 150mM NaCl, 0.1%SDS, 0.5% deoxycholic acid and some protease inhibitors (Roche Diagnostics Corporation, Indianapolis, IN) in.Western blot analyzes the cell protein of 100 μ g, with SDS-PAGE is separated at NuPAGE (4-12% gradient Polyacrylamide, Invitrogen, Carlsbad, CA), passes through electroblotting (Hercules, CA) is transferred on PDVF film, in 10mM TRIS-HCl (pH 8), 150mM NaCl, 10%BSA, 0.05% It is diluted to 1/1000 mouse SOX17 antiserum in Tween-20 (Sigma, St.Louis, MO) to be detected, then to combine alkali Property phosphate anti-rat IgG handle (Jackson ImmunoResearch Laboratories, West Grove, PA), As a result (Vector Laboratories, Burlingame, CA) is shown with Vector Black alkaline phosphate ester enzyme dyeing.Make Albumen size criteria is the color sign object (Sigma, St.Louis, MO) of wide scope.

In Fig. 4, by the Protein Extraction of the human fibroblasts transiently transfected from SOX17, SOX7 or EGFP cDNA Object carries out Western blot by probe of SOX17 antibody.The protein only extracted in the cell of hSOX17 transfection produces about The band of 51Kda, the band are most matched with the 46Kda molecular weight of people's SOX17 protein of prediction.SOX17 antibody is to from people SOX7 Or the extract of EGFP transfection cell does not have reactivity.It is constructed in addition, SOX17 antibody is significantly marked to express with hSOX17 The human fibroblasts core of body transfection, but the cell individually transfected with EGFP is not marked.Similarly, SOX17 antibody, which is shown, passes through The specificity of ICC detection.

Embodiment 4

Confirmation of the SOX17 antibody as definitive endoderm markers

There is specificity and further marks definitive endoderm according to SOX17 antibody on human SOX17 protein, by part point HESCs and SOX17 and the AFP antibody of change mark jointly.It has been proved SOX17, SOX7 and AFP and has respectively been expressed in visceral endoderm one In, SOX7 is the closely related member (Fig. 3) of SOX gene family F subgroup.However, AFP and SOX7 are in definitive endodenn cells Expression in the level that can be detected by ICC, therefore, the negative mark of bonifide definitive endodenn cells can be used as Object.According to display, SOX17 antibody labels cell colony exists with the cell mass dispersed, or mixes with AFP positive cell.In particular, Fig. 5 A shows a small amount of SOX17 cell indicated jointly with AFP；However, it has been found that some regions, wherein in SOX17⁺Cell neck There is on a small quantity in domain or do not have AFP⁺Cell (Fig. 5 B).Similarly, because it is also reported that parietal endoderm express SOX17, can By the antibody indicated jointly with SOX17 and parietal markers SPARC and/or thrombomodulin (TM) together for identifying body wall The SOX17 of entoderm⁺Cell.As shown in attached drawing 6A-C, by the random differentiation of hES cell generate thrombomodulin and The parietal endoderm cells that SOX17 indicates jointly.

According to above-mentioned cell labelling experiments, the characteristic of definitive endodenn cells can be composed SOX17 by marker expression^hi/ AFP^lo/[TM^loOr SPARC^lo] establish.In other words, the expression of SOX17 marker is higher than AFP marker and TM or SPARC mark The expression of object, AFP marker are a kind of feature of visceral endoderm one, TM or SPARC marker is the feature of parietal endoderm.Cause This, those are positive to SOX17 and are negative to AFP and the cell being negative to TM or SPARC is definitive entoderm.

SOX17^hi/AFP^lo/TM^lo/SPARC^loThe specificity of marker expression spectrum can be used as definitive entoderm prediction into one Evidence is walked, it can be quantitatively suitable with the relative populations of antibody labeled cells by SOX17 and AFP gene expression.As shown in Figure 7 A, with Retinoic acid (visceral endoderm inducer) or the hESCs of activin A (definitive endoderm inducer) processing are resulted in 10 times of differences of SOX17mRNA expression.The result reflects 10 times of differences (figure of the cell quantity of SOX17 antibody label 7B).In addition, as shown in Figure 8 A, compared with untreated, the activin A processing of hESCs inhibits 6.8 times of AFP gene expression. The sharply reduction for the cell quantity that AFP is marked in these cultures visually reflects this variation, as shown in Fig. 8 B-C. It further to quantify, is measured with flow cytometer, it was demonstrated that about 7 times of AFP gene expression are reduced to the thin of AFP antibody label Born of the same parents' quantity about reduces by 7 times of result (attached drawing 9A-B).The result is extremely important, shows the gene expression observed in Q-PCR Quantity variation, reflect by antibody dyeing observation cell type specification typically change.

HESCs is cultivated under the conditions of Nodal family member (Nodal, activin A and activin B-NAA) is existing, is caused The cell of SOX17 antibody labels is obviously increased with the time.Continuously with activin processing 5 days, there is the cell quilt more than 50% SOX17 marks (Figure 10 A-F).After activin is handled 5 days, indicated almost without cell with AFP.

In short, 242 antibody aminos of carbon teminal of generated anti-human SOX17 protein, identify in Western blots People SOX17 protein but unidentified SOX7 are the close relative of nearest Sox family.SOX17 antibody identifies the hESC of differentiation Cell subsets in culture, the subgroup are mainly SOX17⁺/AFP^lo/-It is (indicator cell line more than 95%) and a small amount of (< 5%) The cell (visceral endoderm one) that indicates jointly of SOX17, AFP.HESC culture is handled with activin, produces SOX17 gene Expression and SOX17 label cell significantly rise, and significant the cell for inhibiting AFP mRNA to express and mark with AFP antibody Quantity.

Embodiment 5

Q-PCR gene expression analysis

In following tests, real-time quantitative RT-PCR (Q-PCR) is for the various processing of screening to hESC differentiation Main method.In particular, analyzing multiple marker gene at multiple time points with Q-PCR the real time measure gene expression.Evaluation expectation With the Marker genes characteristic of undesirable cell type, to obtain more preferable understanding to cell colony overall dynamics.Q-PCR points The strength of analysis includes its extreme sensitivity and because genome sequence is easy to relatively easily develop necessary marker due to utilization. In addition, Q-PCR high sensitivity allows to detect the gene expression of relatively small amount cell in biggish group.In addition, detection The ability of the gene expression of extremely low level provides the instruction of intragroup " differentiation tendency ".At obvious point of these cell phenotypes Before change, it cannot be identified to the tendentiousness of specific differentiation pathway with immunocytochemical technique.Thus, Q-PCR provides one Kind analysis method, this method are at least the supplement of immunologic cellular activity technology, and are potentially better than immunologic cellular activity technology, Break up processing successful for screening.In addition, Q-PCR provides a kind of mechanism, the mechanism in half high-throughput scale by determining Whether amount assay differentiation method succeeds.

Method relative quantification used herein, at Rotor Gene 3000instrument (Corbett Research) It is upper to be operated using SYBR Green chemistry and two one step RT-PCRs.This method allows to store cDNA sample, to analyze the other of future Therefore marker gene avoids the variability of sample room reverse transcription efficiency.

If may, the primer of design is positioned at exon-exon boundaries or crosses at least introne of 800bp, because This can rule of thumb eliminate the amplification of the genomic DNA of pollution.When mark base of the use without introne or without pseudogene Because when, carry out RNA sample DNA enzymatic I processing.

We measure the gene expression of multiple markers of target and non-target cell type usually using Q-PCR, to mention For describing the wide in range express spectra of cell sample gene expression.By hESC differentiation early stage (specifically, ectoderm, mesoderm, in setting Germinal layer and extraembryonic endoderm) Research of predicting markers and available verifying primer be provided in following table 1.Also these primers have been proved The human specific of group.The fact is critically important, because usually hESCs is grown on Mouse trophoblast.Most commonly, each condition 3 samples are taken, and are independently analyzed twice, to evaluate biologic variability relevant to various quantitative detections.

To prepare pcr template, total serum IgE is separated with RNeasy (Qiagen), and with RiboGreen (Molecular Probes) quantitative.The reverse transcription of 350-500ng total serum IgE, the kit are completed with iScript reverse transcription reagent box (BioRad) Mixture comprising oligomerization dT and random primer.Each 20 μ L reactant is then diluted to 100 μ L total volumes, 3 μ L is taken to be used for respectively 10 μ L Q-PCR reaction, the reaction include 400nM forward primer and reverse primer and 5 μ L 2X SYBR Green master mix(Qiagen).Select two step loop parameters, 85-94 DEG C 5 seconds denaturation (according to the melting temperature of each primer sets amplicon into Row specific choice), then 60 DEG C annealing/extension 45 seconds.Fluorescence data is collected during last 15 seconds of each extended peroid.By 10 Three points of times dilution series are used to generate the standard curve of each wheel, and will recycle threshold (Ct ' s) based on the standard curve and be changed into Quantitative value.The numerical value of each sample is characterized with house-keeping gene and is calibrated, the average and standard deviation of three samples is then calculated.It is tying When beam PCR cycle, the specificity of reaction is determined with melting curve analysis.Single specific product is shown and is closed to PCR amplification Suitable T_mAt simple spike.In addition, not expanded using the reaction for not having reverse transcriptase as negative control.

It is to determine suitable house-keeping gene (HGs) in test system in the first step established in Q-PCR methodology.Due to HG It is significant to make to calibrate for sample room RNA input, RNA integrality and RT efficiency calibration, HG in all samples type at any time Between constant expression level be valuable.We determine Cyclophilin G, hypoxanthine phosphoric acid core in differentiation hESCs Sugared transferase 1 (HPRT), beta-2-microglobulin (microglobulin), hydroxymethylbiane synzyme (HMBS), The expression of TATA- binding protein (TBP) and glucoronidase β (GUS).Our result show that β -2- microballoon egg White expression improves in atomization, and therefore, we eliminate is used to calibrate by the gene.Other gene expression doses with It is consistent with entire treatment process at any time.We usually calculate the school of all samples using Cyclophilin G and GUS simultaneously Quasi- coefficient.The intrinsic variation of calibration process is reduced using multiple HGs simultaneously, increases the reliability of relative gene expression values.

After obtaining the gene for calibration, Q-PCR is used for detection and receives sample after different tests are handled, is determined many The relative gene expression level of marker gene.Marker gene selected to use is rich in the representative specific populations of early stage germinal layer Collection, especially there is differentially expressed multiple groups gene in definitive entoderm and extraembryonic endoderm.By these genes and its related richness Collect feature to highlight in table 1.

Table 1

Because many genes are expressed in more than one germinal layer, the expression water of quantitative comparison many genes in same test Flat is useful.SOX17 is expressed in definitive entoderm, is expressed to lesser extent in internal organ and parietal endoderm.SOX7 and AFP is expressed in visceral endoderm one in the development time point of early stage.SPARC and TM are expressed in parietal endoderm, and Brachyury is expressed in early stage mesoderm.

Predict definitive endodenn cells high level expression SOX17mRNA, low expression level AFP and SOX7 (embryo in internal organ Layer), SPARC (parietal endoderm) and Brachyury (mesoderm).In addition, ZIC1 is used to further exclude early stage by the present invention Ectodermic induction.Finally, GATA4 and HNF3b is expressed in setting and extraembryonic endoderm simultaneously, therefore, shaping with SOX17 Expression in entoderm is related (table 1).Representative test is shown in Figure 11-14, it was confirmed that marker gene described in table 1 is such as What is relative to each other with each sample, thus highlights the spy of definitive entoderm, extraembryonic endoderm, mesoderm and neural cell type Different differentiation model.

Above-mentioned clear data shows activin dose increase, and caused SOX17 gene expression increases.Further, should SOX17 expression main representative definitive entoderm, rather than opposite extraembryonic endoderm.The conclusion of observation is SOX17 gene expression With AFP, SOX7 and SPARC inverse correlation.

Embodiment 6

People's ES cell directional is divided into definitive entoderm

If people ES cell culture is cultivated in the case where not keeping its undifferentiated state initiatively, the culture is by random differentiation. Different differentiation result in extra-embryonic endoderm cells comprising body wall and visceral endoderm one (AFP, SPARC and SOX7 expression) The two, and the ectoderm indicated, mesoderm are expressed as with ZIC1, Nestin (ectoderm) and Brachyury (mesoderm) Derivative.In ES cell culture, due to lacking specific antibody marlcers, definitive endodenn cells appearance is without conventional method It detects or determines.Similarly, due to lacking means, early stage definitive entoderm is generated from the culture of ES cell without carefully grinding Study carefully.Due to the antibody reagent without good, available definitive endodenn cells, overwhelming majority confirmation is concentrated on outside ectoderm and embryo Entoderm.In short, in random differentiation ES cell culture, with SOX17^hiDefinitive endodenn cells are compared, and are had significant a large amount of Embryo it is outer and neurectodermal cell types generate.

When by undifferentiated hESC be cloned in fibroblast nutrient expand when, the edge of clone show with clone in The different morphological feature of portion's cell.Many border cells can be with the high level of its biggish inhomogenous cell body morphology and OCT4 Expression is to distinguish.Have been described, when ES cell starts differentiation, they express OCT4 horizontally relative to undifferentiated ES cell Level is in change up and down.Relative to the OCT4 threshold level of neoblast, changes display up and down and leave the initial of multipotency state Differentiation state.

Surrounding and junction when undifferentiated clone is with SOX17 Immuncytochemical detection, in undifferentiated ESC clone Once in a while can random detection to SOX17 positive cell 10-15 cell form tuftlet.As described above, when clone's volume augmentation So that clone's outer rim is distributed bag-shaped seemingly from first cell of classical ESC Morphological Differentiation when becoming crowded.It is cloning Inside and edge age is smaller, undifferentiated clone (< 1mm of small volume；4-5 days sizes) without SOX17 positive cell, so And older, the larger clone's (1-2mm diameter, > 5 days sizes) of volume within the periphery and edge of some clones contain zero Star, the free sheet SOX17 positive, AFP negative cell, as described above, its hESC Morphological Differentiation from classics.In view of This is the exploitation for the first time of effective SOX17 antibody, the definitive endodenn cells generated in early stage " undifferentiated " the ESC culture Not previously confirm.

The negative correlation of SOX17 and SPARC gene expression dose based on Q-PCR measurement, most SOX17 positives, AFP Negative cells will be negative to the parietal markers that antibody marks altogether.It is thin in the parietal endoderm of Expression of TM as shown in Figure 15 A-B Born of the same parents are specifically confirmed.The play for leading to the number of TM expression intensity and TM positive cell is contacted with Nodal factors Activin A and B Drop.On the culture medium of activin processing, by using three heavy labels of SOX17, AFP and TM antibody, observe to AFP and TM Also the SOX17 positive cell (Figure 16 A-D) being negative.These are all that cell confirms SOX17 for the first time on the culture of differentiation ESC Positive definitive endoderm cell (Figure 16 A-D and 17).

Using above-mentioned SOX17 antibody and Q-PCR tool, we developed it is a series of can effective procedure ESCs become For SOX17^hi/AFP^lo/SPARC/TM^loThe method of definitive endodenn cells.We apply that a series of to be intended to increase these thin The differentiation method of born of the same parents' quantity and proliferative capacity detects SOX17 gene expression using Q-PCR on population level, in individual cells It is upper to be marked using SOX17 protein antibodies.

We analyze for the first time and describe TGF 'beta ' family growth factor, such as Nodal/ activin/BMP, are used for from external thin The effect of definitive endodenn cells is created in the embryonic stem cell of born of the same parents' culture.In typical test, we are by activin A, work Change element B, BMP or combinations thereof addition and starts atomization into undifferentiated human stem cell system hESCyt-25 culture.

As shown in figure 19, the activin A that 100ng/ml concentration is added breaks up 4 days, compared with undifferentiated hESCs, induction 19 times of SOX17 gene expressions.Second member's activin B that activin family is added together with activin A passes through 4 days groups Activin processing is closed, compared with undifferentiated hESCs, induction of 37 times of SOX17 gene expressions.With activin A and activin B one With the third member BMP4 that TGF 'beta ' family Nodal/ activin and BMP subgroup is added, compared with undifferentiated hESCs, induction 57 times of SOX17 gene expressions (Figure 19).When with activin and BMP induction SOX17, and the culture of the factor is not added to photograph Than differentiation leads to 5-, 10- and 15- times of induction for 4 days.It is induced by triple processing of 5 days activin As, B and BMP, SOX17 70 times of hESCs high of multiple ratio or more.These data show, with Nodal/ activin TGF 'beta ' family member higher dosage, longer The processing time causes SOX17 expression to increase.

Nodal and related activation element A, B and BMP molecule promote the expression of SOX17, shape external in definitive entoderm body At.SOX17 induction is caused to increase in addition, BMP is added, it may be possible to pass through the further induction of Nodal co-receptor Cripto.

We have demonstrated that being used in combination in the increase and setting in turn that activin A, B and BMP4 cause SOX17 to induce Germinal layer is formed.It is combined with activin A and B, embryo in can induce body wall and visceral endoderm one for BMP4 (> 4 days) and shaping is added for a long time The SOX17 of layer increases.Therefore, in some embodiments of the present invention, it is important for BMP4 being removed in 4 days that processing is added 's.

In order to determine the effect of TGF β factor treatment on individual cell level, detection is marked to be added one using SOX17 antibody The effect of the TGF β factor of a time-histories.As shown in preceding Figure 10 A-F, as the time carries out, the relative populations of the cell of SOX17 label Increase severely.Relative quantification (Figure 20) shows that the cell of SOX17- label increases by 20 times or more.Should the result shows that, with the TGF β factor The exposed time increases, and cell quantity and gene expression dose increase.As shown in figure 21, with Nodal, activin A, activin After B and BMP4 is contacted 4 days, the level of SOX17 induction is than 168 times of undifferentiated hESCs high.Figure 22 shows SOX17 positive cell Quantity be also in dose dependent.100ng/mL or the activin A of higher doses can forcefully induce the gene table of SOX17 It reaches and cell quantity increases.

Except TGF 'beta ' family member, Wnt family molecule may work in definitive entoderm specificity and/or holding.With It is applied alone activin to compare, is increased using the sample SOX17 gene expression of activin+Wnt3a, show that Wnt molecule breaks up hESCs It is definitive entoderm also beneficial to (Figure 23).

Above-mentioned all tests carry out in the tissue culture medium (TCM) containing 10% serum and the addition factor.It is especially surprising that We have found that serum-concentration has effect to SOX17 expression in the presence of the activin of addition, as shown in Figure 24 A-C.Work as blood Clear water is flat when being down to 2% by 10%, and in the presence of activin A and B, the expression of SOX17 increases by 3 times.

Finally, we demonstrate that activin induces SOX17⁺Cell divides in culture, as shown in Figure 25 A-D.Arrow is aobvious Show and m period is in the cell of SOX17/PCNA/DAPI label, evidence is the mitosis plate of PCNA/DAPI- label Mode and phase difference mitotic figures.

Embodiment 7

The expression of Chemokine receptor4 (CXCR4) it is related to definitive endoderm markers and with mesoderm, ectoderm or Visceral endoderm marker is uncorrelated

As described above, ESCs can by using TGF 'beta ' family and more special activin/nodal subtribe cell factor It is induced to differentiate to definitive endoderm.In addition, we have shown that ratio of the fetal calf serum (FBS) in differential medium Influence the efficiency that definitive entoderm breaks up from ESCs.The effect is, in the medium under conditions of set activin A concentration, The FBS of higher level will inhibit its maximum differentiation to definitive endoderm.When lacking exogenous activin A, ESCs breaks up to fixed The efficiency of shape entoderm pedigree is extremely low, and FBS concentration has weaker effect to the atomization of ESCs.

In these trials, the differentiation of hESCs is in RPMI culture medium (Invitrogen, Carlsbad, CA；cat# Growth 6 days in 61870-036), the culture medium are supplemented with 0.5%, 2.0% or 10%FBS, and packet is activated with or without 100ng/mL Plain A.In addition, the gradient FBS of 0.5%-2.0% is also used in combination with 100ng/mL activin A in the first three days of differentiation.6 days Afterwards, duplicate sample is collected, from each condition of culture with the expression of Real-time PCR Analysis Relative gene.Remaining cell is mixed, With Immunofluorescence test SOX17 albumen.

Under 7 condition of culture used, the expression difference of CXCR4 is huge (Figure 26).Generally, CXCR4 is expressed It is high in the culture medium (A100) of activin A processing, and it is low in the culture medium of no exogenous activin A (NF).In addition, in A100 In the culture medium of processing, when FBS concentration is minimum, CXCR4 expresses highest.Under the conditions of 10%FBS, the horizontal significant drop of CXCR4 It is low, so that the condition of the more identical no activin A (NF) of relative expression.

As described above, the feature phase one of the expression of SOX17, GSC, MIXL1 and HNF3 β gene and definitive endodenn cells It causes.Relative expression of this 4 genes under 7 differentiation conditions has hinted obliquely at the expression of CXCR4 (Figure 27 A-D).This demonstrates CXCR4 is also a kind of definitive endoderm markers.

Ectoderm and mesodermal lineage can be distinguished by the various markers of its expression with definitive entoderm.Early interim embryo Layer expression Brachyury and MOX1 gene, however, nascent neuro-ectoderm expresses SOX1 and ZIC1.Figure 28 A-D is confirmed without external source The culture of activin A is conducive to mesoderm and ectoderm gene expression, in the culture of activin processing, 10%FBS item Part also increases the level of mesoderm and ectoderm marker expression.These expression patterns and CXCR4 mode are on the contrary, be shown in this Time-histories is developed, CXCR4 is not high to be expressed in derived from ESCs mesoderm or ectoderm.

In mammalian development early stage, also broken up to pedigree outside embryo.The differentiation of visceral endoderm one has spy herein Different correlation, the common many genes with definitive entoderm, including SOX17 expression having the same.In order to it will shape in Germinal layer and the outer visceral endoderm one of embryo are distinguished, and the different marker of the two should be detected.SOX7 represents expression in visceral endoderm one, and It is not the marker of definitive entoderm pedigree.Thus, under conditions of no SOX7 is expressed, show the training of strong SOX17 gene expression Supporting object condition may include definitive entoderm, rather than visceral endoderm one.As shown in Figure 28 E, SOX7 is in no activin A culture medium Height expression, SOX7 even existing for the activin A under the conditions of, when FBS includes 10%, also expression increases.The mode and CXCR4 Expression pattern is on the contrary, show CXCR4 in the not high expression of visceral endoderm one.

Also have detected SOX17 immunocompetence (SOX17 under above-mentioned each differentiation condition⁺) cell relative populations.When hESCs exists When breaking up under high dose activin A and low FBS concentration (0.5%-2.0%), SOX17⁺Cell is generally distributed in culture.When Using high dose activin A, and when FBS concentration is 10% (v/v), SOX17⁺The frequency that cell occurs reduces, often with isolated Cluster occur, rather than uniformly publication in culture (Figure 29 A, C, B and E).As no exogenous activin A in use, discovery SOX17⁺Cell further decreases.Under these conditions, SOX17⁺Cell also occurs with tufted, but these clusters are smaller and higher work When changing element A, low FBS processing few (Figure 29 C and F).These results indicate that CXCR4 expression pattern not only meets under various conditions Definitive endoderm gene expression, and meet the quantity of definitive endodenn cells.

Embodiment 8

The differentiation condition for being enriched with definitive entoderm increases the ratio of CXCR4 positive cell

The dosage of activin A also affects definitive entoderm efficiency derived from ESCs.The present embodiment increases activin A Dosage increases CXCR4⁺The ratio of cell in culture.

HESCs (at first 3 days of differentiation, is gradually increased to 1.0% by 0.5%, then extremely being added to 0.5%-2%FBS 2.0%) and in the RPMI culture medium of 0,10 or 100ng/mL activin A break up.After differentiation 7 days, cell is being free of into Ca²⁺/Mg^{2 +}, comprising 2%FBS and 2mM (EDTA) PBS in room temperature dissociate 5 minutes.With 35 μm of nylon filter filtration cells, counts and sink It forms sediment.Precipitating is resuspended in 50% human serum/50% normal donkey serum, 2 minutes blocking nonspecific antibodies is incubated on ice and combines Site.It (include about 10 to every 50 μ L⁵A cell) 1 anti-CXCR4 antibody (Abcam, the cat#ab10403- of μ L mouse of suspension addition 100), then on ice label 45 minutes.It includes the PBS washing cell of 2% human serum (buffer), precipitating that 5mL, which is added,.Again with After 5mL buffer washed once, by cell again with 50 buffer/10 μ L⁵Cell concentration suspends.It is added final concentration of 5 μ g/mL's Secondary antibody (in conjunction with FITC donkey anti-mouse antibody；Jackson Immuno Research, cat#715-096-151), label Again with above-mentioned buffer washing 2 times after 30 minutes.By cell again with 5x10⁶Cell/mL is suspended in buffer, by fluidic cell Instrument equipment operator (The Scripps Research Institute) uses FACS Vantage (Beckton Dickenson it) analyzes, sort.Cell is collected directly from RLT lysis buffer (Qiagen) to separate for subsequent total serum IgE, then Gene expression analysis is carried out with real-time quantitative PCR.

When dosage (Figure 30 A-C) of the activin A in differential medium increases, flow cytometer measurement can be observed CXCR4⁺Cell also increases severely (Figure 30 A-C).CXCR4⁺Cell falls into R4, which is used only secondary antibody conduct and compares, in R4 There are the 0.2% control events.When the dosage of activin A increases, CXCR4⁺The sharp increase of cell quantity and definitive entoderm Gene expression obviously increases related (Figure 31 A-D).

Embodiment 9

Concentration and separation CXCR4 positive cell expresses for definitive endoderm gene, removes expression mesoderm, ectoderm and internal organ The cell of entoderm marker

Collect the CXCR4 that above-described embodiment 8 identifies⁺And CXCR4^-Cell analyzes the expression of its Relative gene, measures simultaneously The gene expression of mother cell group.

When the dosage of activin A increases, CXCR4⁺The relative level of gene expression increases severely (Figure 32).This and activin A Dose dependent increases CXCR4⁺Cell is related good (Figure 30 A-C).It is also obvious that isolating CXCR4 from each cell mass⁺Cell Occupy almost all of CXCR4 gene expression cell in the cell mass.Which demonstrate FACS methods to collect these cellular processes Efficiency.Gene expression analysis shows CXCR4⁺Cell not only includes most of CXCR4 gene expression, but also including other The gene expression of definitive endoderm markers.As shown in Figure 31 A-D, further from the mother of SOX17, GSC, HNF3B and MIXL1 Body A100 cell mass is enriched with CXCR4⁺Cell.In addition, CXCR4^-Part includes these few definitive endoderm markers gene tables It reaches.Moreover, CXCR4⁺And CXCR4^-Cell mass shows that mesoderm, ectoderm and extraembryonic endoderm marker gene expression are opposite Mode.Figure 33 A-D shows to remove CXCR4 relative to A100 mother cell group⁺Cell is to carry out Brachyury, MOX1, ZIC1 And SOX7 gene expression.Relative to condition low or without activin A, which has expressed these markers very It is low.These results indicate that there are the CXCR4 separated under differentiation condition from hESCs for high dose activin A⁺Cell obtains height The substantially pure definitive endodenn cells of enrichment.

Embodiment 10

Use the definitive endodenn cells in CXCR4 quantitating cell populations body

Ratio in order to determine definitive endodenn cells in cell culture or cell mass is quantitative, according to preceding method or in U.S. Provisional Patent Application the 60/532nd, 004 submitted on December 23rd, 2003, side described in entitled " definitive entoderm " Method is published here and is fully incorporated by reference, and expresses the thin of CXCR4 and other definitive endoderm markers with facs analysis Born of the same parents.

Using method described in such as above-described embodiment, hESCs is broken up and generates definitive entoderm.In particular, increasing table Up to the yield and purity in noble cells culture, serum-concentration strict control is as follows in culture medium: first day 0.2%FBS, Two days 1.0%FBS and the 3-6 days 2.0%FBS.Three kinds of cell surface determinants CAM 120/80s are used with FACS (Cadherin), CXCR4 and the culture of thrombomodulin sorting differentiation.Then sorting cell mass is analyzed with true with Q-PCR Determine the marker relative expression levels of definitive entoderm, extraembryonic endoderm and other cell types.It is obtained from best differentiation culture The CXCR4 sorting cell obtained produces the separation of the definitive endodenn cells of > 98% purity.

Table 2 shows the definitive entoderm culture analysis of markers broken up using method of the present invention from hESCs Result

Table 2

The ingredient of definitive entoderm culture

In particular, table 2 shows that CXCR4 and SOX17 positive cell (entoderm) includes in the cell culture of 70%-80% Cell.In the cell of these expression SOX17, it is lower than 2% Expression of TM (parietal endoderm), lower than 1% expression AFP (internal organ Entoderm).When subtracting from SOX17/CXCR4 positive cell ratio, TM is positive and AFP positive cell (body wall and visceral endoderm one Combination；It amounts to 3%), it is possible to find about 67%-77% cell culture is definitive entoderm.About 10% cell is viscous for E- calcium Albumen (ECAD) is positive, is hESCs marker, and the cell of about 10-20% is other cell types.

It has been found that with it is above-mentioned FBS concentration is maintained in differentiation operation in entire 5-6 days < 0.5% low blood Clearing method is compared, and the purity of the definitive entoderm before FACS is separated in noble cells culture can be improved.However, in entire 5- In differentiation operation in 6 days cell culture concentration is maintained at < 0.5%, the definitive endodenn cells for also resulting in generation are total Quantity is reduced.

The definitive endodenn cells of method preparation according to the present invention are kept in culture in the presence of activin Amplification 50 days it is above and without obviously breaking up.In these cases, during culture keep SOX17, CXCR4, MIXL1, GATA4, The expression of HNF3 β.In addition, TM, SPARC, OCT4, AFP, SOX7, ZIC1 and BRACH is not detected in these cultures.It will Definitive endodenn cells kept in the culture in the presence of activin amplification substantially 50 days or more and without obviously break up be can Can.

Embodiment 11

Other markers of definitive endodenn cells

In following tests, RNA is isolated from definitive entoderm and the human embryo stem cell group of purifying.Then with genetic chip Analyze the RNA from each purifying cells group.Further investigate definitive entoderm using Q-PCR and non-embryonic stem cells expression Potentiality of the gene as definitive endoderm markers.

Human embryo stem cell (hESCs) is maintained in DMEM/F12 culture medium, which supplements 20% KnockOut serum replacement, 4ng/mL recombined human basis fibroblast growth factor (bFGF), 0.1mM 2 mercapto ethanol, L- paddy Propylhomoserin, nonessential amino acid and penicillin/streptomycin.HESCs is cultivated to the embryo in shaping of differentiation in 5 days in RPMI culture medium Layer, the culture medium supplement 100ng/mL recombined human activin A, fetal calf serum (FBS) and penicillin/streptomycin.Each day FBS Concentration variation are as follows: 0.1% (first day), 0.2% (second day) and 2% (the 3-5 days)

Gene expression analysis is carried out to obtain cell hESCs and the pure group of definitive entoderm, with Fluorescence Activated Cell point Select (FACS) separation.Using SSEA4 antigen (R&D Systems, cat#FAB1435P) Immunological purification hESCs, CXCR4 is used (R&D Systems, cat#FAB170P) purifies definitive entoderm.Cell dissociation using trypsase/EDTA (Invitrogen, Cat#25300-054), washed, be resuspended in 100% human serum simultaneously with the phosphate buffer (PBS) containing 2% human serum It is placed in 10 minutes blocking non-specific bindings on ice.The antibody of the combination phycoerythrin of 200 μ L is added in the human serum of 800 μ L 5x 10⁶In cell, dye 30 minutes on ice.Twice with 8mL PBS buffer solution washing cell, it is resuspended in 1mL PBS In.FACS separation is carried out with the core equipment of Scripps research institute using FACS Vantage (BD Biosciences).It will Cell is collected directly from RLT lysis buffer, separates RNA according to operating instruction (Qiagen) with RNeasy.

Twice (Durham, NC) by the RNA sample presentation of purifying, using the U133Plus2.0 high density of Affymetrix platform Oligonucleotide arrays generate expression characteristic data and generate expression modal data.The data of presentation are one group and compare, and identify hESCs and determine The gene of two cell mass differential expressions of shape entoderm.The strong raising compared with the gene level that hESCs has found by expression The gene of variation is elected to be new candidate markers, the definitive entoderm feature with height.Q- is used according to the method The selected gene of PCR measurement to verify the changes in gene expression found on genetic chip, and investigates this between hESC differentiation time-histories The expression pattern of a little genes.

Figure 34 A-M shows some marker gene expression results.After 100ng/ml activin A is added, in the 1st, 3 and 5 day Analysis cell culture, the definitive endodenn cells of CXCR4 are expressed in display differentiation operation (CXDE) after 5 days and Human embryo is done The result of cell (HESC).Figure 34 C and G-M relatively show six marker genes FGF17, VWF, CALCR, FOXQ1, CMKOR1 And CRIP1, display substantially identical expression pattern each other, also completely with CXCR4 and SOX17/SOX7 expression pattern phase Together.As described above, SOX17 is expressed in extraembryonic endoderm the two of definitive entoderm and expression SOX7.Since SOX7 is fixed Shape entoderm is not expressed, and the ratio of SOX17/SOX7 reliably has estimated SOX17 in definitive entoderm indicated by entire group The contribution of expression.The similitude of embedding figure G-L and M and embedding figure C shows that FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 can It can be the marker of definitive entoderm, and show it and expressed in extra-embryonic endoderm cells not significantly.

It should be appreciated that Q-PCR result described herein can be confirmed further with ICC.

Method, composition and equipment of the present invention are the representatives of preferred embodiment, are exemplary, and cannot be considered as Limitation of the scope of the invention.Those skilled in the art can make variation to it and make other application, this is included in of the invention It is defined in spirit and by scope of disclosure.It is obvious, therefore, that those skilled in the art are without departing from scope and spirit of the present invention Under, replacement and variation can be made to invention disclosed herein.

As described in following the claims and entire disclosure, phrase " substantially by ... form " refers to including listed after phrase Any ingredient, and be limited to those to the activity illustrated in disclosure or act on the other ingredients not interfered or without contribution.Thus, phrase Whether " substantially by ... form " shows that the ingredient listed is needed or required, but other ingredients are optional, depending on influencing List ingredient activity or effect choosing with or without.

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Sequence table

<110> ViaCyte, Inc.

D'Amour, Kevin A.

Agulnick, Alan D.

Baetge, Emmanuel E.

<120>definitive entoderm

<130> 96183-000242US

<140> US 14/072,642

<141> 2013-11-05

<150> US 12/710,300

<151> 2010-02-22

<150> US 10/584,338

<151> 2007-01-09

<150> WO PCT/US2004/043696

<151> 2004-12-23

<150> US 60/587,942

<151> 2004-07-14

<150> US 60/586,566

<151> 2004-07-09

<150> US 60/532,004

<151> 2003-12-23

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1245

<212> DNA

<213>homo sapiens (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 (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 Gly 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 (10)

1. vitro cell culture, it includes people's cells, wherein at least 15%, at least 20%, at least 25%, at least 30%, until Few 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% people's cell is embryo in people shapes Confluent monolayer cells, people's definitive endodenn cells are the pluripotent cells that can be divided into intestinal tube cell or the organ derived from intestinal tube.

2. cell culture as described in claim 1, also includes:

A) culture medium, the culture medium include the serum less than 2%；Or

B) growth factor of the Nodal/ Activin subgroup of TGF beta superfamily；Or

C) growth factor of Nodal, activin A, activin B and combinations thereof are selected from.

3. cell culture as described in claim 1, wherein people's definitive endodenn cells are adhered to the table of culture vessel Face.

4. cell culture as claimed in claim 3, wherein people's definitive endodenn cells are rendered as single layer.

5. vitro cell culture, it includes culture medium, people's pluripotent cell and fixed from the people of people's pluripotent cell differentiation Shape endoderm cell, in which:

A) for every 5 people's pluripotent cells present in the cell culture, there are at least one people's definitive entoderm is thin Born of the same parents；Or

B) for every 2 people's pluripotent cells present in the cell culture, there are at least one people's definitive entoderm is thin Born of the same parents；Or

C) for every 1 people's pluripotent cell present in the cell culture, there are at least two people's definitive entoderm is thin Born of the same parents；Or

D) for every 1 people's pluripotent cell present in the cell culture, there are at least five people's definitive entoderm is thin Born of the same parents；Or

E) for every 1 people's pluripotent cell present in the cell culture, there are at least ten people's definitive entoderm is thin Born of the same parents；And

The culture medium includes Wnt family member,

Wherein people's pluripotent cell is not obtained from Human embryo or people's pluripotent cell is obtained from commercially available pluripotent cell System.

6. cell culture as claimed in claim 5, in which:

A) culture medium lacks B27；Or

B) culture medium lacks serum substitute；Or

C) culture medium includes the growth factor of the Nodal/ Activin subgroup of TGF beta superfamily；Or

D) culture medium includes the growth factor selected from Nodal, activin A, activin B and combinations thereof；Or

E) culture medium includes activin A；Or

F) the Wnt family member includes Wnt3A.

7. generating the in-vitro method of people's definitive endodenn cells, which comprises

The cell mass comprising human pluripotent stem cells is obtained, wherein the human pluripotent stem cells are not obtained from Human embryo, Huo Zhesuo Human pluripotent stem cells are stated obtained from commercially available pluripotent cell system；And

There is provided TGF beta superfamily growth factor and Wnt family member for the cell mass, thus generated in the cell mass to The definitive endodenn cells of SOX17 and HNF3b are expressed less.

8. the method for claim 7, further including the removing TGF beta superfamily growth factor from the cell mass.

9. the method as described in benefit requires 7, further includes the steps that providing serum for the cell mass.

10. isolated antibody, in conjunction with SOX17.