CN1946838A - Definitive endoderm - Google Patents

Definitive endoderm Download PDF

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Publication number
CN1946838A
CN1946838A CNA2004800387201A CN200480038720A CN1946838A CN 1946838 A CN1946838 A CN 1946838A CN A2004800387201 A CNA2004800387201 A CN A2004800387201A CN 200480038720 A CN200480038720 A CN 200480038720A CN 1946838 A CN1946838 A CN 1946838A
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Prior art keywords
cell
definitive entoderm
mark
expression
sox17
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Inventor
凯文·艾伦·德阿姆尔
艾兰·D·奥戈尔尼克
埃马纽埃尔·E·拜特格
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Cythera Inc
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Cythera Inc
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Priority to CN201410120976.2A priority Critical patent/CN103898047B/en
Priority to CN202110089971.8A priority patent/CN112813019A/en
Priority to CN201410120990.2A priority patent/CN103898045B/en
Priority to CN201910016274.2A priority patent/CN109628371B/en
Publication of CN1946838A publication Critical patent/CN1946838A/en
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Abstract

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

Description

Definitive entoderm
Related application
The application is a non-provisional application, under the regulation of 35U.S.C. § 119 (e), enjoy in the U.S. Provisional Patent Application submitted on December 23rd, 2003 number 60/532,004, name is called the right of priority of " definitive entoderm ", under the regulation of 35U.S.C. § 119 (e), also enjoy in the U.S. Provisional Patent Application submitted on July 9th, 2004 number 60/586,566 name is called the right of priority of " the chemokines cell surface receptor that is used to separate definitive entoderm " item, and under the regulation of 35U.S.C. § 119 (e), enjoy in the U.S. Provisional Patent Application submitted on July 14th, 2004 number 60/587,942, name is called the right of priority of " the chemokines cell surface receptor that is used to separate definitive entoderm ".The open of each priority application that this paper will list above all introduced, for your guidance.
Invention field
The present invention relates to medicine and cytobiology field.Particularly, the present invention relates to composition, comprise Mammals definitive entoderm cell and preparation, separation and use the method for these cells.
Background of invention
In 1994, in not having the alimental culture of inoblast, separated human pluripotent stem cells first, for example the embryo does (ES) cell and embryonic germ (EG) cell (Bongso et al., 1994), these stem cells (Hogan, 1997) in having the alimental culture of inoblast, have been separated then.Afterwards, Thomson, Reubinoff and Shamblott made the mouse trophoderm of mitotic division inactivation set up the continuous culture of people ES and EG cell (Reubinoff et al., 2000; Shamblott et al., 1998; Thomson et al., 1998).
People ES and EG cell (hESCs) provide new chance for research people's early development with to some such as diabetes and parkinsonian disease treatment intervention.For example, use the cell of the generation Regular Insulin that comes from hESCs to provide huge improvement to the present cell therapy method of utilizing the donor pancreas glandular cell.Yet, also do not understand how to produce the β cell that generates Regular Insulin at present from hESCs.Similarly, at present to diabetes be used to be subjected to transplanting the restriction of required high quality islet cells shortage from the cell therapy of the islet cells of donor pancreas.Cell therapy to a type i diabetes patient need transplant about 8 * 10 8Pancreas islet cells (Shapiro et al., 2000; Shapiro et al., 2001a; Shapiro et al., 2001b).Similarly, just need at least two healthy donors organs to obtain the transplanting that enough islet cellss carry out success.HESCs provides a kind of source of parent material, and the high-quality noble cells that fundamental quantity from this material development is used for people's cell therapy.
Two specific characters that make hESCs be very suitable for the cell therapy application are versatility and the ability that keeps these cells in secular cultivation, and the accumulation that heredity changes can not take place.Versatility is meant that hESCs is divided into the ability of the derivative of all 3 kinds of archeocyte layers (entoderm, mesoderm, ectoderm), these archeocyte layers form all somatocyte types of adults and embryo outside organization (as, placenta) and sexual cell subsequently.Though versatility has been given hESCs special availability, this specific character is also given research and is operated these cells and derivative has brought special challenge.Owing to may produce a large amount of cell types in the hESC culture of differentiation, therefore, the efficient that most cell types produce is very low.In addition, the generation key of successfully estimating any set cell type depends on and determines suitable mark.Realize that high efficiency directed differentiation is extremely important for the treatment application of hESCs.
Can be used for the cell that cell therapy is used for hESCs is used for producing as parent material, it is useful overcoming foregoing problems.For example, in order to reach the cell material level that islet cell transplantation treatment needs, in the very early time of differentiation, with hESCs expeditiously directed differentiation be that pancreas islet/β clone is favourable.
Except that the differentiation of the efficiently and directionally of atomization, along differentiation pathway to the separation of the intermediate cell type of pancreas islet/β clone differentiation and qualitative, and with this cell as other step that suitable pedigree precursor is used for breaking up, also be useful.
Summary of the invention
Embodiments more of the present invention relate to the cell culture that comprises the definitive entoderm cell, and wherein said definitive entoderm cell is a pluripotent cell, can be divided into the intestinal tube cell or be derived from the organ of intestinal tube.According to some embodiments, the definitive entoderm cell is a mammalian cell, and in preferred embodiments, definitive entoderm cell behaviour cell.In some embodiments of the present invention, definitive entoderm cell expressing or significantly do not express specific mark.In some embodiments, one or more mark of definitive entoderm cell expressing, described mark is selected from SOX17, CXCR4, MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.In other embodiments, the definitive entoderm cell is not significantly expressed one or more mark, and this mark is selected from OCT4, α-fetoprotein (AFP), thrombomodulin (TM), SPARC and SOX7.
According to other embodiments of the present invention, the method that produces definitive entoderm from pluripotent cell has been described.In some embodiments, pluripotent cell is derived from morula.In some embodiments, multipotential stem cell is a stem cell.The stem cell that is used for these methods can include, but are not limited to embryonic stem cell.Embryonic stem cell can be derived from embryo's inner cell mass or embryo's genital ridge.Embryonic stem cell can originate from various animal species, and these kinds include, but are not limited to various mammal species, comprise the people.In preferred embodiments, human embryo stem cell is used to produce definitive entoderm.
In some embodiments of the present invention, one or more somatomedins are used for atomization from pluripotent cell to the definitive entoderm cell.These one or more somatomedins that are used for atomization can comprise the somatomedin from the TGF beta superfamily.In these embodiments, described one or more somatomedins comprise the BMP subgroup of Nodal/ activin and/or somatomedin TGF beta superfamily.In some embodiments, described one or more somatomedins are selected from Nodal, activin A, the combination of activin B, BMP4, Wnt3a or any of these somatomedin.
Embodiment of the present invention also relates to the cell mass that is enriched in the definitive entoderm cell.In certain embodiments, with described definitive entoderm cellular segregation or basic purifying.In some embodiments, the definitive entoderm cell expressing SOX17 and/or the CXRC4 mark of described separation or basic purifying, its expression degree is higher than OCT4, AFP, TM, SPARC and/or SOX7 mark.
Also provide enrichment to have the method for the cell mass of definitive entoderm.In some embodiments, the definitive entoderm cell can be separated from mixed cell population or basic purifying, by described cell is contacted with a kind of reagent, this reagent combines with a kind of molecule, this molecule is positioned at the definitive entoderm cell surface, rather than other cell surface in the mixed cell population, separate and reagent bonded cell then.In certain embodiments, the molecule that is positioned at described definitive entoderm cell surface is CXCR4.
Other embodiment of the present invention also relates to CXCR4 antibody, other part of SDF-1 part or CXCR4, and it is used to obtain the definitive entoderm cell of enrichment, isolating or basic purified form.For example, the another kind of part of CXCR4 antibody, SDF-1 part or CXCR4 can be used as such as the reagent in affine separation or the isolating method of magnetic, with enrichment, separation or basic purifying and described reagent bonded definitive entoderm cell.
Other embodiment of the present invention as herein described relates to the composition such as cell culture, and it comprises pluripotent cell and definitive entoderm cell.In certain embodiments, cell culture comprises stem cell and definitive entoderm cell simultaneously.Stem cell number in these cultures can greater than, be equal to or less than the definitive entoderm cell number in this culture.In some embodiments, stem cell is a human embryo stem cell.In certain embodiments, hESCs is remained on the trophoderm.In these embodiments, described trophocyte can be from people, mouse or other suitable biological obtain such as fibroblastic cell.
In some embodiments of the present invention, the described composition that comprises definitive entoderm cell and hESCs also comprises one or more somatomedins.These somatomedins can comprise the somatomedin from the TGF beta superfamily.In these embodiments, described one or more somatomedins comprise the BMP subgroup of Nodal/ activin and/or somatomedin TGF beta superfamily.In some embodiments, described one or more somatomedins are selected from the combination of Nodal, activin A, activin B, BMP4, Wnt3a or any of these somatomedin.
Other embodiment of the present invention is described with reference to following numbering paragraph:
1. the cell culture that comprises people's cell is the definitive entoderm cell at least about described people's cell of 10% wherein, and described definitive entoderm cell is for being divided into the intestinal tube cell or being derived from the pluripotent cell of the organ of intestinal tube.
2. paragraph 1 described cell culture is the definitive entoderm cell at least about described people's cell of 50% wherein.
3. paragraph 1 described cell culture is the definitive entoderm cell at least about described people's cell of 80% wherein.
4. paragraph 1 described cell culture, wherein said definitive entoderm cell expressing is selected from the mark of SOX17 and CXCR4.
5. paragraph 4 described cell cultures, wherein in described definitive entoderm cell, the expression that is selected from the described mark of SOX17 and CXCR4 is higher than the expression of the mark that is selected from OCT4, α-fetoprotein (AFP), thrombomodulin (TM), SPARC and SOX7.
6. paragraph 4 described cell cultures, wherein said definitive entoderm cell is not expressed the mark that is selected from OCT4, AFP, TM, SPARC and SOX7.
7. paragraph 4 described cell cultures, wherein said definitive entoderm cell expressing is selected from the mark of MIXL1, GATA4 and HNF3b.
8. paragraph 4 described cell cultures, wherein said definitive entoderm cell expressing is selected from the mark of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.
9. paragraph 1 described cell culture, wherein said definitive entoderm cell expressing SOX17 and CXCR4.
10. paragraph 9 described cell cultures, wherein in described definitive entoderm cell, the expression of described SOX17 and CXCR4 is higher than the expression of OCT4, AFP, TM, SPARC and SOX7.
11. paragraph 9 described cell cultures, wherein said definitive entoderm cell is not expressed OCT4, AFP, TM, SPARC and SOX7.
12. paragraph 9 described cell cultures, wherein said definitive entoderm cell expressing MIXL1, GATA4 and HNF3b.
13. paragraph 9 described cell cultures, wherein said definitive entoderm cell expressing is selected from the mark of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.
14. paragraph 1 described cell culture, wherein in described cell culture, corresponding each pluripotent cell exists at least about 2 definitive entoderm cells.
15. paragraph 14 described cell cultures, wherein said pluripotent cell comprises embryonic stem cell.
16. paragraph 15 described cell cultures, wherein said embryonic stem cell are derived from the tissue that is selected from from morula, embryo's inner cell mass (ICM) and embryo's genital ridge.
17. paragraph 1 described cell culture further comprises substratum, this substratum comprises and is less than about 10% serum.
18. paragraph 1 described cell culture further comprises the somatomedin of the Nodal/ activin subgroup of TGF beta superfamily.
19. paragraph 1 described cell culture further comprises the somatomedin that is selected from Nodal, activin A, activin B and combination thereof.
20. comprise the cell mass of cell, wherein at least about 90% described cell behaviour definitive entoderm cell, described people's definitive entoderm cell is a pluripotent cell, this pluripotent cell can be divided into the intestinal tube cell or be derived from the organ of intestinal tube.
21. paragraph 20 described cell masses are wherein at least about 95% described cell behaviour definitive entoderm cell.
22. paragraph 20 described cell masses are wherein at least about 98% described cell behaviour definitive entoderm cell.
23. paragraph 20 described cell masses, wherein said people's definitive entoderm cell expressing is selected from the mark of SOX17 and CXCR4.
24. paragraph 23 described cell masses, wherein in described people's definitive entoderm cell, the expression that is selected from the described mark of SOX17 and CXCR4 is higher than the expression of the mark that is selected from OCT4, AFP, TM, SPARC and SOX7.
25. paragraph 23 described cell masses, wherein said people's definitive entoderm cell is not expressed the mark that is selected from OCT4, AFP, TM, SPARC and SOX7.
26. paragraph 23 described cell masses, wherein said people's definitive entoderm cell expressing is selected from the mark of MIXL1, GATA4 and HNF3b.
27. paragraph 23 described cell masses, wherein said definitive entoderm cell expressing is selected from the mark of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.
28. paragraph 20 described cell masses, wherein said people's definitive entoderm cell expressing SOX17 and CXCR4.
29. paragraph 28 described cell masses, wherein in described people's definitive entoderm cell, the expression of SOX17 and CXCR4 is higher than the expression of OCT4, AFP, TM, SPARC and SOX7.
30. paragraph 28 described cell masses, wherein said people's definitive entoderm cell is not expressed OCT4, AFP, TM, SPARC and SOX7.
31. paragraph 28 described cell masses, wherein said people's definitive entoderm cell expressing MIXL1, GATA4 and HNF3b.
32. paragraph 28 described cell masses, wherein said definitive entoderm cell expressing is selected from the mark of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.
33. paragraph 20 described cell masses, wherein in described cell colony, corresponding each pluripotent cell exists at least about 2 definitive entoderm cells.
34. paragraph 33 described cell masses, wherein said pluripotent cell comprises embryonic stem cell.
35. paragraph 34 described cell masses, wherein said embryonic stem cell are derived from the tissue of the ICM that is selected from morula, embryo and embryo's genital ridge.
36. produce the method for definitive entoderm cell, described method comprises the steps:
Acquisition comprises the cell mass of people's pluripotent cell;
For described cell mass provides at least a TGF beta superfamily somatomedin, the amount of described somatomedin is enough to promote described pluripotent cell to be divided into the definitive entoderm cell, and described definitive entoderm cell is the pluripotent cell that can be divided into the intestinal tube cell or be derived from the organ of intestinal tube; And
Give time enough and form the definitive entoderm cell, the enough time of wherein said formation definitive entoderm cell with detect definitive entoderm cell in the described cell colony exist determine.
37. paragraph 36 described methods wherein are divided into the definitive entoderm cell at least about 10% described pluripotent cell.
38. paragraph 36 described methods wherein are divided into the definitive entoderm cell at least about 50% described pluripotent cell.
39. paragraph 36 described methods wherein are divided into the definitive entoderm cell at least about 70% described pluripotent cell.
40. paragraph 36 described methods wherein are divided into the definitive entoderm cell at least about 80% described pluripotent cell.
41. paragraph 36 described methods, wherein detect the existence of definitive entoderm cell in described cell colony and be included in the expression that detects the mark of at least a SOX17 of being selected from and CXCR4 in the described cell mass cell, and at least a expression that is selected from the mark of OCT4, AFP, TM, SPARC and SOX7, wherein in described definitive entoderm cell, the expression that is selected from the described mark of SOX17 and CXCR4 is higher than the expression of the described mark that is selected from OCT4, AFP, TM, SPARC and SOX7.
42. paragraph 36 described methods, wherein detect the existence of definitive entoderm cell in described cell mass and be included in the expression that detects the mark of at least a SOX17 of being selected from and CXCR4 in the described cell mass cell, and at least a expression that is selected from the mark of AFP, TM and SOX7, wherein in described definitive entoderm cell, the expression that is selected from the described mark of SOX17 and CXCR4 is higher than the expression of the described mark that is selected from AFP, TM and SOX7.
43. paragraph 42 described methods, wherein the expression of at least a described mark is measured with Q-PCR.
44. paragraph 42 described methods, wherein the expression of at least a described mark is measured with immunocytochemistry.
45. paragraph 36 described methods, the existence that wherein detects definitive entoderm cell in described cell colony is included in the expression that detects the mark of at least a VWF of being selected from, CALCR, FOXQ1, CMKOR1 and CRIP1 in the described cell colony cell, and at least a expression that is selected from the mark of OCT4, AFP, TM, SPARC and SOX7, wherein in described definitive entoderm cell, the expression that is selected from the mark of FGF17, VWF, CALCR, FOXQ1 and CRIP1 is higher than the expression of the mark that is selected from OCT4, AFP, TM, SPARC and SOX7.
46. paragraph 36 described methods, the Nodal/ activin subgroup that wherein said at least a somatomedin is the TGF beta superfamily.
47. paragraph 46 described methods, wherein said at least a somatomedin is selected from Nodal, activin A, activin B and combination thereof.
48. paragraph 47 described methods, wherein said at least a somatomedin is Nodal.
49. paragraph 47 described methods, wherein said at least a somatomedin is activin A.
50. paragraph 47 described methods, wherein said at least a somatomedin is activin B.
51. paragraph 36 described methods wherein provide the multiple somatomedin of TGF beta superfamily.
52. paragraph 51 described methods, wherein said multiple somatomedin comprises Nodal, activin A and activin B.
53. paragraph 36 described methods, the concentration of wherein said at least a somatomedin is at least about 10ng/ml.
54. paragraph 36 described methods, the concentration of wherein said at least a somatomedin is at least about 100ng/ml.
55. paragraph 36 described methods, the concentration of wherein said at least a somatomedin is at least about 500ng/ml.
56. paragraph 36 described methods, the concentration of wherein said at least a somatomedin is at least about 1000ng/ml.
57. paragraph 36 described methods, the concentration of wherein said at least a somatomedin is at least about 5000ng/ml.
58. paragraph 36 described methods, wherein said cell colony grows in and comprises in the substratum that is less than about 10% serum.
59. paragraph 36 described methods, wherein said pluripotent cell comprises stem cell.
60. paragraph 59 described methods, wherein said pluripotent cell comprises embryonic stem cell.
61. paragraph 60 described methods, wherein said embryonic stem cell are derived from the tissue of the ICM that is selected from morula, embryo and embryo's genital ridge.
62. the definitive entoderm cell that produces with the method for paragraph 36.
63. produce the cell mass method of the definitive entoderm cell of enrichment, comprise the steps:
Cell in the differentiation pluripotent human cell colony, to produce the definitive entoderm cell, described definitive entoderm cell is a pluripotent cell, this pluripotent cell can be divided into the intestinal tube cell or be derived from the organ of intestinal tube;
Provide reagent to described cell mass, described reagent combines with mark, and described marker expression is in described definitive entoderm cell and be not expressed in basically in other cell type in the described cell mass; And
Will with the described definitive entoderm cell of described reagent bonded be positioned at that other cell type separates described in the described cell mass, thereby produce the cell mass of the definitive entoderm cell of enrichment.
64. paragraph 63 described methods, wherein differentiation step further comprises,
Acquisition comprises the cell mass of pluripotent human cell,
Provide described at least a TGF beta superfamily somatomedin to described cell colony, the amount of this somatomedin is enough to promote described pluripotent cell is divided into the definitive entoderm cell, described definitive entoderm cell is a pluripotent cell, this pluripotent cell can be divided into the intestinal tube cell or be derived from the organ of intestinal tube, and
Give time enough and form the definitive entoderm cell, the enough time of wherein said formation definitive entoderm cell with detect definitive entoderm cell in the described cell colony exist determine.
65. paragraph 63 described methods, wherein detection comprises,
In the cell of described cell colony, detect at least a expression that is selected from the mark of SOX17 and CXCR4, and at least a expression that is selected from the mark of OCT4, AFP, TM, SPARC and SOX7, wherein in described definitive entoderm cell, the expression that is selected from the mark of SOX17 and CXCR4 is higher than the expression of the mark that is selected from OCT4, AFP, TM, SPARC and SOX7.
66. paragraph 63 described methods, wherein detect and comprise, in the cell of described cell colony, detect at least a expression that is selected from the mark of SOX17 and CXCR4, and at least a expression that is selected from the mark of AFP, TM and SOX7, wherein in described definitive entoderm cell, the expression that is selected from the mark of SOX17 and CXCR4 is higher than the expression of the mark that is selected from AFP, TM and SOX7.
67. paragraph 63 described methods, wherein detect and comprise, in the cell of described cell colony, detect at least a expression that is selected from the mark of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1, and at least a expression that is selected from the mark of OCT4, AFP, TM, SPARC and SOX7, wherein in described definitive entoderm cell, the expression that is selected from the mark of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 is higher than the expression of the mark that is selected from OCT4, AFP, TM, SPARC and SOX7.
68. paragraph 63 described methods are the definitive entoderm cell at least about 95% described cell wherein.
69. paragraph 63 described methods are the definitive entoderm cell at least about 98% described cell wherein.
70. paragraph 63 described methods, wherein said mark is CXCR4.
71. paragraph 63 described methods, wherein said reagent is antibody.
72. paragraph 71 described methods, wherein said antibody has affinity to CXCR4.
73. the cell mass of the enrichment definitive entoderm cell that paragraph 63 described methods produce.
74. the described cell culture of each of paragraph 4 or 9, wherein said definitive entoderm cell are not significantly expressed the mark that is selected from OCT4, AFP, TM, SPARC and SOX7.
75. the cell mass of arbitrary paragraph of paragraph 23 or 28, wherein said definitive entoderm cell are not significantly expressed the mark that is selected from OCT4, AFP, TM, SPARC and SOX7.
Should understand, above-mentioned method and composition relates to cell in vitro and cultivates.Yet above-mentioned vitro differentiation cell composition can be used in body.
Other embodiment of the present invention also can be found in the following U.S. Provisional Patent Application, No. the 60/532nd, 004, the U.S. Provisional Patent Application of submitting on December 23rd, 1, " definitive entoderm " by name; No. the 60/586th, 566, the U.S. Provisional Patent Application of submitting on July 9th, 2004, " being used to separate the chemokine cell surface receptor of definitive entoderm " by name; And on July 14th, 2004 No. the 60/587th, 942, the U.S. Provisional Patent Application submitted to, " being used to separate the cell surface receptor of definitive entoderm " by name, this paper openly all introduces these, for your guidance.
The accompanying drawing summary
Fig. 1 produces the synoptic diagram of the differentiation pathway of beta cell for what propose from hESCs.The first step in this approach is for to change the ES cell into definitive entoderm clone, and its representative further is one of pancreas entoderm, incretory gland entoderm or pancreas islet/beta cell known steps the earliest with the ES cytodifferentiation.Some factors that can be used for mediating this transformation are the member of TGF 'beta ' family, and these members include, but are not limited to activin, nodals and BMPs.The representative mark of determining the definitive entoderm target cell is SOX17, GATA4, HNF3b, MIX1 and CXCR4.
Fig. 2 SOX17 cDNA diagram of behaving, it has shown the position of conservative motif and the zone of having given prominence to the immunization method that is used for GENOVAC.
Fig. 3 is a kind of dependency dendrogram, shows that the dependency of SOX17 and SOX7 is the strongest, with the dependency of SOX18 slightly a little less than.SOX17 protein in allied species is more much better than than other member's of the SOX group F subtribe in the same species dependency.
Fig. 4 is for being the western hybridization (Western blot) of probe with rat anti SOX17 antibody.This hybridization has proved the people SOX17 proteinic specificity (swimming lane 1) of this antibody to overexpression in the inoblast, and EGFP (swimming lane 2) or maximally related SOX family member SOX7 (swimming lane 3) are lacked immunoreactivity.
Fig. 5 A-B is for showing SOX17 +The Photomicrograph of cell cluster shows the AFP of a large amount of common marks +Cell (A).This and other SOX17 +Cell cluster (B) is observed a small amount of AFP +Cell or do not observe AFP +Cell forms sharp contrast.
Fig. 6 A-C is for showing the Photomicrograph of body wall entoderm and SOX17.Embedding figure A shows the immunocytochemistry to people's thrombomodulin (TM), and this protein is arranged in the body wall endoderm cell's of the hES cell culture of differentiation cell surface at random.Embedding figure B be TM and SOX17 duplication of marking show with the identical zone of embedding figure A.Embedding figure C is the phase difference image with the same area of DAPI mark nuclear.Notice that DAPI mark nuclear is relevant fully with the SOX17 mark.
Fig. 7 A-B is the histogram of the anti-SOX17 positive cell of the SOX17 genetic expression that shows quantitative PCR (Q-PCR) and SOX17 specific antibody.For the control medium (SR20) of undifferentiated, embedding figure A shows that activin A increases SOX17 genetic expression, and vitamin A acid (RA) strongly inhibited SOX17 expresses.Embedding figure B shows, the reacting condition of model identical and similarity degree is on the SOX17+ cell number, and the Q-PCR test that shows SOX17 genetic expression is highstrung to the variation of unicellular level.
Fig. 8 A is a histogram, be presented at activin A and exist the hESCs culture of differentiation down to keep low-level AFP genetic expression, and in 10% foetal calf serum (FBS), cell breaks up at random, shows that AFP seriously raises.Difference on the expression level is approximately 7 times.
Fig. 8 B-C is two Photomicrograph images, has shown activin A to the AFP expression inhibiting in unicellular level also clearly, for only using 10% FBS (top), and (bottom) observed AFP in the condition that activin A handles +Cell cluster seldom and very little.
Fig. 9 A-B is contrast figure, shows and uses the quantitative AFP of flow cytometer +Cell count.This figure shows, AFP changes in gene expression amplitude (Fig. 8 A) and AFP when having (right embedding figure) or do not have (left embedding figure) activin A +Cell count is very consistent, shows that further Q-PCR analyzes being presented at the practicality of the variation that occurs on the unicellular level.
Figure 10 A-F was a Photomicrograph, shows hESCs is exposed to nodal, activin A and activin B (NAA), had produced the remarkable increase (A-C) of cell count 5 day time.By comparing SOX17 +The relative quantity of the cell total amount that cell and this zone exist, shown in DAPI colour developing nuclear (D-F), all cells of about 30-50% has immunoreactivity to SOX17 after handling 5 days with NAA as can be seen.
Figure 11 is a histogram, shows that activin A (0,10,30 or 100ng/mL) dose-dependently ground increases the SOX17 genetic expression of the hESCs that breaks up.To adhering to the culture processing after 3 days, continue resuspending and cultivated 3～5 days, expressing increases clearly.
Figure 12 A-C is a histogram, has confirmed the effect of activin A to MIXL1 (embedding figure A), GATA4 (embedding figure B) and HNF3b (embedding figure C) expression.In other three definitive entoderm mark MIXL1, GATA4 and HNF3b, also observed the increase that activin is dose-dependently.The expression amplitude that the activin dose-dependently is increased is extremely similar to SOX17's, has shown that strongly activin A specific action is in all four gene (SOX17 of coexpression +, MIXL1 +, GATA4 +AndHNF3b +) cell mass.
Figure 13 A-C is a histogram, has confirmed that activin A is to the effect in AFP (embedding figure A), SOX7 (embedding figure B) and SPARC (the embedding figure C) expression.Activin A dose-dependently ground reduces the expression of internal organ entoderm mark AFP.Primitive endoderm (SOX7) and body wall entoderm (SPARC) mark are still constant or only show to suppress at some point, show activin A not specific action in these extraembryonic endoderm cell types.This supports that further the expression increase of SOX17, MIXL1, GATA4 and HNF3b is because activin A causes the increase of definitive entoderm cell quantity.
Figure 14 A-B is a histogram, has shown the effect of activin A to ZIC1 (embedding figure A) and Brachyury (embedding figure B) expression.The continuous expression of neural mark ZIC1 shows that activin A does not have the effect of dose-dependently to Neural Differentiation.Express reduction as can be seen from brachyury, 100ng/mL activin A handles has significant inhibition to mesoblastic differentiation.This may be the result from the definitive entoderm specificity increase of mesendoderm precursor.Compare with the blank culture of non-processor, low-level activin A handles (10 and 30ng/mL) and has kept the brachyury expression at differentiation later stage time point.
Figure 15 A-B is a Photomicrograph, and activin is handled and caused that the differentiation of body wall entoderm reduces.In only with the culture (A) of serum differentiation, find TM HiBody wall entoderm zone, however when comprising activin (B), seldom be divided into TM +Cell and total immunoreactive intensity are lower.
Figure 16 A-D is a Photomicrograph, shows that activin A and activin B handle the marker expression that causes.Handled hESCs4 days with activin A and activin B continuously, carry out three marks with SOX17, AFP and TM antibody.Embedding figure A-SOX17; Embedding figure B-AFP; Embedding figure C-TM; And embedding figure D-Phase/DAPI.Attention is visible a large amount of SOX17 positive cell (A) when not having AFP (B) and TM (C) immunoreactivity fully.
Figure 17 is a displaing micro picture, shows external definitive entoderm and internal organ entoderm to occur from hESCs.Internal organ entoderm zone is with AFP Hi/ SOX17 Lo/-Identify, yet definitive entoderm has shown antipodal feature, SOX17 Hi/ AFP Lo/-Select this territory to be because these two zones are closer to each other, yet, have repeatedly at complete isolating AFP HiCell compartment is observed SOX17 Hi/ AFP Lo/-The zone shows that the part definitive entoderm is cell-derived in the internal organ endoderm cell.
Figure 18 is for describing the chart of TGF 'beta ' family part and acceptor.The factor of activation AR-Smads and BR-Smads helps producing definitive entoderm (referring to, J CellPhysiol.187:265-76) from human embryo stem cell.
Figure 19 is a histogram, shows that handling inductive SOX17 with the single or multiple TGF-β factor expresses in time.
Figure 20 is a histogram, shows with multiple TGF-β factor processing to cause SOX17 +Cell quantity increase in time.
Figure 21 is a histogram, shows that handling inductive SOX17 with the multiple TGF-β factor expresses in time.
Figure 22 is a histogram, shows that activin A dose-dependently ground increases SOX17 +Cell quantity.
Figure 23 is a histogram, and showing that Wnt3a is added to increases SOX17 in the culture that activin A and activin B handle and express, and is higher than activin A and activin B uses the inductive level separately.
Figure 24 A-C is a histogram, shows under low FBS condition, and being divided into definitive entoderm increases.Handle hESCs and handle ratio under the similarity condition in 10%FBS (10AA) (embedding figure A) with activin A and B in the substratum that comprises 2%FBS (2AA), the high 2-3 of SOX17 expression level doubly.Definitive entoderm mark MIXL1 (embedding figure B) also is affected in an identical manner, and 2%FBS is high under the 10%FBS condition to the rejection ratio of AFP (internal organ entoderm) (embedding figure C).
Figure 25 A-D is a Photomicrograph, shows the SOX17 in cultivating +Cell fission.The SOX17 immunoreactive cell is in hESC clone dividing edge, and (C D), with proliferative cell cell nuclear antigen (PCNA) (embedding figure B) mark, and does not indicate with OCT4 (embedding figure C) altogether.In addition, with DAPI mark nuclear, at SOX17 +Cell (arrow) and OCT4 +, undifferentiated hESCs (arrow) can clearly see the mitotic division picture in (D).
Figure 26 is a histogram, is presented in the various substratum relative expression level of CXCR4 in the hESCs that is breaking up.
Figure 27 A-D is a histogram, by with Figure 26 same treatment, show how a series of definitive entoderm marks have closely similar CXCR4 expression pattern.
Figure 28 A-E is a histogram, show mesoderm (BRACHYURY, MOX1), ectoderm (SOX1, ZIC1) and internal organ entoderm (SOX7) mark how to express by having opposite CXCR4 with Figure 26 same treatment.
Figure 29 A-F is a Photomicrograph, is presented at the relative different of SOX17 immunoreactive cell under Figure 26-28 3 kind of culture medium condition.
Figure 30 A-C is the flow cytometer point diagram, confirms along with the concentration that is added to the activin A in the division culture medium increases CXCR4 +Cell quantity increases.
Figure 31 A-D is a histogram, shows that high dosage activin A handles the isolating CXCR4 in (A100-CX+) back +The definitive entoderm mark quantity of cell is than its mother cell group (A100) horn of plenty more.
Figure 32 is a histogram, shows to use the isolating CXCR4 of fluorescent activation cell sorter (FACS) +And CXCR4 -Cell and mother cell group's genetic expression.This confirms CXCR4 +Cell comprises all CXCR4 genetic expression that exists, CXCR4 substantially on each mother cell group -Then comprise and seldom or not comprise CXCR4 genetic expression.
Figure 33 A-D is a histogram, confirms the CXCR4 that high dosage activin A handles +The mesoderm of cell (BRACHYURY, MOX1), (SOX1, ZIC1) and internal organ entoderm (SOX7) genetic expression disappearance, the expression of these non-definitive entoderm marks is suppressed ectoderm.
Figure 34 A-M is a histogram, shows the marker gene expression pattern, and it can be used for identifying the definitive entoderm cell.The expression analysis of definitive entoderm mark FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 is respectively shown in embedding figure G-L.Aforementioned pedigree marker gene SOX17, SOX7, SOX17/SOX7, TM, ZIC1 and MOX1 are respectively shown in embedding figure A-F.Embedding figure M shows the expression analysis of CXCR4.About embedding figure A-M, mark hESC one hurdle shows the human embryo stem cell genetic expression of purifying; 2NF shows the cell of handling with 2%FBS, does not add activin; 0.1A100 show the cell of handling with 0.1%FBS, 100ng/ml activin A; 1A100 shows the cell of handling with 1%FBS, 100ng/ml activin A; 2A100 shows the cell of handling with 2%FBS, 100ng/ml activin A.
Detailed Description Of The Invention
A critical stage of people's early development, promptly the term gastrula forms and occurs in fertilization 2-3 after week.Gastrula forms extremely important, because in this phase three original embryos at first Focus and ordering (Lu et al., 2001; Schoenwolf and Smith, 2000).Ectoderm is responsible for body covering and whole neural formation, yet heart, blood, bone, skeletal muscle and other reticular tissue all are derived from mesoderm.Definitive entoderm is defined as is responsible for the germinal layer that whole intestinal tubes form, this whole intestinal tube comprises esophagus, stomach, small intestine and large intestine, and from enteron aisle deutero-organ, as lung, liver, thymus gland, parathyroid gland and Tiroidina, 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).Have very significantly different between definitive entoderm and the complete isolated cells system that is called primitive endoderm.Primitive endoderm mainly forms embryo outside organization, mainly is the body wall of placenta yolk sac and the cell epimatrix material of internal organ entoderm part and Reichert ' s film.
In the gastrula forming process, the definitive entoderm forming process starts from cell and migrates incident, and wherein, mesendoderm cell (can form mesoderm or endoblastic cellular component) passes a structure that is called former line.Definitive entoderm is derived from the cell that passes former line front portion and knot (ad hoc structure that is positioned at former line foremost part).When migrating generation, the foremost part that definitive entoderm at first forms intestinal tube finishes when forming the intestinal tube rear end.
The body inner analysis that definitive entoderm forms is as Conlon et al., 1994; Feldman et al., 1998; Zhou et al., 1993; Aoki et al., 2002; Dougan et al., 2003; Tremblay etal., 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., the research of 2002 zebra fishs that carry out and African toad, these researchs growth that end user's embryonic stem cell is finished specific germinal layer cell type in culture dish for how is laid a good foundation.Cultivate two relevant aspects with external ESC and constituted the main bottleneck of in culture dish, restarting growth.At first, can not produce orderly germinal layer or organ structure.In the hESC culture systems of breaking up, the special genetic marker of most of germinal layer and organ can be expressed with allogenic form.Therefore, owing to lack the organ specificity boundary, be difficult to estimate the formation of specific tissue or cell type.Nearly all gene of expressing in the cell type of a concrete germinal layer or types of organization is also expressed in the cell type of other germinal layer or types of organization.Do not have special boundary, be difficult to give the specificity of genetic expression with 1-3 cdna sample.Thereby, must carefully detect considerable gene, some gene should also can be expressed on the concrete cell type of the organ or tissue of loseing interest in.Secondly, the opportunity of genetic expression type is movable most important to what grow along special modality.
As for more complicated incident, should be pointed out that external differentiation of stem cells is quite nonsynchronous and may be than much remarkable in the body.Like this, one group of cell may expressed when forming relevant gene with gastrula, and another group may begin final differentiation.And, when having or not having the extrinsic factor participation, handle the significant difference that hESC individual layer or embryoid (EBs) may cause full gene expression pattern or differentiation state.Thereby in order to advance along specific differentiation passage effectively, according to the gene expression pattern of foreign cell mixture, the use of exogenous factor must be carried out time control.The morphology relation of considering these cells in culture vessel also is useful.With in medium container, grow up or when being divided into individual layer and/or hESC clone ability compare, the ability of the hESCs when the homogeneous influence forms so-called embryoid is far from the most desirable.
As handling above-mentioned heterogeneous and nonsynchronous effective ways, embodiments more of the present invention consider with the method for noble cells and enrichment, separate and/or purifying differentiation passage in the method for intermediate cell type unite.
Embodiment of the present invention relates to the new definite method that generates the definitive entoderm cell in culture, and it will be by being divided into multipotency definitive entoderm cell such as the pluripotent cell of stem cell." multipotency " used herein or " pluripotent cell " refer to produce the cell type of other concrete cell type of limited quantity.As mentioned above, the definitive entoderm cell is not divided into and comes from ectoderm or mesoblastic tissue, yet, can be divided into intestinal tube and come from the organ of intestinal tube.In some preferred embodiments, definitive entoderm is cell-derived in hESCs.These methods provide effective generation people entoderm deutero-tissue, as pancreas, liver, lung, stomach, intestines and thyroid basis.For example, but the generation the first step differentiated stem cells of definitive entoderm is the β cell of functional generation Regular Insulin.For the β cell of the generation Regular Insulin that obtains consumption, before reaching pancreas islet/β cell, expect that each differentiation step all is a differentiation step efficiently.Perhaps represent the initial step (as shown in Figure 1) of systematic function pancreas islet/β cell because differentiation of stem cells is the definitive entoderm cell, needing this step differentiation efficient especially.
In view of the differentiation pluripotent cell to the needs of definitive entoderm cell, some aspects of the present invention relate in vitro method to be learned, and changes the pluripotent cell of about 50-80% into the definitive entoderm cell.Typically, these methods comprise and defining and temporary transient specified mode is used culture and somatomedin.Use can with definitive entoderm cell-specific bonded reagent, from cell mass,, can obtain more enrichments of definitive entoderm cell mass with definitive entoderm cell and other cellular segregation and/or purifying.Like this, the present invention relates to that definitive entoderm cell and preparing separates and/or the method for these cells of purifying.
In order to determine the quantity of setting endoderm cell in cell culture or the cell mass, need from culture or cell mass, distinguish the method for this class cell and other cell.Therefore, embodiment of the present invention relates to the cell sign thing, and its existence, disappearance and/or relative expression's level are special to definitive entoderm, and detects the method for determining these marker expression." expression " of Shi Yonging herein refers to the generation of material or material, perhaps the level or the content of the generation of material or material.Thereby, the expression of determining special mark refer to detect expression mark relatively or absolute content, or only detect mark and whether exist.Refer to can observed or detected any molecule for " mark " of Shi Yonging herein.For example, mark can be including, but not limited to nucleic acid, as the transcript of specific gene, polypeptide product, non-genomic polypeptide product, glycoprotein, sugar, glycolipid, lipid, lipoprotein or the small molecules of gene.
In embodiments more of the present invention, the existence of marker expression whether and/or its level determine by quantitative PCR (Q-PCR).For example, the transcript amount of some genetic marker deposits yields, for example SOX17, CXCR4, OCT4, AFP, TM, SPARC, SOX7, MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1, CRIP1 and other mark as herein described are determined by quantitative Q-PCR.In other embodiments, Q-PCR and immunohistochemistry technique are used to discern and determine the amount or the relative proportion of these marks.
By using as the method for above-mentioned definite one or more suitable marker expression, the ratio of identification definitive entoderm cell and definite definitive entoderm cell is possible in cell culture or cell mass.For example, in some embodiments of the present invention, the definitive entoderm cell of generation or cell mass are expressed the level of SOX17 and/or CXCR4 gene than the non-definitive entoderm cell type or high about 2 orders of magnitude of cell mass.In other embodiments, the definitive entoderm cell of generation or cell mass are expressed the level of SOX17 and/or CXCR4 gene than the non-definitive entoderm cell type or high 2 the above orders of magnitude of cell mass.In other other embodiments, the definitive entoderm cell or the cell mass that produce are expressed the mark that one or more are selected from SOX17, CXCR4, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1, than the high about order of magnitude more than 2 or 2 of the expression of non-definitive entoderm cell type or cell mass.
The present invention relates to cell culture, comprise the cell mass of the definitive entoderm cell that has a large amount of definitive entoderms and comprise enrichment.Thus, some embodiments relate to the cell culture that comprises the definitive entoderm cell, and the cell at least about 50-80% in the wherein said culture is the definitive entoderm cell.A preferred embodiment relates to the cell culture that comprises people's cell, is the definitive entoderm cell at least about 50-80% people's cell in the culture wherein.Because the effect of differentiation method can be by changing some parameter regulation, include but not limited to the time of cell growth conditions, somatomedin concentration and culturing step, differentiation method of the present invention 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 be converted into definitive entoderm greater than 95% pluripotent cell.In other embodiment of the present invention, will be converted into pure substantially definitive entoderm cell such as the pluripotent cell group of stem cell colony.
Composition of the present invention and method have several useful features.For example, comprise the cell culture and the cell mass of definitive entoderm, and the method for preparing these cell cultures and cell mass can be used for mould and builds the commitment that the people grows.In addition, composition of the present invention and method also can be used for the therapeutic intervention as the disease of diabetes.For example, because definitive entoderm only is the tissue-derived of limited quantity, thereby it can be used for growing pure tissue or cell type.
Prepare definitive entoderm from pluripotent cell
Comprise that the definitive entoderm cell culture of definitive entoderm cell as herein described and composition prepare in the pluripotent cell of embryonic stem cell freely." embryo " used herein refers to organism etap scope, and it begins to finish to the multi-cellular structure that no longer comprises multipotency or totipotent cell except that the gametid [cell of growing from one zygote.Remove and be derived from the embryo that gamete is merged, term " embryo " refers to be derived from the embryo of SCNT.The method of definitive entoderm cell of preferably deriving is utilized the parent material of human embryo stem cell as the preparation definitive entoderm.The embryonic stem cell that this method is used can be the cell that is derived from morula, embryo's inner cell mass or obtains from embryo's genital ridge.Use art methods, human stem cell can keep the multipotency state and not break up substantially in substratum.Described method, for example, United States Patent (USP) the 5th, 670,5,690,9265,843,6,200,806 and 6,251, disclosed method in No. 671, this paper all introduces, for your guidance.
In some embodiments of the present invention, hESCs remains on trophoderm.In these embodiments, any trophoderm that can make hESCs remain on the multipotency state can be used in the method for the present invention.The trophoderm of a cultivator embryonic stem cell commonly used is one deck l cell.Recently, human fibroblasts's trophoderm has also developed and has been used for cultivating hESCs (referring to Application No. 2002/0072117 disclosed method, this paper all introduces, for your guidance).Other embodiment of the method for the invention allows not use trophoderm to keep multipotency hESCs.These methods are as described in the Application No. 2003/0175956, and it is disclosed in this whole introducings, for your guidance.
Human embryo stem cell of the present invention can remain on to have or not to have in the substratum of serum.In some embodiments, use the blood serum substituting product.In other embodiment, the substratum technology of serum-free, as described in Application No. 2003/0190748, it is disclosed in this whole introducings, for your guidance.
In substratum, keep the stem cell of multipotency state to go down to posterity, up to being divided into definitive entoderm according to routine.In some embodiments, breaking up to finishing of definitive entoderm is that the amount of its adding is enough to promote to break up to definitive entoderm by adding TGF beta superfamily somatomedin in stem cell media.The TGF beta superfamily somatomedin that is used to prepare definitive entoderm is selected from Nodal/ activin or BMP subgroup.In some embodiments of differentiation method of the present invention, somatomedin is selected from Nodal, activin A, activin B and BMP4.In addition, somatomedin Wnt3a and other Wnt family member can be used for preparing the definitive entoderm cell.In some embodiments of the present invention, can use above-mentioned any somatomedin of mentioning.
In some embodiments of differentiation method of the present invention, embryo in the above-mentioned somatomedin that adds in cell, its concentration in substratum are enough to promote to the small part differentiation of stem cells to setting.In some embodiments of the present invention, in the substratum concentration of above-mentioned somatomedin at least about 5ng/ml, at least about 10ng/ml, at least about 25ng/ml, at least about 50ng/ml, at least about 75ng/ml, at least about 100ng/ml, at least about 200ng/ml, at least about 300ng/ml, at least about 400ng/ml, at least about 500ng/ml, at least about 1000ng/ml, at least about 2000ng/ml, at least about 3000ng/ml, at least about 4000ng/ml, at least about 5000ng/ml or greater than 5000ng/ml.
In some embodiments of the present invention, above-mentioned somatomedin needs to remove from cell culture after adding substratum.For example, the time of removing of somatomedin about add back 1 day, 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 time of removing of somatomedin is about adding back 4 days.
The cultivation of definitive entoderm cell can be in the substratum that comprises a small amount of serum or serum-free.In some embodiments of the present invention, the concentration range of serum is that about 0.05%v/v is to about 20%v/v.For example, in some embodiments, the concentration of serum can be lower than about 0.05% (v/v) in the substratum, be lower than about 0.1% (v/v), be lower than about 0.2% (v/v), be lower than about 0.3% (v/v), be lower than about 0.4% (v/v), be lower than about 0.5% (v/v), be lower than about 0.6% (v/v), be lower than about 0.7% (v/v), be lower than about 0.8% (v/v), be lower than about 0.9% (v/v), be lower than about 1% (v/v), be lower than about 2% (v/v), be lower than about 3% (v/v), be lower than about 4% (v/v), be lower than about 5% (v/v), be lower than about 6% (v/v), be lower than about 7% (v/v), be lower than about 8% (v/v), be lower than about 9% (v/v), be lower than about 10% (v/v), be lower than about 15% (v/v) or be lower than about 20% (v/v).In some embodiments, the definitive entoderm cell is grown under serum-free.In other embodiments, the definitive entoderm cell is grown in the presence of the blood serum substituting product.In other embodiments, the definitive entoderm cell is grown in the presence of B27.In these embodiments, the concentration range of B27 additive is about 0.2% to about 20%v/v.
HESC is cultured to definitive entoderm can be by determining the mark feature representation monitoring of definitive entoderm.In some embodiments, whether the expression of some marks can by existing mark to determine.Selectively, the expression of some marks can be determined by measuring the level of mark in cell culture or cell mass cell.In these embodiments, can qualitatively or quantitatively determine the expression of mark.The method of the expression mark that the quantitative marker gene produces can be used quantitative PCR (Q-PCR).The method of carrying out Q-PCR is a prior art.Other method of prior art also can be used for the quantitative marker expression of gene.For example, the expression of marker gene product can detect by using the antibody at interested special marker gene product.In some embodiments of the present invention, determine to have the expression of the mark characterizing gene of definitive entoderm, and lacked the expression of the mark characterizing gene of the hESCs that significantly expresses and other cell type.
As following embodiment further as described in, a reliable markers thing of definitive entoderm is the SOX17 gene.Like this, the definitive entoderm cell expressing SOX17 marker gene of the method for the invention preparation produces the SOX17 gene product thus.The mark of other definitive entoderm is MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.In some embodiments of the present invention, the level of definitive entoderm cell expressing SOX17 marker gene is higher than the level of SOX7 marker gene, and it has original and internal organ entoderm feature (referring to table 1).In addition, in some embodiments, the SOX17 marker gene is expressed the expression that is higher than the OCT4 marker gene, and it has the feature of hESCs.In other embodiment of the present invention, the level of definitive entoderm cell expressing SOX17 marker gene is higher than the level of AFP, SPARC or thrombomodulin (TM) marker gene.In some embodiments of the present invention, the PDX1 (PDX1-feminine gender) that does not express conspicuous level or amount according to the definitive entoderm cell of the method for the invention expression SOX17.
Other mark of definitive entoderm is the CXCR4 gene.CXCR4 genes encoding cell surface Chemokine Receptors, its part is chemoattractant SDF-1.The main effect that comprises the cell of CXCR4 acceptor among the grownup it is believed that to moving hematopoietic cell to marrow, delivery lymphocyte, the various B cells of differentiation and the huge blood cell line [Kim that bites, C., and Broxmeyer, H.J.LeukocyteBiol.65,6-15 (1999)].The CXCR4 acceptor also plays HIV-1 and enters co-receptor effect [Feng, Y., et al.Science, 272,872-877 (1996)] in the T cell.At a series of [McGrath, K.E.etal.Dev.Biology213,442-456 (1999)] in the research carried out, expression [the Kim of in adult mice and early development thereof Chemokine Receptors CXCR4 and unique part SDF-1 thereof has been described, C., andBroxmyer, H., J.Leukocyte Biol.65,6-15 (1999)].CXCR4/SDF1 is clear in developmental interaction, in transgenic mice, when arbitrary gene disruption [Nagasawa et al.Nature, 382 wherein, 635-638 (1996)], Ma, Q., et alImmunity, 10,463-471 (1999)] all cause late embryo death.McGrath etc. use ribonuclease protecting and in-situ hybridization method to confirm that at early stage (E7.5) that form gastrula, CXCR4 is the abundantest Chemokine Receptors messenger RNA(mRNA).As if be in the gastrula stage, the CXCR4/SDF-1 signal is mainly induced the migration of former line cell, and be expressed on definitive entoderm, mesoderm and the ectoderm that exists this moment.In the E7.2-7.8 mice embryonic, CXCR4 and alpha fetal protein are mutually exclusive, are presented at the internal organ entoderm and lack expression [McGrath, K.E.et al.Dev.Biology 213,442-456 (1999)].
In some embodiments of the present invention, the definitive entoderm cell expressing CXCR4 marker gene for preparing according to the method for the invention.In other embodiment, definitive entoderm cell expressing CXCR4 marker gene and other definitive entoderm mark according to the method for the invention preparation include but not limited to SOX17, MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.In some embodiments of the present invention, the level of definitive entoderm cell expressing CXCR4 marker gene is higher than the SOX7 marker gene.In addition, in some embodiments, the CXCR4 marker gene is expressed and is higher than OCT4 marker gene expression levels.In other embodiment of the present invention, the level of definitive entoderm cell expressing CXCR4 marker gene is higher than the level of AFP, SPARC or thrombomodulin (TM) marker gene.In some embodiments of the present invention, the PDX1 (PDX1-feminine gender) that the definitive entoderm cell of CXCR4 is not expressed conspicuous level or amount is expressed in preparation according to method of the present invention.
Should be appreciated that the expression of CXCR4 in the endoderm cell do not repel the expression of SOX17.Correspondingly, in some embodiments of the present invention, the level of definitive entoderm cell expressing SOX17 and CXCR4 marker gene all is higher than the level of SOX7 marker gene.In addition, in some embodiments, the expression of SOX17 and CXCR4 marker gene all is higher than the expression of OCT4 marker gene.In other embodiments of the present invention, the level of definitive entoderm cell expressing SOX17 and CXCR4 marker gene is higher than the level of AFP, SPARC or thrombomodulin (TM) marker gene.In some embodiments of the present invention, the PDX1 (PDX1-feminine gender) that the definitive entoderm cell of SOX17/CXCR4 is not expressed conspicuous level or amount is expressed in preparation according to method of the present invention.
Should be appreciated that according to differentiation condition, in the definitive entoderm cell, induce the SOX17 and/or the CXCR4 marker expression of different levels scope.Like this, in some embodiments of the present invention, SOX17 mark and/or CXCR4 mark the expression ratio SOX17 mark of definitive entoderm cell or cell mass and/or CXCR4 mark such as the non-definitive entoderm cell of multipotential stem cell or the expression in the cell mass high at least about 2 times at least about 10,000 times.In other embodiment of the present invention, SOX17 mark and/or CXCR4 mark are expressed high at least about 4 times at the expression ratio SOX17 mark and/or the CXCR4 mark of definitive entoderm cell or cell mass at non-definitive entoderm cell or cell mass such as multipotential stem cell, at least about 6 times, 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 times, 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 mark and/or CXCR4 mark unrestrictedly are higher than the SOX17 mark and/or the CXCR4 mark is expressed at non-definitive entoderm cell or cell mass such as multipotential stem cell in the expression of definitive entoderm cell or cell mass.
Be to be understood that, in some embodiments of the present invention, compare with the expression of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 mark in non-definitive entoderm cell or cell mass, the expression of the GATA4 in definitive entoderm cell or cell colony, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 mark increases.
Also should be appreciated that in the definitive entoderm cell expression level of SOX17 mark and OCT4, SPARC, AFP, TM and/or SOX7 marker expression level existence difference scope.Similarly, in the definitive entoderm cell, the expression level of CXCR4 mark and OCT4, SPARC, AFP, TM and/or SOX7 marker expression level existence difference scope.Similarly, in some embodiments of the present invention, the expression height of expression ratio OCT4, the SPARC of SOX17 mark or CXCR4 mark, AFP, TM and/or SOX7 mark at least about 2 times at least about 10,000 times.In other embodiment of the present invention, the expression height of expression ratio OCT4, the SPARC of SOX17 mark or CXCR4 mark, AFP, TM and/or SOX7 mark at least about 4 times, at least about 6 times, 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 times, 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 mark are not remarkable at the definitive entoderm cell expressing.
Be to be understood that, in some embodiments of the present invention, in the definitive entoderm cell, to compare with OCT4, SPARC, AFP, TM and/or SOX7, the expression that is selected from the mark of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 increases.
The composition that comprises definitive entoderm
Some aspects of the present invention relate to the composition such as cell colony and cell culture, and it comprises such as pluripotent cell of stem cell and definitive entoderm cell.For example, use method of the present invention, can prepare the composition that comprises hESCs mixture and definitive entoderm cell.In some embodiments, comprise whenever in the composition of preparation that 95 pluripotent cells just comprise at least about 5 definitive entoderm cells.In other embodiments, comprise that whenever 5 pluripotent cells just comprise at least about 95 definitive entoderm cells.In addition, composition comprises the definitive entoderm cell and the pluripotent cell of other ratio.For example, in composition, whenever comprise 1,000,000 pluripotent cell just comprises at least about 1 definitive entoderm cell, whenever comprise 100,000 pluripotent cell just comprises at least about 1 definitive entoderm cell, whenever comprise 100,000 pluripotent cell just comprises at least about 1 definitive entoderm cell, whenever comprise 10,000 pluripotent cell just comprises at least about 1 definitive entoderm cell, whenever comprise 1,000 pluripotent cell just comprises at least about 1 definitive entoderm cell, whenever comprise that 500 pluripotent cells just comprise at least about 1 definitive entoderm cell, whenever comprise that 100 pluripotent cells just comprise at least about 1 definitive entoderm cell, whenever comprise that 10 pluripotent cells just comprise at least about 1 definitive entoderm cell, whenever comprise that 5 pluripotent cells just comprise at least about 1 definitive entoderm cell, whenever comprise that 2 pluripotent cells just comprise at least about 1 definitive entoderm cell, whenever comprise that 1 pluripotent cell just comprises at least about 2 definitive entoderm cells, whenever comprise that 1 pluripotent cell just comprises at least about 5 definitive entoderm cells, whenever comprise that 1 pluripotent cell just comprises at least about 10 definitive entoderm cells, whenever comprise that 1 pluripotent cell just comprises at least about 20 definitive entoderm cells, whenever comprise that 1 pluripotent cell just comprises at least about 50 definitive entoderm cells, whenever comprise that 1 pluripotent cell just comprises at least about 100 definitive entoderm cells, whenever comprise that 1 pluripotent cell just comprises at least about 1,000 definitive entoderm cell, whenever comprise that 1 pluripotent cell just comprises at least about 10,000 definitive entoderm cell, whenever comprise that 1 pluripotent cell just comprises at least about 100,000 definitive entoderm cell, whenever comprise that 1 pluripotent cell just comprises at least about 1,000,000 definitive entoderm cell.In some embodiments of the present invention, pluripotent cell is a human pluripotent stem cells.In some embodiments, stem cell-derived in morula, embryo's inner cell mass or embryo's genital ridge.In some of the other embodiments, pluripotent cell is derived from the sexual gland or the germinal tissue of the multi-cellular structure of process embryo stage growth.
Some aspects of the present invention relate to cell culture or cell mass, comprise at least about 5% definitive entoderm cell extremely at least about 95% definitive entoderm cell.In some embodiments, cell culture or cell mass comprise mammalian cell.In preferred embodiments, cell culture or cell mass comprise people's cell.For example, relate to cell culture in some specific embodiments, comprise people's cell, wherein at least about 5% to being the definitive entoderm cell at least about 95% people's cell.Other embodiment of the present invention relates to cell culture, comprise 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 be the definitive entoderm cell greater than people's cell of 90%.
Other embodiment of the present invention relates to the composition such as cell culture or cell colony, it comprises the people's cell such as people's definitive entoderm cell, wherein in the people's cell about 5% at least the expression of SOX17 or CXCR4 mark all greater than the expression of OCT4, SPARC, alpha fetal protein (AFP), thrombomodulin (TM) and/or SOX7 mark.In other embodiments, wherein in the people's cell about 10% at least, in people's cell of 15%, in people's cell of 20%, in people's cell of 25%, in people's cell of 30%, in people's cell of 35%, in people's cell of 40%, in people's cell of 45%, in people's cell of 50%, in people's cell of 55%, in people's cell of 60%, in people's cell of 65%, in people's cell of 70%, in people's cell of 75%, in people's cell of 80%, in people's cell of 85%, in people's cell of 90%, at least about in 95% people's cell or greater than in 95% people's cell, the expression of SOX17 or CXCR4 mark is all greater than OCT4, SPARC, AFP, the expression of TM and/or SOX7 mark.
Be to be understood that, some embodiments of the present invention relate to the composition such as cell culture or cell mass, it comprises the people's cell as people's definitive entoderm cell, wherein at least about 5% to greater than at least about 95% people's cell, the expression that wherein is selected from one or more marks of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 is higher than the expression of OCT4, SPARC, AFP, TM and/or SOX7 mark.
Other embodiment of the present invention relates to the composition as cell culture or cell mass, it comprises the people's cell as people's definitive entoderm cell, and wherein the expression of SOX17 and CXCR4 mark all is higher than OCT4, SPARC, AFP, TM and/or SOX7 marker expression at least about people's cell of 5%.In other embodiments, in at least about 10% people's cell, in people's cell of 15%, in at least about 20% people's cell, in at least about 25% people's cell, in at least about 30% people's cell, in at least about 40% people's cell, in at least about 50% people's cell, in at least about 55% people's cell, in at least about 60% people's cell, in at least about 65% people's cell, in at least about 70% people's cell, in at least about 75% people's cell, in at least about 80% people's cell, in at least about 85% people's cell, in at least about 90% people's cell, in at least about 95% people's cell or greater than in 95% people's cell, the expression of SOX17 and CXCR4 mark all is higher than OCT4, SPARC, AFP, the expression of TM and/or SOX7 mark.
Be to be understood that, some embodiments of the present invention relate to the composition as cell culture or cell mass, it comprises the people's cell as people's definitive entoderm cell, at least about 5% to greater than at least about 95% people's cell, wherein the expression of the mark of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 is higher than the expression of OCT4, SPARC, AFP, TM and/or SOX7 mark.
Other embodiment of the present invention relates to the composition such as cell culture or cell mass, comprise Mammals endoderm cell such as people's definitive entoderm cell, wherein at least in about 5% endoderm cell the expression of SOX17 or CXCR4 mark all greater than the expression of OCT4, SPARC, alpha fetal protein (AFP), thrombomodulin (TM) and/or SOX7 mark.In other embodiments, wherein at least in about 10% endoderm cell, in 15% endoderm cell, in 20% endoderm cell, in 25% endoderm cell, in 30% endoderm cell, in 35% endoderm cell, in 40% endoderm cell, in 45% endoderm cell, in 50% endoderm cell, in 55% endoderm cell, in 60% endoderm cell, in 65% endoderm cell, in 70% endoderm cell, in 75% endoderm cell, in 80% endoderm cell, in 85% endoderm cell, in 90% endoderm cell, at least about among 95% the endoderm cell or greater than among 95% the endoderm cell, the expression of SOX17 or CXCR4 mark is all greater than OCT4, SPARC, AFP, the expression of TM and/or SOX7 mark.
Be understood that, some embodiments of the present invention relate to the composition as cell culture or cell mass, it comprises the Mammals endoderm cell, wherein at least about 5% to greater than at least about 95% described endoderm cell, one or more expression that are selected from the mark of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 are higher than the expression of OCT4, SPARC, AFP, TM and/or SOX7 mark.
Other further embodiment of the present invention relates to the composition as cell culture or cell mass, comprise Mammals endoderm cell such as people's definitive entoderm cell, wherein in the described endoderm cell about 5% at least the expression of SOX17 and CXCR4 mark all greater than the expression of OCT4, SPARC, alpha fetal protein (AFP), thrombomodulin (TM) and/or SOX7 mark.In other embodiments, wherein at least about 10% endoderm cell, in 15% endoderm cell, in 20% endoderm cell, in 25% endoderm cell, in 30% endoderm cell, in 35% endoderm cell, in 40% endoderm cell, in 45% endoderm cell, in 50% endoderm cell, in 55% endoderm cell, in 60% endoderm cell, in 65% endoderm cell, in 70% endoderm cell, in 75% endoderm cell, in 80% endoderm cell, in 85% endoderm cell, in 90% endoderm cell, at least about among 95% the endoderm cell or greater than among 95% the endoderm cell, the expression of SOX17 and CXCR4 mark is all greater than OCT4, SPARC, AFP, the expression of TM and/or SOX7 mark.
Be to be understood that, some embodiments of the present invention relate to the composition such as cell culture or cell mass, comprise the Mammals endoderm cell, wherein at least about 5% to greater than at least about 95% endoderm cell, the expression of the mark of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 is higher than the expression of OCT4, SPARC, AFP, TM and/or SOX7 mark.
Use method of the present invention, can prepare the composition that comprises the definitive entoderm cell that does not contain other cell type substantially.About the cell in cell culture or the cell mass, term " do not contain substantially " refer to the specific cell in cell culture or the cell mass do not exist or less than cell culture or cell mass in total cell count about 5%.In some embodiments of the present invention, use the definitive entoderm cell mass of method preparation of the present invention or cell culture not to contain special remarkable cell of expressing OCT4, SOX7, AFP, SPARC, TM, ZIC1 or BRACH marker gene substantially.
In one embodiment of the invention, based on the expression of marker gene, the definitive entoderm cell be described as SOX17 height, MIXL1 height, AFP is low, SPARC is low, thrombomodulin is low, SOX7 is low, CXCR4 is high.
The enrichment of definitive entoderm, separation and/or purifying
About others of the present invention, the definitive entoderm cell can use special affinity labelling at these cells to come enrichment, separation and/or purifying.The example of special affinity labelling at the definitive entoderm cell is that antibody, part or other are special at the binding reagents as the marker molecules of polypeptide, this marker molecules is present in the surface of cell definitive entoderm cell, but is not present in substantially in other cell type of finding in the cell culture medium for preparing according to method of the present invention.In some embodiments, with CXCR4 bonded antibody be used for definitive entoderm enrichment, separate and/or the affinity labelling of purifying.In other embodiments, chemokine SDF-1 or other molecule based on SDF-1 also can be used for affinity labelling.These molecules include, but are not limited to the SDF-1 segment, SDF-1 fusions or SDF-1 stand-in.
The method for preparing antibody and be used for cellular segregation is known in the art, and these methods can utilize antibody of the present invention and cell to implement.In one embodiment, will combine with magnetic bead, in cell culture medium, combine then with the definitive entoderm cell in conjunction with the antibody of CXCR4, after enzyme is handled, reduced iuntercellular and with the adhesion of substrate.Cell/antibody/magnetic bead mixture is exposed in the shifting magnetic field so that magnetic bead bonded definitive entoderm cell is separated with unconjugated cell.In case the definitive entoderm cell separates with other cell physics in substratum, destroy bonded antibody, cell is placed suitable tissue culture medium (TCM) again.
Embodiment of the present invention has been considered enrichment, separation and/or the purification process of other definitive entoderm cell culture or cell mass.For example, in some embodiments, CXCR4 antibody is hatched in comprising the cell culture of definitive entoderm, reduce after treatment iuntercellular and with the adhesion of substrate.Then with cell washing, centrifugal and resuspending.The cell suspension thing and then and second antibody, as closing antibody incubation with first antibody bonded FITC yoke.And then with in cell washing, the centrifugal and resuspending damping fluid.Subsequent analysis cell suspension thing uses fluorescent activation cell sorter (FACS) sorting.With CXCR4 -Negative cells separates, and collects CXCR4 -Positive cell has separated the type cell thus.When needing, the isolated cells composition can further use the affine method of alternate or increase sorting for several times, uses special identical or different mark at definitive entoderm.
In other embodiment of the present invention, use with CXCR4 bonded part or other molecule enrichment, separate and/or the purifying definitive entoderm.In some embodiments, described molecule is SDF-1 or its fragment, fusions or its stand-in.
In preferred embodiments, induced after definitive entoderm system differentiation enrichment in other non-definitive entoderm cell, separation and/or purifying definitive entoderm cell when stem cell culture.Should be understood that above-mentioned enrichment, separation and/or purification process can utilize this culture of any differential period.
Remove above-mentioned method, the definitive entoderm cell also can separate by other cell separation technology.In addition, the definitive entoderm cell also can be by enrichment of a series of cultured method again or separation under growth conditions, and this growth conditions helps described definitive entoderm cell selective survival or selective amplification.
Use method of the present invention, can be external from pass through at least some differentiation as the pluripotent cell culture of stem cell culture or cell mass or cell mass in enrichment, separation and/or purifying definitive entoderm cell.In some embodiments, described cell breaks up at random.Yet in certain preferred embodiments, described cell mainly is divided into definitive entoderm.Some preferred enrichments, separation and/or purification process relate to and externally prepare definitive entoderm from human embryo stem cell.Use method of the present invention, compare with untreated cell mass or cell culture, the cell mass of enrichment or cell culture are at least about 2 times to about 1000 times in the definitive entoderm.In some embodiments, compare with untreated cell mass or cell culture, the definitive entoderm of enrichment is at least about 5 times to about 500 times.In other embodiments, compare with untreated cell mass or cell culture, the definitive entoderm of enrichment is at least about 10 times to about 200 times.In other embodiments, compare with untreated cell mass or cell culture, the definitive entoderm of enrichment is at least about 20 times to about 100 times.In other embodiments, compare with untreated cell colony or cell culture, the definitive entoderm of enrichment is at least about 40 times to about 80 times.In certain embodiments, compare with untreated cell colony or cell culture, the definitive entoderm of enrichment is at least about 2 times to about 20 times.
The present invention has been carried out general description, can further understand the present invention with reference to some specific embodiments, these embodiment of the present invention are the purpose of exemplary illustration only, but not are intended to limit the present invention.
Embodiment
Following many embodiment have described the use of pluripotent human cell.The method for preparing the pluripotent human cell is that persons skilled in the art are known, and a large amount of technical presses comprise U.S. Patent number 5,453,357,5,670,372,5,690,926,6,090,622,6,200,806 and 6,251,671 and U.S. Patent Application Publication No. 2004/0229350 description is all arranged, this paper is with its whole introducings, as a reference.
Embodiment 1
People ES cell
Grow for the research entoderm, we utilize human embryo stem cell, as if it is a multipotential stem cell, can infinitely divide in substratum, keeps normal karyotype simultaneously.ES is cell-derived in embryo's inner cell mass of 5 days sizes, uses immunology or mechanical means to separate.Particularly, the human embryonic stem cell hESCyt-25 extra freezing embryo in the cycle in vitro fertilization that the patient agrees that hangs oneself.After the thawing, the blastaea of hatching is inoculated on the mouse embryo fibroblasts (MEF), uses ES substratum (DMEM, 20%FBS, non-essential amino acid, beta-mercaptoethanol, ITS conditioning agent).After about two weeks, the embryo adheres in the culture dish, will not break up the hESCs zone-transfer to MEFs.Transfer is finished with cut mechanically, after the simple digestion of use neutral protease, removes cell cluster with machinery, washs, inoculates.After self-derivedization, continuous passage hESCyt-25 100 times.We utilize the hESCyt-25 human embryonic stem cell to prepare definitive entoderm as parent material.
Persons skilled in the art should be understood that stem cell or other pluripotent cell also can be used as the parent material of differentiation method of the present invention.For example, embryo's genital ridge cell can separate according to methods known in the art, as the pluripotent cell parent material.
Embodiment 2
The sign of hESCyt-25
Human embryonic stem cell keeps normal morphology, karyotype, growth and self characteristic 18 middle of the month cultivating always.This clone has shown the intensive immunoreactivity to OCT4, SSEA-4 and TRA-1-60 antigen, and above-mentioned antigen all is the feature of not breaking up hESCs, and shows that other hESC that has set up is identical alkaline phosphate ester activity and morphology.In addition, when human stem cell is the hESCyt-25 suspension culture, also be easy to form class embryoid body (EBs).Because the proof of its multipotency essence, hESCyT-25 is divided into the different cell types of representing three kinds of main germinal layers.Detect ZIC1 and immunocytochemistry (ICC) detection nidogen and how sophisticated neural mark with Q-PCR and prove conclusively ectodermic generation.The immunocytochemical stain of β-III microtubule can be observed in the cell cluster of elongation, has early stage neuronic feature.Before this, we handle the internal organ entoderm (VE) that the EBs induced multipotent stem cells is divided into system outside the embryo in comprising the suspension of vitamin A acid.Through 54 hours processing, two VE marks of the high-caliber alpha fetal protein of the cell expressing of processing (AFP), SOX7.Immunocytochemical stain shows that the cell expressing AFP with the individual layer differentiation is odd sheet.As described below, hESCyT-25 clone also can form definitive entoderm, proves conclusively by the immunocytochemistry that real-time quantitative polymerase chain reaction (Q-PCR) and detection SOX17, no AFP express.In order to confirm to be divided into mesoderm, the EB that is breaking up in several time point analyses is to detect short-tail (Brachyury) genetic expression.In process of the test, the increase of carrying out property of short-tail genetic expression.As previously mentioned, hESCyT-25 is polyenergic, can form the cell of representing three germinal layers.
Embodiment 3
The preparation of SOX17 antibody
The main bottleneck of identification definitive entoderm is for lacking proper implements in hESC cultivates.We thus prepare the proteic antibody of anti-SOX17.
When forming during gastrula forms, mark SOX17 is expressed in whole definitive entoderm, and its expression remains on intestinal tube (axle is variant although expression level prolongs A-P) and begins to form until organ.SOX17 also is expressed on a series of extraembryonic endoderm cells.This protein does not have expression at mesoderm or ectoderm.When combining with other mark when getting rid of outside the embryo pedigree, have now found that SOX17 is the suitable mark of definitive entoderm pedigree.
Describe in detail as this paper,, SOX17 antibody is used for the effect of various processing of special detection and differentiation method in order to prepare SOX17 male definitive entoderm cell.The antibody of other and AFP, SPARC and thrombomodulin reaction also is used to get rid of the generation of internal organ and body wall entoderm (extraembryonic endoderm).
In order to prepare the antibody of anti-SOX17, according to the GENOVAC (Freiberg of Antibody Preparation company, Germany) Kai Fa method, the groups of people SOX17 cDNA (SEQIDNO:1) corresponding with the carboxylic terminal amino acid 172-414 (SEQNO:2) of SOX17 albumen (accompanying drawing 2) is used for the inherited immunity of rat.The method of inherited immunity is found in U.S. Patent number 5,830, and 876,5,817,637,6,165,993 and 6,261,281 and International Patent Application Publication No. WO 00/29442 and WO99/13915, it openly is incorporated herein, for your guidance.
Other method of inherited immunity also is found in non-patent literature.For example, Barry etc. are described to prepare monoclonal antibody according to inherited immunity, Biotechniques 16:616-620, and 1994, it is disclosed in this whole introducings, for your guidance.Prepare the concrete grammar of anti-differential protein antibody according to inherited immunity, for example, the inherited immunity of Costaglia etc. (1998) human thyrotropin receptor causes the monoclonal antibody of thyroiditis and preparation identification autoreceptor, J.Immunol.160:1458-1465; The quick preparation that people such as Kilpatrick (1998) particle gun transmits based on the mouse monoclonal antibody of the immune-mediated Flt-3 acceptor of DNA, Hybridoma 17:569-576; People such as Schmolke, (1998), the E2-monoclonal antibody specific that in human serum, produces identification hepatitis G virion J, Virol.72:4541-4545 by dna immunization; People such as Krasemann, (1999) use unconventional nucleic acid immunization strategy to produce at proteic monoclonal antibody, J.73:119-129; And people (1996) such as Ulivieri produces the monoclonal antibody of helicobacter pylori vacuolating cytotoxin determining section by dna immunization, and 51:191-194 above-mentionedly is disclosed in this whole introducings, for your guidance.
Shown in the relational tree of Fig. 3, in Sox family, SOX7 and SOX18 are the most close with SOX17.We utilize people SOX7 polypeptide is special with confirmation SOX17 antibody to SOX17 as negative control, not with the most close family member's reaction.Particularly, in order to prove that the antibody that inherited immunity produces is special to SOX17, SOX7 and other protein expression on the human fibroblasts, are analyzed total reaction activity with SOX17 antibody by Western blot and ICC then.For example, use following method to prepare the expression vector of SOX17, SOX7 and EGFP, it is transfected into the human fibroblasts also analyzes with Western blot.The expression vector that is used to prepare SOX17, SOX7 and EGFP is respectively pCMV6 (OriGene Technologies, Inc., Rockville, MD), pCMV-SPORT6 (Invitrogen, Carlsbad, CA) and pEGFP-N1 (Clonetech, Palo Alto, CA).Be preparation albumen, use Lipofectamine 2000 with the telomerase immortalized MDX human fibroblasts of super coiled DNA transient transfection (Invitrogen, Carlsbad, CA).After the transfection 36 hours, collect total cell lysate in 50mM TRIS-HCl (pH 8), 150mMNaCl, 0.1%SDS, 0.5% Septochol and some proteinase inhibitor (Roche DiagnosticsCorporation, Indianapolis, IN) in.Western blot analyzes the cell protein of 100 μ g, with SDS-PAGE at NuPAGE (4-12% gradient polyacrylamide, Invitrogen, Carlsbad, CA) separate, be transferred to (Hercules on the PDVF film by electroblotting, CA), with in 10mMTRIS-HCl (pH 8), 150mM NaCl, 10%BSA, 0.05%Tween-20 (Sigma, St.Louis, MO) be diluted to 1/1000 mouse SOX17 antiserum(antisera) in and survey, handle (Jackson ImmunoResearch Laboratories, West Grove with the mouse IgG of the Chinese People's Anti-Japanese Military and Political College then in conjunction with alkaline phosphatase, PA), the result with Vector Black alkaline phosphatase dyeing show (VectorLaboratories, Burlingame, CA).The albumen size criteria of using as the color sign thing of wide region (Sigma, St.Louis, MO).
In Fig. 4, will be that probe carries out Western blot with SOX17 antibody from the human fibroblasts's of SOX17, SOX7 or EGFP cDNA transient transfection protein extract.Only the protein that extracts in the hSOX17 cells transfected has produced the band of about 51Kda, and this band mates with the proteinic 46Kda molecular weight of people SOX17 of prediction most.SOX17 antibody does not have reactivity to the extract from people SOX7 or EGFP transfectional cell.In addition, SOX17 antibody significantly mark with the human fibroblasts of hSOX17 expression construct transfection nuclear, but not mark with the independent cells transfected of EGFP.Similarly, SOX17 antibody shows the specificity that detects by ICC.
Embodiment 4
SOX17 antibody is as the conclusive evidence of definitive entoderm mark
According to SOX17 antibody people SOX17 protein is had specificity and further indicates definitive entoderm, with hESCs and the SOX17 and the common mark of AFP antibody of part differentiation.Proved that SOX17, SOX7 and AFP are expressed in the internal organ entoderm separately, SOX7 is the closely-related member of SOX gene family F subgroup (Fig. 3).Yet AFP and the SOX7 expression in the definitive entoderm cell is in the level that can be detected by ICC, therefore, and can be used as the negative mark of bonifide definitive entoderm cell.According to the show, SOX17 antibody indicator cell line colony exists with the dispersive cell mass, or mixes with the AFP positive cell.Particularly, Fig. 5 A shows the common a small amount of SOX17 cell that indicates with AFP; Yet, also found some zones, wherein at SOX17 +Have in the cell field and have AFP on a small quantity or not +Cell (Fig. 5 B).Similarly, express SOX17, the antibody with the common sign of SOX17 can be used from the endoblastic SOX17 of discriminating body wall with body wall mark SPARC and/or thrombomodulin (TM) because also reported the body wall entoderm +Cell.Shown in accompanying drawing 6A-C, produce the body wall endoderm cell of thrombomodulin and the common sign of SOX17 by the differentiation at random of hES cell.
According to above-mentioned cell sign test, the characteristic of definitive entoderm cell can be composed SOX17 by marker expression Hi/ AFP Lo/ [TM LoOr SPARC Lo] set up.In other words, the expression of SOX17 mark is higher than the expression of AFP mark and TM or SPARC mark, and the AFP mark is the endoblastic a kind of features of internal organ, and TM or SPARC mark are the endoblastic feature of body wall.Therefore, those are positive to SOX17 and AFP are negative and are definitive entoderm to the cell that TM or SPARC are negative.
SOX17 Hi/ AFP Lo/ TM Lo/ SPARC LoThe specificity of marker expression spectrum can be used as the further evidence of definitive entoderm prediction, and the relative populations with the antibody labeling cell is suitable quantitatively with SOX17 and AFP genetic expression.Shown in Fig. 7 A, the hESCs that handles with vitamin A acid (internal organ entoderm inductor) or activin A (definitive entoderm inductor) has caused 10 times of differences at SOX17 mRNA expression level.This result reflects 10 times of differences (Fig. 7 B) of the cell quantity of SOX17 antibody labeling.In addition, shown in Fig. 8 A, compare with being untreated, the activin A of hESCs handles and has suppressed 6.8 times AFP genetic expression.The rapid minimizing of the cell quantity of AFP mark is from visually having reflected this variation, shown in Fig. 8 B-C in these cultures.For further quantification,, proved that about 7 times cell quantity that is reduced to the AFP antibody labeling of AFP genetic expression reduces 7 times result (accompanying drawing 9A-B) approximately with cells were tested by flow cytometry.This result is extremely important, has shown the number change of observed genetic expression among the Q-PCR, has reflected that specialization of cell type of observing by antibody staining typically changes.
HESCs cultivates under the condition that Nodal family member (Nodal, activin A and activin B-NAA) exists, and the cell that causes SOX17 antibody sign is along with the time obviously increases.Handled 5 days with activin continuously, have more than 50% cell by SOX17 mark (Figure 10 A-F).Activin was handled after 5 days, did not almost have cell to indicate with AFP.
In a word, 242 antibody aminos of the proteinic carbon teminal of anti-people SOX17 that produced have been differentiated people SOX17 protein but unidentified SOX7 at Western blots, and it is the close relative of nearest Sox family.The SOX17 antibody recognition differentiation hESC cells in culture subgroup, this subgroup is mainly SOX17 +/ AFP Lo/-The cell (internal organ entoderm) of the common sign of SOX17, the AFP of (indicator cell line) and a small amount of (＜5%) more than 95%.Handle the hESC culture with activin, produced the remarkable rising of SOX17 genetic expression and SOX17 labeled cell, and suppressed AFP mRNA significantly and expressed the cell quantity that reaches with the AFP antibody labeling.
Embodiment 5
The Q-PCR gene expression analysis
In following test, real-time quantitative RT-PCR (Q-PCR) is for being used for the main method of the various processing of examination to the hESC Differentiation.Particularly, with the genetic expression of Q-PCR The real time measure, analyze a plurality of marker gene at a plurality of time points.The marker gene feature of the cell type of estimating expectation and not expecting is to obtain the overall dynamic (dynamical) better understanding of pair cell colony.The strength that Q-PCR analyzes comprises its extreme sensitivity and relatively easily can develop necessary mark because of genome sequence is easy to utilize.In addition, the high sensitivity of Q-PCR allows to detect the relatively small amount gene expression of cells in bigger colony.In addition, the ability of the extremely low-level genetic expression of detection provides the indication of intragroup " differentiation tendency ".Before the obvious differentiation of these cell phenotypes, its proneness to concrete differentiation pathway can not be discerned with immunocytochemical technique.Thereby Q-PCR provides a kind of analytical procedure, and this method is at least replenishing of immunologic cellular activity technology, and more is better than the immunologic cellular activity technology potentially, is used for the examination differentiation and handles success.In addition, Q-PCR provides a kind of mechanism, and whether this mechanism is successful by the quantitative analysis evaluate differentiation method on the half high-throughput scale.
The method relative quantification of Shi Yonging is gone up use SYBR Green chemistry and the operation of two one step RT-PCRs at Rotor Gene 3000 instrument (CorbettResearch) herein.This method allows to store the cDNA sample, to analyze other following marker gene, therefore, avoids the variability of sample room reverse transcription efficient.
If may, the primer of design is positioned exon-exon border or crosses the intron of 800bp at least, but because this rule of thumb amplification of the genomic dna of decontamination.When use did not contain intron or do not have the marker gene of pseudogene, the DNA enzyme I that carries out the RNA sample handled.
We use the genetic expression of a plurality of marks of Q-PCR mensuration target and non-target cell type usually, to provide a description the wide in range express spectra of cell sample genetic expression.Early stage (ectoderm, mesoderm, definitive entoderm and extraembryonic endoderm particularly) correlating markings thing of hESC differentiation and available checking primer are provided in the following table 1.Also proved the human specific of these primer sets.This fact is very important, because usually hESCs is grown on the mouse trophoderm.Modal is that each condition is got 3 samples, and analyzes twice independently, to estimate the biomutation relevant with various detection by quantitative.
For the preparation pcr template, total RNA is separated with RNeasy (Qiagen), and quantitative with RiboGreen (Molecular Probes).Finish the reverse transcription of the total RNA of 350-500ng with iScript reverse transcription test kit (BioRad), this test kit comprises the mixture of oligomerization dT and random primer.Subsequently each 20 μ L reactant is diluted to 100 μ L cumulative volumes, gets 3 μ L and be used for each 10 μ L Q-PCR reaction, this reaction comprises 400nM forward primer and reverse primer and 5 μ L 2X SYBR Greenmaster mix (Qiagen).Select two step loop parameters for use, in 85-94 ℃ of sex change in 5 seconds (melting temperature (Tm) according to each primer sets amplicon is specifically selected), then 60 ℃ of annealing/extensions 45 seconds.During last 15 seconds of each extended peroid, collect fluorescence data.Three points of 10 times of dilution series are used to produce the typical curve of each wheel, and change circulation threshold (Ct ' s) into quantitative value based on this typical curve.The numerical value of each sample is characterized calibration with house-keeping gene, calculate the average and standard deviation of three samples then.Finish the PCR circulation time, determining the specificity of reaction with the melting curve analysis.Single special product is presented at the suitable T of pcr amplification mThe simple spike place.In addition, the reaction that do not have ThermoScript II as negative control, is not increased.
The first step in setting up the Q-PCR methodology is a house-keeping gene (HGs) suitable in the confirmed test system.Because HG is used for sample room RNA input, RNA integrity and RT efficiency calibration, for making calibration meaningful, HG constant in time expression level in all samples type is valuable.We have measured the expression level of Cyclophilin G, hypoxanthine phosphoribosyltransferase 1 (HPRT), beta-2-microglobulin (microglobulin), hydroxymethylbiane synthetic enzyme (HMBS), TATA-conjugated protein (TBP) and glucoronidase β (GUS) in differentiation hESCs.Our result has shown that the beta-2-microglobulin expression level improves in atomization, therefore, we have got rid of this gene is used for calibration.Other gene expression dose is with consistent with the entire treatment process in time.We use Cyclophilin G and GUS to calculate the calibration factor of all samples usually simultaneously.Use a plurality of HGs to reduce the variation of calibration process inherent simultaneously, increase the reliability of relative genetic expression value.
After obtaining the gene that is used to calibrate, Q-PCR is used for detection accepts different tests processing back sample, determine the relative gene expression dose of many marker gene.The marker gene of select using is enrichment in the representative special colony of germinal layer in early days, and differentially expressed many groups gene is particularly arranged in definitive entoderm and extraembryonic endoderm.These genes and relevant enrichment characteristics thereof are highlighted in table 1.
Table 1
Germinal layer Gene The expression structure territory
Entoderm extraembryonic ectoderm mesoderm SOX17 MIXL1 GATA4 HNF3b GSC SOX7 AFP SPARC TM ZIC1 BRACH Setting, internal organ and body wall entoderm entoderm and mesoderm setting and primitive endoderm definitive entoderm and primitive endoderm, mesoderm, neural plate entoderm and mesoderm internal organ entoderm internal organ entoderm, liver body wall entoderm body wall entoderm/trophectoderm neurocele, the neural system precursor mesoderm of coming into being
Because many genes are not only being expressed in a germinal layer, the many expression of gene levels of quantitative comparison are useful in same test.SOX17 expresses in definitive entoderm, and express on less degree ground in internal organ and body wall entoderm.SOX7 and AFP in early days development time point in the internal organ entoderm is expressed.SPARC and TM express in the body wall entoderm, and Brachyury expresses in the mesoderm in early days.
Prediction setting endoderm cell high level expression SOX17 mRNA, low expression level AFP and SOX7 (internal organ entoderm), SPARC (body wall entoderm) and Brachyury (mesoderm).In addition, the present invention is used for further getting rid of early stage ectodermic inducing with ZIC1.At last, GATA4 and HNF3b express in setting and extraembryonic endoderm simultaneously, therefore, and with the expression of SOX17 in definitive entoderm relevant (table 1).Representative test is shown among Figure 11-14, has confirmed how the described marker gene of table 1 is relative to each other with each sample, thereby has emphasized the special differentiation model of definitive entoderm, extraembryonic endoderm, mesoderm and neural cell type.
The clear activin dosage that shown of above-mentioned data increases, and the SOX17 genetic expression that causes increases.Further, this SOX17 expresses and mainly represents definitive entoderm, rather than opposite extraembryonic endoderm.The conclusion of observing is SOX17 genetic expression and AFP, SOX7 and SPARC inverse correlation.
Embodiment 6
People ES cell directional is divided into definitive entoderm
If people ES cell culture is cultivated under its undifferentiated state not keeping on one's own initiative, this culture will break up at random.Different differentiation causes forming the extraembryonic endoderm cell, it comprise body wall and internal organ entoderm (AFP, SPARC and SOX7 express) both, and with the ectoderm that is expressed as sign, the mesoderm derivative of ZIC1, Nestin (ectoderm) and Brachyury (mesoderm).In the ES cell culture, owing to lack the specific antibody mark, definitive entoderm cell outward appearance is without traditional technique in measuring or definite.Similarly, because the shortage means produce early stage definitive entoderm without scrutinizing from the culture of ES cell.Because no antibody reagent good, available definitive entoderm cell, most conclusive evidences concentrate on ectoderm and extraembryonic endoderm.In a word, in differentiation ES cell culture at random, with SOX17 HiThe definitive entoderm cell is compared, and having significantly, a large amount of embryos reaches the generation of neuroderm cell type outward.
When undifferentiated hESC being cloned in the expansion of inoblast nutrient, clone's edge presents and the different morphological feature of the inner cell of clone.Many border cells can distinguish with its bigger inhomogenous cell build and the high level expression of OCT4.The existing description, when the ES cell begins differentiation phase, they are expressed the level of OCT4 and are up and down with respect to the level of not breaking up the ES cell and change.With respect to the OCT4 threshold level of undifferentiated cell, change the initial differentiation state that the multipotency state is left in demonstration up and down.
When undifferentiated clone detects with the SOX17 immunocytochemistry, do not break up ESC clone around and the junction can detect the tuftlet of 10-15 cell composition of SOX17 positive cell once in a while at random.As mentioned above, when the amplification of clone's volume consequently becomes crowded, first cell that the bag shape that clone's outer rim distributes seemingly breaks up from the ESC form of classics.Clone (＜1mm in clone's the inside and the not differentiation that the age is less, volume is less at edge; 4-5 days sizes) no SOX17 positive cell, yet at some clones' periphery and edge with interior older, the bigger clone of volume (1-2mm diameter,＞5 days sizes) contain odd, the free sheet SOX17 positive, AFP negative cells, as mentioned above, its hESC form from classics differentiates.In view of this is the exploitation first of effective SOX17 antibody, the definitive entoderm cell that generates in this early stage " not differentiation " ESC culture does not confirm before this as yet.
Based on the SOX17 of Q-PCR mensuration and the negative correlation of SPARC gene expression dose, most SOX17 positives, AFP negative cells are negative the body wall mark that antagonist is total to mark.Shown in Figure 15 A-B, obtain special confirmation the body wall endoderm cell of Expression of TM.Contact the sharp fall of the number that causes TM expression intensity and TM positive cell with Nodal factor activin A and B.On the substratum that activin is handled,, observe the SOX17 positive cell (Figure 16 A-D) that AFP and TM also are negative by using three heavy labels of SOX17, AFP and TM antibody.These all are the positive definitive entoderm cells (Figure 16 A-D and 17) of the SOX17 of cell confirmation first on the culture of differentiation ESC.
Use above-mentioned SOX17 antibody and Q-PCR instrument, we have developed and a series ofly can become SOX17 by effective procedure ESCs Hi/ AFP Lo/ SPARC/TM LoThe method of definitive entoderm cell.We have used a series of differentiation method that is intended to increase these cell quantities and multiplication capacity, use Q-PCR to detect SOX17 genetic expression on population level, use SOX17 protein antibodies mark on individual cells.
We analyze and have described TGF 'beta ' family somatomedin first, and as Nodal/ activin/BMP, the embryonic stem cell that is used for cultivating from cell in vitro is created the effect of definitive entoderm cell.In typical test, we are added to undifferentiated human stem cell with activin A, activin B, BMP or its combination is to begin atomization in the hESCyt-25 culture.
As shown in figure 19, the activin A that adds 100ng/ml concentration broke up 4 days, compared with undifferentiated hESCs, had induced 19 times of SOX17 genetic expressions.Second the member's activin B that adds activin family with activin A handles by 4 days combination activin, compares with undifferentiated hESCs, induced 37 times of SOX17 genetic expressions.Together add the 3rd member BMP4 of TGF 'beta ' family Nodal/ activin and BMP subgroup with activin A and activin B, compare, induced 57 times of SOX17 genetic expressions (Figure 19) with undifferentiated hESCs.When inducing SOX17 with activin and BMP, compare with the culture contrast that does not add the factor, break up and caused 5-in 4 days, 10-and 15-times induce.By triple processing of 5 days activin A, B and BMP, SOX17 inductive multiple is higher more than 70 times than hESCs.These data presentation cause that with Nodal/ activin TGF 'beta ' family member higher dosage, long treatment time SOX17 expresses increase.
Nodal and relevant activin A, B and BMP molecule promote the expression of SOX17, the formation of definitive entoderm vivo and vitro.In addition, adding BMP and cause that SOX17 induces rising, may be further inducing by Nodal co-receptor Cripto.
We confirmed to unite use activin A, B and BMP4 to cause that the SOX17 inductive increases and and then definitive entoderm form.With activin A and B combination, add BMP4 (＞4 days) for a long time and can induce the SOX17 of body wall and internal organ entoderm and definitive entoderm to increase.Therefore, in some embodiments of the present invention, it is important removing BMP4 in 4 days of adding processing.
In order on unicellular level, to determine the effect that the TGF β factor is handled, use the SOX17 antibody labeling to detect the effect of the TGF β factor that adds a time-histories.Shown in preceding Figure 10 A-F, along with the time carries out, increasing severely appears in the relative populations of the cell of SOX17 mark.Relative quantification (Figure 20) shows that the cell of SOX17-mark increases more than 20 times.This result shows that the time that exposes with the TGF β factor increases, and cell quantity and gene expression dose all increase.As shown in figure 21, after Nodal, activin A, activin B and BMP4 contacted 4 days, SOX17 inductive level was higher 168 times than undifferentiated hESCs.Figure 22 shows that the quantity of SOX17 positive cell also is dose-dependently.100ng/mL or more the activin A of high dosage can induce the genetic expression of SOX17 and cell quantity to increase forcefully.
Remove TGF 'beta ' family member, the Wnt family molecule works on may and/or keeping in the definitive entoderm specificity.Compare with activin with single, use the sample SOX17 genetic expression of activin+Wnt3a to increase, show that the Wnt molecule is divided into definitive entoderm also useful (Figure 23) to hESCs.
Above-mentioned all tests are all carried out in the tissue culture medium (TCM) that contains 10% serum and the interpolation factor.It is shocking that we find that serum-concentration has effect to the SOX17 expression level, shown in Figure 24 A-C in the presence of the activin that adds.When serum level reduced to 2% by 10%, in the presence of activin A and B, the expression of SOX17 increased by 3 times.
At last, we confirm that activin induces SOX17 +Cell divides in culture, shown in Figure 25 A-D.Arrow shows that the cell with the SOX17/PCNA/DAPI mark is in m period, and evidence is that the mitotic division plate mode of PCNA/DAPI-mark in time differs the mitotic division feature.
Embodiment 7
The expression of Chemokine Receptors 4 (CXCR4) is relevant with the definitive entoderm mark and uncorrelated with mesoderm, ectoderm or internal organ entoderm mark
As mentioned above, reach the cytokine of more special activin/nodal subtribe by using the TGF 'beta ' family, ESCs can be induced to break up to the definitive entoderm germinal layer.In addition, we have shown the efficient of the scale effect definitive entoderm of foetal calf serum (FBS) in division culture medium from the ESCs differentiation.This effect is that under the condition of set activin A concentration, the FBS of higher level will suppress its maximum and break up to the definitive entoderm germinal layer in substratum.When lacking external source activin A, ESCs breaks up to the efficient of definitive entoderm pedigree extremely low, and FBS concentration has more weak effect to the atomization of ESCs.
In these trials, the differentiation of hESCs is at RPMI substratum (Invitrogen, Carlsbad, CA; Cat#61870-036) growth is 6 days in, and this culture medium supplemented has 0.5%, 2.0% or 10%FBS, comprises or do not contain 100ng/mL activin A.In addition, in first three day of differentiation, the gradient FBS of 0.5%-2.0% also unites use with 100ng/mL activin A.After 6 days, in each culture condition, collect the replica sample, with the relative genetic expression of real-time quantitative PCR analysis.Remaining cell is mixed, with immune fluoroscopic examination SOX17 albumen.
Under 7 culture condition that use, the expression level difference of CXCR4 huge (Figure 26).Usually, CXCR4 is expressed in the middle height of substratum (A100) that activin A handles, and low in the substratum of no external source activin A (NF).In addition, in the substratum that A100 handles, when FBS concentration was minimum, CXCR4 expressed the highest.Under the 10%FBS condition, the CXCR4 level significantly reduces, so that the condition of the more identical no activin A of relative expression (NF).
As mentioned above, SOX17, GSC, MIXL1, and HNF3 β expression of gene consistent with the feature of definitive entoderm cell.The relative expression of these 4 genes under 7 differentiation condition hinted obliquely at the expression of CXCR4 (Figure 27 A-D).This has confirmed that also CXCR4 also is a kind of definitive entoderm mark.
Ectoderm and mesoderm pedigree can distinguish by the various marks and the definitive entoderm of its expression.Early stage mesoderm is expressed Brachyury and MOX1 gene, yet nascent neuroderm is expressed SOX1 and ZIC1.Figure 28 A-D confirms that the culture of no external source activin A helps mesoderm and ectoderm genetic expression, and in the culture that activin is handled, the 10%FBS condition has also increased the level of mesoderm and ectoderm marker expression.These expression patterns are opposite with the CXCR4 pattern, are presented at this growth time-histories, CXCR4 not high expression level in being derived from ESCs mesoderm or ectoderm.
Grow in early days Mammals, pedigree to embryo has also taken place to break up.The endoblastic differentiation of internal organ has special dependency at this, and itself and the many genes of definitive entoderm common comprise that SOX17 has identical expression.For definitive entoderm and the outer internal organ entoderm of embryo are distinguished, should detect both different marks.SOX7 is expressed in the internal organ entoderm, rather than the mark of definitive entoderm pedigree.Thereby, under the condition that no SOX7 expresses, show that the culture condition of strong SOX17 genetic expression may comprise definitive entoderm, but not the internal organ entoderm.Shown in Figure 28 E, SOX7 is high expression level in no activin A substratum, SOX7 even under the condition that activin A exists, and when FBS comprised 10%, also expressing increased.This pattern is opposite with the CXCR4 expression pattern, shows that CXCR4 is at internal organ entoderm high expression level not.
Also detected SOX17 immunocompetence (SOX17 under above-mentioned each differentiation condition +) relative populations of cell.As hESCs differentiation phase under high dosage activin A and low FBS concentration (0.5%-2.0%), SOX17 +Cell is widespread distribution in culture.When using high dosage activin A, and FBS concentration is when being 10% (v/v), SOX17 +The frequency that cell occurs reduces, and often with isolated bunch of appearance, rather than evenly is distributed on (Figure 29 A, C, B and E) in the culture.When no external source activin A uses, find SOX17 +Cell further reduces.Under these conditions, SOX17 +Cell also occurs with bunch shape, but the less and higher activin A of these bunches, when low FBS handles few (Figure 29 C and F).These results show that the CXCR4 expression pattern not only meets definitive entoderm genetic expression under various conditions, and meet the quantity of definitive entoderm cell.
Embodiment 8
The differentiation condition of enrichment definitive entoderm increases the ratio of CXCR4 positive cell
The dosage of activin A has also influenced definitive entoderm from ESCs deutero-efficient.The dosage that present embodiment increases activin A has increased CXCR4 +The ratio of cell in culture.
With hESCs added 0.5%-2%FBS (in differentiation preceding 3 days, increase to 1.0% by 0.5% gradually) again to 2.0% and 0,10 or the RPMI substratum of 100ng/mL activin A in break up.Break up after 7 days, cell is not being contained Ca 2+/ Mg 2+, comprise among the PBS of 2%FBS and 2mM (EDTA) room temperature and dissociated 5 minutes.With 35 μ m NF filtration cells, counting and precipitation.To precipitate resuspending in the normal donkey serum of 50% human serum/50%, hatch 2 minutes blocking-up nonspecific antibody binding sites on ice.(comprise about 10 to per 50 μ L 5Individual cell) suspension add 1 μ L mouse anti CXCR4 antibody (Abcam, cat#ab10403-100), again ice marking 45 minutes.Adding 5mL comprises PBS washed cell, the precipitation of 2% human serum (damping fluid).Again with after the 5mL damping fluid washing once, with cell again with 50 μ L damping fluid/10 5Cell concn suspends.Adding final concentration is second antibody (the anti-mouse antibodies of bonded FITC donkey of 5 μ g/mL; Jackson Immuno Research, cat#715-096-151), mark washs 2 times with above-mentioned damping fluid after 30 minutes again.With cell again with 5 * 10 6Cell/mL is suspended in the damping fluid, uses FACS Vantage (Beckton Dickenson) analysis, sorting by flow cytometer device operator (The ScrippsResearch Institute).Cell directly is collected in RLT lysis buffer (Qiagen) supplies total RNA subsequently to separate, carry out gene expression analysis with real-time quantitative PCR again.
When the dosage (Figure 30 A-C) of activin A in division culture medium increases, can be observed the CXCR4 of cells were tested by flow cytometry +Cell also increase severely (Figure 30 A-C).CXCR4 +Cell falls into the R4 door, and this door only uses second antibody in contrast, has this contrast incident of 0.2% in the R4 door.When the dosage of activin A increases, CXCR4 +The sharp increase of cell quantity obviously increases relevant (Figure 31 A-D) with definitive entoderm genetic expression.
Embodiment 9
Concentration and separation CXCR4 positive cell supplies definitive entoderm genetic expression, removes the cell of expressing mesoderm, ectoderm and internal organ entoderm mark
Collect the CXCR4 of the foregoing description 8 identifications +And CXCR4 -Cell has been analyzed its relative genetic expression, has measured mother cell group's genetic expression simultaneously.
When the dosage of activin A increases, CXCR4 +The sharp increase of level relatively (Figure 32) of genetic expression.This and activin A dose-dependently increase CXCR4 +Cell relevant good (Figure 30 A-C).Also clearly, isolate CXCR4 from each cell mass +Cell has occupied CXCR4 genetic expression cell nearly all in this cell mass.This has confirmed that the FACS method collects the efficient of these cell methods.Gene expression analysis shows, CXCR4 +Cell not only comprises most of CXCR4 genetic expression, and comprises the genetic expression of other definitive entoderm mark.Shown in Figure 31 A-D, further from SOX17, GSC, HNF3B, and the parent A100 cell mass enrichment CXCR4 of MIXL1 +Cell.In addition, CXCR4 -Part comprises these few definitive entoderm marker genes expression.And, CXCR4 +And CXCR4 -Cell mass has shown mesoderm, ectoderm and extraembryonic endoderm marker gene expression reverse mode.Figure 33 A-D shows, with respect to A100 mother cell group, removes CXCR4 +Cell is to carry out Brachyury, MOX1, ZIC1 and SOX7 genetic expression.With respect to low or do not have the condition of activin A, it is very low that this A100 mother cell group expresses these marks.These results show, there is under the differentiation condition isolating CXCR4 in hESCs in high dosage activin A +Cell obtains highly enriched pure substantially definitive entoderm cell.
Embodiment 10
Use the definitive entoderm cell in the quantitative cell colony of CXCR4
For the ratio of determining definitive entoderm cell in cell culture or the cell mass quantitative, according to preceding method or in the U.S. Provisional Patent Application the 60/532nd of submission on December 23rd, 2003, No. 004, exercise question is " definitive entoderm " described method, it is disclosed in this and all introduces for your guidance, expresses the cell of CXCR4 and other definitive entoderm mark with facs analysis.
The described method of use such as the foregoing description produces definitive entoderm with the hESCs differentiation.Particularly, increase the productive rate and the purity that are expressed in the noble cells culture, the strict control of serum-concentration is as follows in the substratum: first day 0.2%FBS, second day 1.0%FBS and 3-6 days 2.0%FBS.Use the culture of three kinds of cell-surface antigens determinant E-cadherins (Cadherin), CXCR4 and thrombomodulin sorting differentiation with FACS.Analyze the sorting cells group to determine the mark relative expression level of definitive entoderm, extraembryonic endoderm and other cell type with Q-PCR then.The CXCR4 sorting cells that obtains from best differentiation culture thing produced＞separation of the definitive entoderm cell of 98% purity.
Table 2 has shown and has used the result of method of the present invention from the definitive entoderm culture analysis of markers of hESCs differentiation
Table 2
The composition of definitive entoderm culture
Mark The % culture The % definitive entoderm The % extraembryonic endoderm The %hES cell
SOX17 thrombomodulin AFP CXCR4 ECAD other (ECAD feminine gender) 70-80 ＜2 ＜1 70-80 10 10-20 100 0 0 100 0 ? ? 75 25 0 ? ? ? ? ? ? 100 ?
Amount to 100 100 100 100
Particularly, table 2 shows that CXCR4 and SOX17 positive cell (entoderm) comprise the cell in the cell culture of 70%-80%.Express in the cell of SOX17 at these, be lower than 2% Expression of TM (body wall entoderm), be lower than 1% and express AFP (internal organ entoderm).When from SOX17/CXCR4 positive cell ratio, deducting the TM positive and AFP positive cell (body wall and the combination of internal organ entoderm; Amount to 3%), can find that about 67%-77% cell culture is a definitive entoderm.About 10% cell is E-cadherin (ECAD) positive, and it is the hESCs mark, and the cell of about 10-20% is other cell type.
We also find, with aforesaidly in whole 5-6 days differentiation operation, FBS concentration is remained on≤0.5% low serological method compares, the purity of the definitive entoderm before FACS separates in the noble cells culture can improve.Yet, in whole 5-6 days differentiation operation, the cell cultures substrate concentration is remained on≤0.5%, the total quantity of definitive entoderm cell that has also caused producing reduces.
Keep amplification not have obviously differentiation more than 50 days in the culture of definitive entoderm cell in the presence of activin according to method preparation of the present invention.In these cases, the expression that keeps SOX17, CXCR4, MIXL1, GATA4, HNF3 β between incubation period.In addition, in these cultures, do not detect TM, SPARC, OCT4, AFP, SOX7, ZIC1 and BRACH.In the culture in the presence of the activin, keep amplification basically more than 50 days and do not have obviously that differentiation is possible in the definitive entoderm cell.
Embodiment 11
Other mark of definitive entoderm cell
In following test, RNA separates definitive entoderm and the human embryo stem cell group from purifying.Then with the RNA of gene chip analysis from each purifying cells group.Adopt Q-PCR further to investigate definitive entoderm and gene that non-embryonic stem cells is expressed as the potentiality of definitive entoderm mark.
Human embryo stem cell (hESCs) is remained in the DMEM/F12 substratum, this culture medium supplemented 20%KnockOut blood serum substituting product, 4ng/mL recombinant human basis fibroblast growth factor (bFGF), 0.1mM 2 mercapto ethanol, L-L-glutamic acid, non-essential amino acid and penicillin/streptomycin.HESCs cultivated in the RPMI substratum broke up to definitive entoderm in 5 days, this culture medium supplemented 100ng/mL recombinant human activin A, foetal calf serum (FBS) and penicillin/streptomycin.The change in concentration of FBS each day is: 0.1% (first day), 0.2% (second day) and 2% (3-5 days).
Carry out gene expression analysis for obtaining cell hESCs and the pure colony of definitive entoderm, separate with fluorescent activation cell sorting (FACS).Use SSEA4 antigen (R﹠amp; D Systems, cat#FAB1435P) immune purifying hESCs uses CXCR4 (R﹠amp; D Systems, cat# FAB170P) the purifying definitive entoderm.Cell dissociation uses trypsinase/EDTA, and (Invitrogen cat#25300-054), phosphate buffered saline buffer (PBS) washing to contain 2% human serum, is suspended in 100% human serum again and places 10 minutes blocking-up non-specific binding on ice.The antibody in conjunction with phycoerythrin of 200 μ L is added 5 * 10 in the human serum of 800 μ L 6In the cell, dyeed 30 minutes on ice.With 8mL PBS damping fluid washed cell twice, resuspending is in 1mL PBS.FACS separates the nucleus equipment with Scripps institute, uses FACS Vantage (BDBiosciences) to carry out.Cell directly is collected in the RLT lysis buffer, according to operation instructions (Qiagen) with the RNeasy isolation of RNA.
Twice (Durham NC), uses the U133Plus2.0 high density oligonucleotide array of Affymetrix platform to generate expression characteristic data generation express spectra data with the purified RNA sample presentation.The data that present are one group and compare, differentiate the gene of hESCs and two cell mass differential expressions of definitive entoderm.The gene that expression level and the gene level that hESCs finds are compared strong rising variation is elected to be new candidate markers, and it has the definitive entoderm feature of height.Use Q-PCR to measure selected gene according to described method, with the changes in gene expression of finding on the checking gene chip, and these expression of gene patterns between investigation hESC differentiation time-histories.
Figure 34 A-M shows some marker gene expressions of results.After adding 100ng/ml activin A, analyzed cell culture in the 1st, 3 and 5 day, demonstration differentiation operation (CXDE) finished the definitive entoderm cell of back expression CXCR4 and the result of human embryo stem cell (HESC) in 5 days.Figure 34 C and G-M comparison shows that and six marker gene FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 show almost completely identical each other expression pattern that it is also fully identical with CXCR4 and SOX17/SOX7 expression pattern.As mentioned above, SOX17 all expresses among both at the definitive entoderm and the extraembryonic endoderm of expressing SOX7.Because SOX7 does not express at definitive entoderm, the ratio of SOX17/SOX7 has been estimated the contribution that SOX17 expresses in the indicated definitive entoderm of whole colony reliably.The similarity of embedding figure G-L and M and embedding figure C shows that FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 may be the mark of definitive entoderm, and has shown that it expresses not remarkable in the extraembryonic endoderm cell.
Should be appreciated that Q-PCR result described herein can further prove conclusively with ICC.
Method of the present invention, composition and equipment are the representatives of preferred embodiment, are exemplary, can not be considered as limitation of the scope of the invention.Those skilled in the art can make variation and do other application it, and this is included in the spirit of the present invention and by the scope of disclosure and defines.Therefore, clearly, those skilled in the art are not departing under the scope of the invention and the spirit, can make invention disclosed herein and replacing and change.
As following claim and as described in all disclose, phrase " basically by ... form " refer to comprise listed any composition behind the phrase, and be limited to those to the active of explanation in open or act on other composition that does not disturb or do not have contribution.Thereby, phrase " basically by ... form " show that the composition of listing is needs or essential, but whether other composition is optional, look to influence and list the active of composition or act on and selecting for use or need not.
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Sequence table
＜110〉Novocell Inc.
D′Amour，Kevin?Allen
Agulnick，Alan?D.
Baetge，Emmanuel?E.
＜120〉Ding Xing entoderm
<130>CYTHERA.045VPC
<150>60/532004
<151>2003-12-23
<150>60/586566
<151>2004-07-09
<150>60/587942
<151>2004-07-14
<160>2
<170>FastSEQ?for?Windows?Version?4.0
<210>1
<211>1245
<212>DNA
<213>Homo?sapiens
<400>1
atgagcagcc?cggatgcggg?atacgccagt?gacgaccaga?gccagaccca?gagcgcgctg 60
cccgcggtga?tggccgggct?gggcccctgc?ccctgggccg?agtcgctgag?ccccatcggg 120
gacatgaagg?tgaagggcga?ggcgccggcg?aacagcggag?caccggccgg?ggccgcgggc 180
cgagccaagg?gcgagtcccg?tatccggcgg?ccgatgaacg?ctttcatggt?gtgggctaag 240
gacgagcgca?agcggctggc?gcagcagaat?ccagacctgc?acaacgccga?gttgagcaag 300
atgctgggca?agtcgtggaa?ggcgctgacg?ctggcggaga?agcggccctt?cgtggaggag 360
gcagagcggc?tgcgcgtgca?gcacatgcag?gaccacccca?actacaagta?ccggccgcgg 420
cggcgcaagc?aggtgaagcg?gctgaagcgg?gtggagggcg?gcttcctgca?cggcctggct 480
gagccgcagg?cggccgcgct?gggccccgag?ggcggccgcg?tggccatgga?cggcctgggc 540
ctccagttcc?ccgagcaggg?cttccccgcc?ggcccgccgc?tgctgcctcc?gcacatgggc 600
ggccactacc?gcgactgcca?gagtctgggc?gcgcctccgc?tcgacggcta?cccgttgccc 660
acgcccgaca?cgtccccgct?ggacggcgtg?gaccccgacc?cggctttctt?cgccgccccg 720
atgcccgggg?actgcccggc?ggccggcacc?tacagctacg?cgcaggtctc?ggactacgct 780
ggccccccgg?agcctcccgc?cggtcccatg?cacccccgac?tcggcccaga?gcccgcgggt 840
ccctcgattc?cgggcctcct?ggcgccaccc?agcgcccttc?acgtgtacta?cggcgcgatg 900
ggctcgcccg?gggcgggcgg?cgggcgcggc?ttccagatgc?agccgcaaca?ccagcaccag 960
caccagcacc?agcaccaccc?cccgggcccc?ggacagccgt?cgccccctcc?ggaggcactg 1020
ccctgccggg?acggcacgga?ccccagtcag?cccgccgagc?tcctcgggga?ggtggaccgc 1080
acggaatttg?aacagtatct?gcacttcgtg?tgcaagcctg?agatgggcct?cccctaccag 1140
gggcatgact?ccggtgtgaa?tctccccgac?agccacgggg?ccatttcctc?ggtggtgtcc 1200
gacgccagct?ccgcggtata?ttactgcaac?tatcctgacg?tgtga 1245
<210>2
<211>414
<212>PRT
<213>Homo?sapiens
<400>2
Met?Ser?Ser?Pro?Asp?Ala?Gly?Tyr?Ala?Ser?Asp?Asp?Gln?Ser?Gln?Thr
1 5 10 15
Gln?Ser?Ala?Leu?Pro?Ala?Val?Met?Ala?Gly?Leu?Gly?Pro?Cys?Pro?Trp
20 25 30
Ala?Glu?Ser?Leu?Ser?Pro?Ile?Gly?Asp?Met?Lys?Val?Lys?Gly?Glu?Ala
35 40 45
Pro?Ala?Asn?Ser?Gly?Ala?Pro?Ala?Gly?Ala?Ala?Gly?Arg?Ala?Lys?Gly
50 55 60
Glu?Ser?Arg?Ile?Arg?Arg?Pro?Met?Asn?Ala?Phe?Met?Val?Trp?Ala?Lys
65 70 75 80
Asp?Glu?Arg?Lys?Arg?Leu?Ala?Gln?Gln?Asn?Pro?Asp?Leu?His?Asn?Ala
85 90 95
Glu?Leu?Ser?Lys?Met?Leu?Gly?Lys?Ser?Trp?Lys?Ala?Leu?Thr?Leu?Ala
100 105 110
Glu?Lys?Arg?Pro?Phe?Val?Glu?Glu?Ala?Glu?Arg?Leu?Arg?Val?Gln?His
115 120 125
Met?Gln?Asp?His?Pro?Asn?Tyr?Lys?Tyr?Arg?Pro?Arg?Arg?Arg?Lys?Gln
130 135 140
Val?Lys?Arg?Leu?Lys?Arg?Val?Glu?Gly?Gly?Phe?Leu?His?Gly?Leu?Ala
145 150 155 160
Glu?Pro?Gln?Ala?Ala?Ala?Leu?Gly?Pro?Glu?Gly?Gly?Arg?Val?Ala?Met
165 170 175
Asp?Gly?Leu?Gly?Leu?Gln?Phe?Pro?Glu?Gln?Gly?Phe?Pro?Ala?Gly?Pro
180 185 190
Pro?Leu?Leu?Pro?Pro?His?Met?Gly?Gly?His?Tyr?Arg?Asp?Cys?Gln?Ser
195 200 205
Leu?Gly?Ala?Pro?Pro?Leu?Asp?Gly?Tyr?Pro?Leu?Pro?Thr?Pro?Asp?Thr
210 215 220
Ser?Pro?Leu?Asp?Gly?Val?Asp?Pro?Asp?Pro?Ala?Phe?Phe?Ala?Ala?Pro
225 230 235 240
Met?Pro?Gly?Asp?Cys?Pro?Ala?Ala?Gly?Thr?Tyr?Ser?Tyr?Ala?Gln?Val
245 250 255
Ser?Asp?Tyr?Ala?Gly?Pro?Pro?Glu?Pro?Pro?Ala?Gly?Pro?Met?His?Pro
260 265 270
Arg?Leu?Gly?Pro?Glu?Pro?Ala?Gly?Pro?Ser?Ile?Pro?Gly?Leu?Leu?Ala
275 280 285
Pro?Pro?Ser?Ala?Leu?His?Val?Tyr?Tyr?Gly?Ala?Met?Gly?Ser?Pro?Gly
290 295 300
Ala?Gly?Gly?Gly?Arg?Gly?Phe?Gln?Met?Gln?Pro?Gln?His?Gln?His?Gln
305 310 315 320
His?Gln?His?Gln?His?His?Pro?Pro?Gly?Pro?Gly?Gln?Pro?Ser?Pro?Pro
325 330 335
Pro?Glu?Ala?Leu?Pro?Cys?Arg?Asp?Gly?Thr?Asp?Pro?Ser?Gln?Pro?Ala
340 345 350
Glu?Leu?Leu?Gly?Glu?Val?Asp?Arg?Thr?Glu?Phe?Glu?Gln?Tyr?Leu?His
355 360 365
Phe?Val?Cys?Lys?Pro?Glu?Met?Gly?Leu?Pro?Tyr?Gln?Gly?His?Asp?Ser
370 375 380
Gly?Val?Asn?Leu?Pro?Asp?Ser?His?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
Received from the disk form sequence table of JLH with
Unanimity among the CYTHERA.045A that submits to
With A: the PCT version submitted to of CYTHERA045V.TXT
122204

Claims (75)

1. the cell culture that comprises people's cell is the definitive entoderm cell at least about described people's cell of 10% wherein, and described definitive entoderm cell is for being divided into the intestinal tube cell or being derived from the pluripotent cell of the organ of intestinal tube.
2. cell culture as claimed in claim 1 is the definitive entoderm cell at least about described people's cell of 50% wherein.
3. cell culture as claimed in claim 1 is the definitive entoderm cell at least about described people's cell of 80% wherein.
4. cell culture as claimed in claim 1, wherein said definitive entoderm cell expressing is selected from the mark of SOX17 and CXCR4.
5. cell culture as claimed in claim 4, wherein in described definitive entoderm cell, the expression that is selected from the described mark of SOX17 and CXCR4 is higher than the expression of the mark that is selected from OCT4, α-fetoprotein (AFP), thrombomodulin (TM), SPARC and SOX7.
6. cell culture as claimed in claim 4, wherein said definitive entoderm cell is not expressed the mark that is selected from OCT4, AFP, TM, SPARC and SOX7.
7. cell culture as claimed in claim 4, wherein said definitive entoderm cell expressing is selected from the mark of MIXL1, GATA4 and HNF3b.
8. cell culture as claimed in claim 4, wherein said definitive entoderm cell expressing is selected from the mark of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.
9. cell culture as claimed in claim 1, wherein said definitive entoderm cell expressing SOX17 and CXCR4.
10. cell culture as claimed in claim 9, wherein in described definitive entoderm cell, the expression of described SOX17 and CXCR4 is higher than the expression of OCT4, AFP, TM, SPARC and SOX7.
11. cell culture as claimed in claim 9, wherein said definitive entoderm cell is not expressed OCT4, AFP, TM, SPARC and SOX7.
12. cell culture as claimed in claim 9, wherein said definitive entoderm cell expressing MIXL1, GATA4 and HNF3b.
13. cell culture as claimed in claim 9, wherein said definitive entoderm cell expressing is selected from the mark of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.
14. cell culture as claimed in claim 1, wherein in described cell culture, corresponding each pluripotent cell exists at least about 2 definitive entoderm cells.
15. cell culture as claimed in claim 14, wherein said pluripotent cell comprises embryonic stem cell.
16. cell culture as claimed in claim 15, wherein said embryonic stem cell are derived from the tissue that is selected from from morula, embryo's inner cell mass (ICM) and embryo's genital ridge.
17. cell culture as claimed in claim 1 further comprises substratum, described substratum comprises and is less than about 10% serum.
18. cell culture as claimed in claim 1 further comprises the somatomedin of the Nodal/ activin subgroup of TGF beta superfamily.
19. cell culture as claimed in claim 1 further comprises the somatomedin that is selected from Nodal, activin A, activin B and combination thereof.
20. comprise the cell mass of cell, wherein at least about 90% described cell behaviour definitive entoderm cell, described people's definitive entoderm cell is a pluripotent cell, described pluripotent cell can be divided into the intestinal tube cell or be derived from the organ of intestinal tube.
21. cell mass as claimed in claim 20 is wherein at least about 95% described cell behaviour definitive entoderm cell.
22. cell mass as claimed in claim 20 is wherein at least about 98% described cell behaviour definitive entoderm cell.
23. cell mass as claimed in claim 20, wherein said people's definitive entoderm cell expressing is selected from the mark of SOX17 and CXCR4.
24. cell mass as claimed in claim 23, wherein in described people's definitive entoderm cell, the expression that is selected from the described mark of SOX17 and CXCR4 is higher than the expression of the mark that is selected from OCT4, AFP, TM, SPARC and SOX7.
25. cell mass as claimed in claim 23, wherein said people's definitive entoderm cell is not expressed the mark that is selected from OCT4, AFP, TM, SPARC and SOX7.
26. cell mass as claimed in claim 23, wherein said people's definitive entoderm cell expressing is selected from the mark of MIXL1, GATA4 and HNF3b.
27. cell mass as claimed in claim 23, wherein said definitive entoderm cell expressing is selected from the mark of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.
28. cell mass as claimed in claim 20, wherein said people's definitive entoderm cell expressing SOX17 and CXCR4.
29. cell mass as claimed in claim 28, wherein in described people's definitive entoderm cell, the expression of SOX17 and CXCR4 is higher than the expression of OCT4, AFP, TM, SPARC and SOX7.
30. cell mass as claimed in claim 28, wherein said people's definitive entoderm cell is not expressed OCT4, AFP, TM, SPARC and SOX7.
31. cell mass as claimed in claim 28, wherein said people's definitive entoderm cell expressing MIXL1, GATA4 and HNF3b.
32. cell mass as claimed in claim 28, wherein said definitive entoderm cell expressing is selected from the mark of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.
33. cell mass as claimed in claim 20, wherein in described cell colony, corresponding each pluripotent cell exists at least about 2 definitive entoderm cells.
34. cell mass as claimed in claim 33, wherein said pluripotent cell comprises embryonic stem cell.
35. cell mass as claimed in claim 34, wherein said embryonic stem cell are derived from the tissue of the ICM that is selected from morula, embryo and embryo's genital ridge.
36. produce the method for definitive entoderm cell, described method comprises the steps:
Acquisition comprises the cell mass of people's pluripotent cell;
For described cell mass provides at least a TGF beta superfamily somatomedin, the amount of described somatomedin is enough to promote described pluripotent cell to be divided into the definitive entoderm cell, and described definitive entoderm cell is the pluripotent cell that can be divided into the intestinal tube cell or be derived from the organ of intestinal tube; And
Give time enough and form the definitive entoderm cell, the enough time of wherein said formation definitive entoderm cell with detect definitive entoderm cell in the described cell mass exist determine.
37. method as claimed in claim 36 wherein is divided into the definitive entoderm cell at least about 10% described pluripotent cell.
38. method as claimed in claim 36 wherein is divided into the definitive entoderm cell at least about 50% described pluripotent cell.
39. method as claimed in claim 36 wherein is divided into the definitive entoderm cell at least about 70% described pluripotent cell.
40. method as claimed in claim 36 wherein is divided into the definitive entoderm cell at least about 80% described pluripotent cell.
41. method as claimed in claim 36, wherein detect the existence of definitive entoderm cell in described cell mass and be included in the expression that detects the mark of at least a SOX17 of being selected from and CXCR4 in the described cell mass cell, and at least a expression that is selected from the mark of OCT4, AFP, TM, SPARC and SOX7, wherein in described definitive entoderm cell, the expression that is selected from the described mark of SOX17 and CXCR4 is higher than the expression of the described mark that is selected from OCT4, AFP, TM, SPARC and SOX7.
42. method as claimed in claim 36, wherein detect the existence of definitive entoderm cell in described cell mass and be included in the expression that detects the mark of at least a SOX17 of being selected from and CXCR4 in the described cell mass cell, and at least a expression that is selected from the mark of AFP, TM and SOX7, wherein in described definitive entoderm cell, the expression that is selected from the described mark of SOX17 and CXCR4 is higher than the expression of the described mark that is selected from AFP, TM and SOX7.
43. method as claimed in claim 42, wherein the expression of at least a described mark is measured with Q-PCR.
44. method as claimed in claim 42, wherein the expression of at least a described mark is measured with immunocytochemistry.
45. method as claimed in claim 36, the existence that wherein detects definitive entoderm cell in described cell colony is included in the expression that detects the mark of at least a VWF of being selected from, CALCR, FOXQ1, CMKOR1 and CRIP1 in the described cell colony cell, and at least a expression that is selected from the mark of OCT4, AFP, TM, SPARC and SOX7, wherein in described definitive entoderm cell, the expression that is selected from the mark of FGF17, VWF, CALCR, FOXQ1 and CRIP1 is higher than the expression of the mark that is selected from OCT4, AFP, TM, SPARC and SOX7.
46. method as claimed in claim 36, the Nodal/ activin subgroup that wherein said at least a somatomedin is the TGF beta superfamily.
47. method as claimed in claim 46, wherein said at least a somatomedin is selected from Nodal, activin A, activin B and combination thereof.
48. method as claimed in claim 47, wherein said at least a somatomedin is Nodal.
49. method as claimed in claim 47, wherein said at least a somatomedin is activin A.
50. method as claimed in claim 47, wherein said at least a somatomedin is activin B.
51. method as claimed in claim 36 wherein provides the multiple somatomedin of TGF beta superfamily.
52. method as claimed in claim 51, wherein said multiple somatomedin comprises Nodal, activin A and activin B.
53. method as claimed in claim 36, the concentration of wherein said at least a somatomedin is at least about 10ng/ml.
54. method as claimed in claim 36, the concentration of wherein said at least a somatomedin is at least about 100ng/ml.
55. method as claimed in claim 36, the concentration of wherein said at least a somatomedin is at least about 500ng/ml.
56. method as claimed in claim 36, the concentration of wherein said at least a somatomedin is at least about 1000ng/ml.
57. method as claimed in claim 36, the concentration of wherein said at least a somatomedin is at least about 5000ng/ml.
58. method as claimed in claim 36, wherein said cell mass grow in and comprise in the substratum that is less than about 10% serum.
59. method as claimed in claim 36, wherein said pluripotent cell comprises stem cell.
60. method as claimed in claim 59, wherein said pluripotent cell comprises embryonic stem cell.
61. method as claimed in claim 60, wherein said embryonic stem cell are derived from the tissue of the ICM that is selected from morula, embryo and embryo's genital ridge.
62. the definitive entoderm cell that produces with the method for claim 36.
63. produce the cell mass method of the definitive entoderm cell of enrichment, comprise the steps:
Cell in the differentiation pluripotent human cell colony, to produce the definitive entoderm cell, described definitive entoderm cell is a pluripotent cell, this pluripotent cell can be divided into the intestinal tube cell or be derived from the organ of intestinal tube;
Provide reagent to described cell mass, described reagent combines with mark, and described marker expression is in described definitive entoderm cell and be not expressed in basically in other cell type in the described cell mass; And
To separate with described other cell type that is arranged in described cell mass with the described definitive entoderm cell of described reagent bonded, thus the cell mass of the definitive entoderm cell of generation enrichment.
64. as the described method of claim 63, wherein differentiation step further comprises,
Acquisition comprises the cell mass of pluripotent human cell,
Provide described at least a TGF beta superfamily somatomedin to described cell colony, the amount of described somatomedin is enough to promote described pluripotent cell is divided into the definitive entoderm cell, described definitive entoderm cell is a pluripotent cell, described pluripotent cell can be divided into the intestinal tube cell or be derived from the organ of intestinal tube, and
Give time enough and form the definitive entoderm cell, the enough time of wherein said formation definitive entoderm cell with detect definitive entoderm cell in the described cell colony exist determine.
65. as the described method of claim 63, wherein detection comprises,
In the cell of described cell colony, detect at least a expression that is selected from the mark of SOX17 and CXCR4, and at least a expression that is selected from the mark of OCT4, AFP, TM, SPARC and SOX7, wherein in described definitive entoderm cell, the expression that is selected from the mark of SOX17 and CXCR4 is higher than the expression of the mark that is selected from OCT4, AFP, TM, SPARC and SOX7.
66. as the described method of claim 63, wherein detect and comprise, in the cell of described cell colony, detect at least a expression that is selected from the mark of SOX17 and CXCR4, and at least a expression that is selected from the mark of AFP, TM and SOX7, wherein in described definitive entoderm cell, the expression that is selected from the mark of SOX17 and CXCR4 is higher than the expression of the mark that is selected from AFP, TM and SOX7.
67. as the described method of claim 63, wherein detect and comprise, in the cell of described cell colony, detect at least a expression that is selected from the mark of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1, and at least a expression that is selected from the mark of OCT4, AFP, TM, SPARC and SOX7, wherein in described definitive entoderm cell, the expression that is selected from the mark of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 is higher than the expression of the mark that is selected from OCT4, AFP, TM, SPARC and SOX7.
68., be the definitive entoderm cell wherein at least about 95% described cell as the described method of claim 63.
69., be the definitive entoderm cell wherein at least about 98% described cell as the described method of claim 63.
70. as the described method of claim 63, wherein said mark is CXCR4.
71. as the described method of claim 63, wherein said reagent is antibody.
72. as method as described in the claim 71, wherein said antibody has affinity to CXCR4.
73. the cell mass of the enrichment definitive entoderm cell that the described method of claim 63 produces.
74. as the described cell culture of arbitrary claim of claim 4 or 9, wherein said definitive entoderm cell is not significantly expressed the mark that is selected from OCT4, AFP, TM, SPARC and SOX7.
75. as the cell mass of arbitrary claim of claim 23 or 28, wherein said definitive entoderm cell is not significantly expressed the mark that is selected from OCT4, AFP, TM, SPARC and SOX7.
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