CN101023163A - Methods for identifying factors for differentiating definitive endoderm - Google Patents

Methods for identifying factors for differentiating definitive endoderm Download PDF

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Publication number
CN101023163A
CN101023163A CNA2005800281150A CN200580028115A CN101023163A CN 101023163 A CN101023163 A CN 101023163A CN A2005800281150 A CNA2005800281150 A CN A2005800281150A CN 200580028115 A CN200580028115 A CN 200580028115A CN 101023163 A CN101023163 A CN 101023163A
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cell
pdx1
expression
mark
differentiation
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凯文·艾伦·达穆尔
艾伦·D·阿古尼克
苏珊·埃利泽
伊曼纽尔·贝特格
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Cythera Inc
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Cythera Inc
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Priority to CN201210419046.8A priority Critical patent/CN102925406B/en
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Abstract

The invention discloses a method for identifying one or a plurality of differentiation factors. The differentiation factor can be used for differentiating cells in a cell cluster comprising committed endoderm cells to be cells capable of forming tissues and/or organs from intestinal canals.

Description

Evaluation is used for the method for the factor of differentiating definitive endoderm
Related application
The application be on April 26th, 2005 submit to be entitled as the 11/115th of PDX1 EXPRESSINGENDODERM (expressing the entoderm of PDX1), the part continuation application of No. 868 U.S. Patent applications, it requires to enjoy the right of priority of following three temporary patent applications as formal application based on 35 U.S.C. § 119 (e): the 60/566th, No. 293 U.S. Provisional Patent Application of submitting on April 27th, 2004 that is entitled as PDX1EXPRESSING ENDODERM (expressing the entoderm of PDX1); The 60/587th, No. 942 U.S. Provisional Patent Application of submitting on July 14th, 2004 that is entitled as CHEMOKINE CELLSURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVEENDODERM (being used to separate the chemokine cell surface receptor of definitive endoderm); With the 60/586th, No. 566 U.S. Provisional Patent Application of submitting on July 9th, 2004 that is entitled as CHEMOKINE CELL SURFACE RECEPTOR FOR THE ISOLATION OFDEFINITIVE ENDODERM (being used to separate the chemokine cell surface receptor of definitive endoderm); The application still on December 23rd, 2004 submit to be entitled as the 11/021st of DEFINITIVE ENDODERM (definitive endoderm), the part continuation application of No. 618 U.S. Patent applications, it requires to enjoy the right of priority of following three temporary patent applications as formal application according to 35 U.S.C. § 19 (e): the 60/587th, No. 942 U.S. Provisional Patent Application of submitting on July 14th, 2004 that is entitled as CHEMOKINE CELL SURFACE RECEPTOR FOR THEISOLATION OF DEFINITIVE ENDODERM (being used to separate the chemokine cell surface receptor of definitive endoderm); With the 60/586th, No. 566 U.S. Provisional Patent Application of submitting on July 9th, 2004 that is entitled as CHEMOKINE CELL SURFACE RECEPTOR FORTHE ISOLATION OF DEFINITIVE ENDODERM (being used to separate the chemokine cell surface receptor of definitive endoderm); With the 60/532nd, No. 004 U.S. Provisional Patent Application of submitting on December 23rd, 2003 that is entitled as DEFINITIVE ENDODERM (definitive endoderm).
Technical field
The present invention relates to medical science and cytobiology field, relate in particular to and identify that can be used for the differentiating definitive endoderm cell is the factor of other cell type.
Background of invention
People's multipotent stem cells (pluripotent stem cell), as embryonic stem cell (ES) and embryonic genital cell (EG), at first from the culture of no inoblast feeder layer, separated (Bongso et al. in 1994,1994), from the culture that contains the inoblast feeder layer, separated in 1997 (Hogan, 1997).Thomson, Reubinoff and Shamblott have set up and have adopted the mouse feeder layer cultured continuously people ES of mitotic division inactivation and method (Reubinoffet al., 2000 of EG cell subsequently; Shamblott et al., 1998; Thomson et al., 1998).
People ES and EG cell (hESC) provide chance scarcely ever for the human early development of research with for therapeutic intervention some diseases state (as diabetes and Parkinson's disease).For example, in the process that adopts cell therapy treatment diabetes, use can have very much progress than the cell therapy diabetes that are used at present from donor pancreas derived from the β cell of the generation Regular Insulin of hSEC.But also do not know at present how to obtain producing the β cell of Regular Insulin from hSEC.Therefore, being used to cell therapy from the islet cells of donor pancreas treatment diabetes at present is subject to lack and transplants required high quality islet cells.Cell therapy to single type i diabetes people need transplant about 8 * 10 8Islet cells (Shapiro et al., 2000; Shapiro et al., 2001a; Shapiro et al., 2001b).Once successfully transplanting required islet cells needs the organ of two healthy donors to provide at least.And a large amount of high quality noble cells that human embryo stem cell is used for human cell treatment for exploitation provides the source of parent material.
Versatility and can keep the hSEC cell long period and cultivate these two character and make it be particularly suitable for cell therapy.Versatility is meant that hESC can be divided into the derivative of all 3 kinds of main germinal layers (entoderm, mesoderm and ectoderm), and then also forms the ability of all somatocyte types of ripe organ organize (as placenta) and sexual cell except that the embryo outside.Although versatility has given hSEC special purposes, this character brings challenges also for the research and the control of these cells and derivative thereof.Owing in differentiation hSEC substratum, can produce a large amount of cell types, so most of cell type generation efficient is very low.And the generation key of successfully estimating arbitrary designated cell type depends on the mark that definition is suitable.Efficiently, Ding Xiang differentiation is used extremely important to the therapeutic of hESCs.
Address the above problem to utilize hSEC as parent material production to be used for the cell that cell therapy uses favourable.In addition, it also helps and identifies the factor that promotes to be divided into derived from the precursor cell of hESCs the cell type that is used for cell therapy.
Brief summary of the invention
Embodiment of the present invention relate to the method for identifying one or more differentiation factors, the cell that this differentiation factor can be used in the differentiated cell population becomes the cell that can be used for cell therapy, this cell mass comprises the PDX1-positive (expressing PDX1's) endoderm cell and/or the negative endoderm cell's (significantly not expressing the endoderm cell of PDX1) of PDX1-, as definitive endoderm.For instance, some embodiments of methods described herein relate to evaluation and can promote definitive endoderm to be divided into the factor of the precursor cell of tissue and/or organ, and these tissues and/or organ include but not limited to pancreas, liver, lung, stomach, intestines, Tiroidina, thymus gland, pharynx, gall-bladder and bladder.In some embodiments, these precursor cells are PDX1-positive entoderm cells.In other embodiments, these precursor cells are the endoderm cells that significantly do not express PDX1.
In some embodiments of methods described herein, the cell culture of definitive endoderm or cell mass contact with candidate's (test) differentiation factor or provide candidate's (test) differentiation factor with other method.In preferred embodiments, definitive endoderm is people's definitive endoderm.In a more preferred embodiment, this people's definitive endoderm is can be divided into intestinal tube or derived from the multipotential cell of the cell of the organ of intestinal tube.
In other embodiment of methods described herein, the cell culture of PDX1-positive entoderm cell or cell mass contact with candidate's (test) differentiation factor or provide candidate's (test) differentiation factor with other method.In preferred embodiments, the PDX1-positive entoderm cell is a people PDX1-positive entoderm cell.In certain embodiments, this people PDX1-positive entoderm cell is PDX1-male anterior intestine/midgut endoderm cell.In a more preferred embodiment, this people PDX1-positive entoderm cell is PDX1-male anterior intestine endoderm cell.In other embodiment preferred, this people PDX1-positive entoderm cell is the PDX1-positive entoderm cell at anterior intestine rear portion.In the embodiment of special choosing, this people PDX1-is positive, and the anterior intestine endoderm cell is the multipotential cell that can be divided into derived from intestinal tube forward cell, tissue or organ.
When relating to methods described herein, candidate's differentiation factor may be the known factor or the unknown factor that causes cytodifferentiation that causes cytodifferentiation.In certain embodiments, candidate's differentiation factor can be polypeptide, for example somatomedin.In some embodiments, this somatomedin includes but not limited to FGF10, FGF4, FGF2, Wnt3A or Wnt3B.In other embodiments, candidate's differentiation factor can be a small molecules.In specific embodiments, this small molecules is the retinoid compound, as vitamin A acid.Perhaps, in some embodiments, candidate's differentiation factor is not a retinoid, is not the anterior intestine differentiation factor or is not the member of TGF beta superfamily.In other embodiments, candidate's differentiation factor is any molecule except that retinoid compound, anterior intestine differentiation factor or TGF beta superfamily member, as activin (activin) A and B.In other embodiments, candidate's differentiation factor is the unknown before factor that causes the definitive endoderm differentiation.
Other embodiment of methods described herein relates to test candidate differentiation factor under numerous concentration.For example, candidate's differentiation factor may only cause the differentiation of definitive endoderm and/or PDX1-positive entoderm cell when concentration is higher than specific threshold.In addition, when providing with lower concentration, candidate's differentiation factor can make cytodifferentiation become first cell type, and can make same cytodifferentiation become second cell type when providing with greater concn.In some embodiments, candidate's differentiation factor provides with one or more concentration of about 0.1ng/ml to about 10mg/ml scope.
Before making the contact of the cell culture that comprises definitive endoderm and/or PDX1-positive entoderm cell or cell mass or candidate's differentiation factor otherwise is provided or approximately simultaneously, selected and assess at least a mark to determine its expression.This step can be described as the first mark appraisal procedure.Perhaps, this step can be described as at very first time point and determines the expression of mark.Mark can be any mark that can be used for monitoring cytodifferentiation, but, preferred mark includes but not limited to sex-determining region Y-box 17 (SOX17), pancreas-duodenum homology frame the factor-1 (PDX1), albumin, liver cell specific antigens (HSA), Prospero-relevant homology frame 1 (prospero-related homeobox 1, PROX1), Tiroidina transcription factor 1 (TITF1), villin, α-Jia Taidanbai (AFP), Cytochrome P450 7A (CYP7A), tyrosine aminotransferase (TAT), hepatocyte neclear factor 4a (HNF4a), CXC type Chemokine Receptors 4 (CXCR4), vWF ELISA (VMF), vascular cell adhesion molecule (VACM1), aPoA 1 (APOA1), glucose transporter (GLUT2), α-1-antitrypsin (AAT), glucokinase (GLUKO) and artificial blood express the homology frame (humanhematopoietically expressed homeobox, hHEX) and CDX2.
From making cell culture or the cell mass contact that comprises definitive endoderm and/or PDX1-positive entoderm cell or otherwise providing candidate's differentiation factor to count, assess the expression of at least one mark in cell culture or cell mass once more through behind the time enough.This step can be described as the second mark appraisal procedure.Perhaps, this step can be described as the expression of determining mark at second time point.In preferred embodiments, the mark in the assessment of first and second time points is identical mark.
In some embodiments of methods described herein, whether further definite this at least one mark is compared with its expression at very first time point in the expression of second time point increases or reduces.The increase of this at least one marker expression or minimizing show that this candidate's differentiation factor can promote the differentiation of definitive endoderm and/or PDX1-positive entoderm cell.Make the cell culture or the cell mass that comprise definitive endoderm and/or PDX1-positive entoderm cell contact or otherwise provide candidate's differentiation factor and determine that the enough timed interval of this at least one mark between the expression of second time point can be from being as short as 1 hour to growing to 10 days.In some embodiments, make the cell culture that comprises definitive endoderm and/or PDX1-positive entoderm cell or cell mass contact or the expression of repeatedly assessing this at least one mark after candidate's differentiation factor otherwise is provided.In certain embodiments, assess the expression of mark with Q-PCR.In other embodiments, assess the expression of mark with immunocytochemistry.
Other embodiment of the present invention relates to evaluation can promote the method for the negative definitive endoderm of PDX1-to the differentiation factor of the positive anterior intestine endoderm cell differentiation of PDX1-.In these methods, the negative definitive endoderm contact of PDX1-candidate differentiation factor, and whether the expression of PDX1 in cell mass is compared and is increased before determining behind the contact candidate differentiation factor expression of PDX1 in cell mass and contacting candidate's differentiation factor.The expression increase of PDX1 in cell mass shows that this candidate's differentiation factor can promote the differentiation of the negative definitive endoderm of PDX1-to the positive anterior intestine endoderm cell of PDX1-.In some embodiments, determine the expression of PDX1 with quantitative polyase chain reaction (Q-PCR).Some embodiments of preceding method further comprise the step of the expression of determining before and after the cell mass contact candidate differentiation factor HOXA13 wherein and/or HOXC16.In some embodiments, candidate's differentiation factor is a small molecules, and retinoid for example is as RA.In other embodiments, candidate's differentiation factor is a polypeptide, and somatomedin for example is as FGF-10.
Other embodiment of the present invention relates to the method that evaluation can promote the differentiation factor of the positive anterior intestine endoderm cell differentiation of PDX1-.In these methods, PDX1-is positive, and the anterior intestine endoderm cell contacts candidate's differentiation factor, and whether this identical mark expression in cell mass increases or reduce before determining behind the contact candidate differentiation factor expression of mark in cell mass and contacting candidate's differentiation factor.The increase of marker expression or minimizing show that this candidate's differentiation factor can promote the positive anterior intestine endoderm cell's of PDX1-differentiation.In some embodiments, determine the expression of mark with Q-PCR.In some embodiments, candidate's differentiation factor is a small molecules, and retinoid for example is as RA.In other embodiments, candidate's differentiation factor is a polypeptide, and somatomedin for example is as FGF-10.
Other embodiment of the present invention relates to the cell with the methods described herein differentiation.These cells include but not limited to the precursor cell of pancreas, liver, lung, stomach, intestines, Tiroidina, thymus gland, pharynx, gall-bladder and bladder.In some embodiments, this cell may be end differentiation eventually.Other embodiment as herein described relates to cell culture or the cell mass that comprises above-mentioned cell.
In some authorities, term " comprises " may not have any definition of being accepted usually." to comprise " be open to the term of Shi Yonging herein, can comprise any other element.Consider this point, other embodiments of the present invention are described with reference to the paragraph of following numbering:
1. identify the differentiation factor of the differentiation of people's definitive endoderm in the cell mass can promote to comprise people's cell, described method comprises step: the cell mass that (a) obtains to comprise people's definitive endoderm; (b) provide the candidate differentiation factor to described cell mass; (c) determine that mark is in the expression of very first time point in the cell mass; (d) determine that identical mark is in the expression of second time point in the described cell mass, wherein said second time point is after described very first time point, and wherein said second time point is after providing described candidate's differentiation factor to described cell mass; And (e) determine that this mark in the expression of described second time point and described cell mass of mark in the described cell mass is compared in the expression of described very first time point and whether increase or reduce that the increase of marker expression or minimizing described in the wherein said cell mass show that described candidate's differentiation factor can promote the differentiation of described people's definitive endoderm.
2. the method for paragraph 1, wherein said people's definitive endoderm account for people's cell in the described cell mass at least about 10%.
3. the method for paragraph 1, wherein people's feeder cell are present in the described cell mass, and are definitive endoderms at least about 10% the people's cell except described feeder cell wherein.
4. the method for paragraph 1, wherein said people's definitive endoderm account for people's cell in the described cell mass at least about 90%.
5. the method for paragraph 1, wherein said people's feeder cell are present in the described cell mass, and are definitive endoderms at least about 90% the people's cell except described feeder cell wherein.
6. the method for paragraph 1, wherein said people's definitive endoderm responds described candidate's differentiation factor and is divided into cell, tissue or the organ that is derived from intestinal tube.
7. the method for paragraph 1, wherein said people's definitive endoderm responds described candidate's differentiation factor and is divided into the pancreas precursor cell.
8. the method for paragraph 7, wherein said mark is selected from pancreas-duodenum homology frame factor-1 (PDX1), homology frame A13 (HOXA13) and homology frame C6 (HOXC6).
9. the method for paragraph 1, wherein said people's definitive endoderm responds described candidate's differentiation factor and is divided into liver precursor.
10. the method for paragraph 9, wherein said mark are selected from albumin, Prospero-relevant homology frame 1 (PROX1) and liver cell specific antigens (HSA).
11. the method for paragraph 1, wherein said people's definitive endoderm responds described candidate's differentiation factor and is divided into the lung precursor cell.
12. the method for paragraph 11, wherein said mark are Tiroidina transcription factor 1 (TITF1).
13. the method for paragraph 1, wherein said people's definitive endoderm responds described candidate's differentiation factor and is divided into the intestines precursor cell.
14. as the method for paragraph 13, wherein said mark be selected from villin and tail type homeobox transcription factor 2 (caudal type homeobox transcription factor 2, CDX2).
15. the method for paragraph 1, wherein said very first time point is before providing described candidate's differentiation factor to described cell mass.
16. the method for paragraph 1, the wherein said very first time puts and provides described candidate's differentiation factor roughly simultaneously to described cell mass.
17. the method for paragraph 1, wherein said very first time point is after providing described candidate's differentiation factor to described cell mass.
18. the method for paragraph 1, the expression of wherein said mark increases.
19. the method for paragraph 1, the expression decreased of wherein said mark.
20. the method for paragraph 1, the expression of wherein said mark is determined by quantitative polyase chain reaction (Q-PCR).
21. the method for paragraph 1, the expression of wherein said mark is determined by immunocytochemistry.
22. the method for paragraph 1, wherein said mark are selected from pancreas-duodenum homology frame factor-1 (PDX1), homology frame A13 (HOXA13) and homology frame C6 (HOXC6).
23. the method for paragraph 1, wherein said mark are selected from albumin, Prospero-relevant homology frame 1 (PROX1) and liver cell specific antigens (HSA).
24. the method for paragraph 1, wherein said mark are selected from villin and tail type homeobox transcription factor 2 (CDX2).
25. the method for paragraph 1, wherein said mark are Tiroidina transcription factor 1 (TITF1).
26. the method for paragraph 1, wherein said differentiation factor comprises the anterior intestine differentiation factor.
27. the method for paragraph 1, wherein said differentiation factor comprises small molecules.
28. the method for paragraph 1, wherein said differentiation factor comprises retinoid.
29. the method for paragraph 1, wherein said differentiation factor comprises vitamin A acid.
30. the method for paragraph 1, wherein said differentiation factor comprises polypeptide.
31. the method for paragraph 1, wherein said differentiation factor comprises somatomedin.
32. the method for paragraph 1, wherein said differentiation factor comprises FGF-10.
33. the method for paragraph 1, wherein said differentiation factor comprises FGF-2.
34. the method for paragraph 1, wherein said differentiation factor comprises Wnt3B.
35. the method for paragraph 1, wherein said differentiation factor are not the anterior intestine differentiation factors.
36. the method for paragraph 1, wherein said differentiation factor are not retinoides.
37. the method for paragraph 1, wherein said differentiation factor are not vitamin A acids.
38. the method for paragraph 1, wherein said differentiation factor offers described cell mass with about 0.1ng/ml to the concentration of about 10mg/ml.
39. the method for paragraph 1, wherein said differentiation factor offers described cell mass with about 1ng/ml to the concentration of about 1mg/ml.
40. the method for paragraph 1, wherein said differentiation factor offers described cell mass with about 10ng/ml to the concentration of about 100 μ g/ml.
41. the method for paragraph 1, wherein said differentiation factor offers described cell mass with about 100ng/ml to the concentration of about 10 μ g/ml.
42. the method for paragraph 1, wherein said differentiation factor offers described cell mass with the concentration of about 1 μ g/ml.
43. the method for paragraph 1, wherein said differentiation factor offers described cell mass with the concentration of about 100ng/ml.
Should be appreciated that above-mentioned method and composition is relevant with the cell of vitro culture.But the cell composition of above-mentioned vitro differentiation can be used for purposes in the body.
Other embodiments of the present invention can be referring to following patent application: the 60/532nd, No. 004 U.S. Provisional Patent Application of submitting on December 23rd, 2003 that is entitled as DEFINITIVE ENDODERM (definitive endoderm); The 60/566th, No. 293 U.S. Provisional Patent Application of submitting on April 27th, 2004 that is entitled as PDX1EXPRESSING ENDODERM (expressing the entoderm of PDX1); The 60/586th, No. 566 U.S. Provisional Patent Application of submitting on July 9th, 2004 that is entitled as CHEMOKINE CELLSURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVEENDODERM (being used to separate the chemokine cell surface receptor of definitive endoderm); The 60/587th, No. 942 U.S. Provisional Patent Application of submitting on July 14th, 2004 that is entitled as CHEMOKINE CELL SURFACE RECEPTOR FOR THE ISOLATION OFDEFINITIVE ENDODERM (being used to separate the chemokine cell surface receptor of definitive endoderm); The 11/021st, No. 618 U.S. Patent application of submitting on December 23rd, 2004 that is entitled as DEFINITIVE ENDODERM (definitive endoderm); The 11/115th, No. 868 U.S. Patent application of submitting on April 26th, 2005 that is entitled as PDX1 EXPRESSINGENDODERM (expressing the definitive endoderm of PDX1).
Description of drawings
Fig. 1 is the imaginary differentiation pathway synoptic diagram that produces beta cell from hESC.The first step of approach is responsible for ES cell to definitive entoderm, also represents the first step before the further differentiation incident of pancreas entoderm, internal secretion entoderm or pancreas islet/beta cell.Second step of approach shows that the definitive entoderm of the SOX17-positive/PDX1-feminine gender is to the endoblastic conversion of the positive anterior intestine of PDX1-.These factors that work in transforming are represented with italics in mediation.The correlating markings thing of definition target cell is represented with underscore.
Fig. 2 is the synoptic diagram of people SOX17cDNA, has shown the position of conserved regions and has highlighted the zone that is used for by the immune step of GENOVAC.
Fig. 3 concerns dendrogram, shows that SOX17 and SOX7 relation are nearest, and is far away slightly with the SOX18 relation.SOX17 protein is more much better than than other member's of the SOX group F subtribe in the same species dependency in allied species
Fig. 4 is a Westem trace of making probe with the anti-SOX17 antibody of rabbit, this trace proves that this antibody has specificity to crossing the SOX17 that expresses in the inoblast (1 road), and the immunoreactivity of shortage and EGFP (2 road) or immediate SOX family member SOX7 (3 road).
Fig. 5 A-B shows a large amount of AFP +Be total to the SOX17 of labeled cell +The Photomicrograph of cell cluster.This and other SOX17 +Bunch (B) forms significantly contrast, because at other SOX17 +Can only observe seldom in bunch or just do not have AFP at all +Cell.
Fig. 6 A-C is the Photomicrograph that shows body wall entoderm and SOX17.Figure A shows that the immunocytochemistry of the human thrombomodulin adjusting albumen (TM) on body wall endoderm cell surface in the hES cell culture that breaks up at random detects.Figure B shows the identical zone with figure A, to TM and SOX17 double-tagging.Figure C is the figure that differs with the same area of DAPI mark nuclear.Attention: the nuclear of DAPI mark and SOX17 mark relevant fully.
Fig. 7 A-B shows the SOX17 genetic expression of quantitative PCR (Q-PCR) detection and the bar graph of the anti-SOX17 positive cell that the SOX17-specific antibody detects.Figure A shows with respect to undifferentiated control medium (SR20) activin A increases SOX17 genetic expression, and vitamin A acid (RA) strongly inhibited SOX17 expresses.Figure B is presented at SOX17 +The similar multiple that has reflected similar pattern and these variations on the cell quantity shows that Q-PCR detects SOX17 genetic expression and can reflect variation in the individual cells level.
Fig. 8 A is a bar graph, shows that activin A exists the hESC culture that is breaking up down to keep low-level AFP genetic expression, and the strong rise of the cell of differentiation demonstration AFP at random in 10% calf serum (FBS).The difference of expression level approximately is 7 times.
Fig. 8 B-C is two displaing micro pictures, shows that activin A is also very remarkable to the individual cells level that is suppressed at that AFP expresses, because with respect to single 10%FBS (top) that uses, can only observe considerably less and little AFP down in the condition (bottom) that activin A handles +Cell cluster.
Fig. 9 A-B shows to use the quantitative AFP of flow cytometer +The contrast picture of cell count.This figure proof exists at activin A under the condition of (left figure) or disappearance (right figure), and the change multiple of AFP genetic expression (Fig. 8 A) is fully corresponding to AFP +The quantity of cell has further been supported to use Q-PCR to analyze and has been indicated the variation that takes place on the individual cells level.
Figure 10 A-F shows to make hESC be exposed to nodal, activin A and activin B (NAA) SOX17 after 5 days +Cell number significantly increases the displaing micro picture of (A-C).By comparing each region S OX17 +Cell accounts for the relative abundance of whole cell numbers (shown in the painted nuclear of DAPI (D-F)), and the cell of about 30-50% shows immunoreactivity to SOX17 after handling 5 days with NAA as can be seen.
Figure 11 is the bar graph of SOX17 genetic expression among the hESC that breaking up of the dose-dependent increase of proof activin A (0,10,30 or 100ng/ml).It is very high to handle the expression that increases after 3 days when adhere to cultivating, and lasts till 1,3 and 5 day of suspension culture subsequently always.
Figure 12 A-C is the bar graph of proof activin A to the influence of the expression of MIXL1 (figure A), GATA4 (figure B) and HNF3b (figure C).Also observe the dose-dependent increase definitive entoderm of activin A 3 kinds of other marks: MIXL1, GATA4 and HNF3b.The multiple that the expression corresponding with activin dosage increases shows all 4 kinds of gene (SOX17 of specialization of activin A coexpression with viewed closely similar to SOX17 +, MIXL1 +, GATA4 +And HNF3b +) cell mass.
Figure 13 A-C is the bar graph of proof activin A to the influence of the expression of AFP (figure A), SOX7 (figure B) and SPARC (figure C).The expression of internal organ entoderm mark AFP is to the dependent reduction of activin A show dose.Primitive endoderm mark (SOX7) and body wall entoderm mark (SPARC) remain unchanged or show inhibition at some time points, show that activin A does not play these extraembryonic endoderm cell types of specialization.This has further supported the increase of the increase of the following fact: SOX17, MIXL1, GATA4 and HNF3b expression owing to the definitive entoderm cell number of corresponding activin A.
Figure 14 A-B shows the bar graph of activin A to the influence of the expression of ZIC1 (figure A) and Brachyury (figure B).The expression proof activin A that neural mark ZIC1 is stable does not have dose-dependent effect to Neural Differentiation.The expression of Brachyury reduces demonstration 100ng/ml activin A processing and has significantly suppressed the mesoderm differentiation.This may be from the result of mesendoderm precursor to the increase of specialization of definitive entoderm.With respect to untreated control cultures, low-level activin A (10 and 30ng/ml) handles the expression that keeps brachyury than the later stage in differentiation.
Figure 15 A-B shows that the body wall entoderm that the response activin is handled breaks up the displaing micro picture that reduces.Having only differentiation phase under the condition of serum, body wall entoderm Tm HiThe zone is present in (A) in all cultures, and when comprising activin A, to TM +The differentiation of cell is considerably less, and the immunoreactive total intensity of TM is lower.
Figure 16 A-D is the displaing micro picture that shows the marker expression of response activin A and activin B processing.HESC handled 4 days continuously with activin A and activin B, with SOX17, AFP and TM antibody three heavy labels.Figure A-SOX17; Figure B-AFP; Figure C-TM; Figure D-Phase/DAPI.Notice that many SOX17 positive cells are accompanied by AFP (B) and the immunoreactive disappearance fully of TM (C).
Figure 17 is presented at externally definitive entoderm and the endoblastic displaing micro picture of internal organ to occur from hESC.Pass through AFP Hi/ SOX17 Lo/-Identify internal organ entoderm zone, and definitive entoderm shows antipodal Mode S OX17 Hi/ AFP Lo/-Because these two zones are adjacent to each other, so selected this zone.But, from AFP HiObserve SOX17 through regular meeting in the complete isolate of the arbitrary region of cell Hi/ AFP Lo/-The zone shows the independent origin from the endoblastic definitive entoderm of internal organ.
Figure 18 describes the part of TGF 'beta ' family and the chart of acceptor.The factor that activates AR Smads and BR Smads can the process that produces definitive entoderm from human embryo stem cell, work (referring to: J Cell Physiol.187:265-76).
Figure 19 shows as what the SOX17 through the TGF β factor result of single or combination expressed to induce time dependent bar graph.
Figure 20 is the SOX17 that shows as the TGF β factor result through making up +The time dependent bar graph of cell number.
Figure 21 shows as what the SOX17 through the TGF β factor result of combination expressed to induce time dependent bar graph.
Figure 22 induces SOX17 with showing activin A dose-dependently +The bar graph that cell number increases.
Figure 23 is a bar graph, shows that adding Wnt3a in the culture of activin A and activin B processing impels the expression level of SOX17 to be higher than only with activin A and activin B inductive expression level.
Figure 24 A-C shows that low FBS condition strengthens the bar graph to the definitive entoderm differentiation.In containing the substratum of 2%FBS, handle (2AA) hESC with activin A and activin B, the expression level that can cause SOX17 than in containing the substratum of 10%FBS through the expression level high 2 to 3 times (figure A) of the SOX17 of same treatment (10AA).Inducing of definitive entoderm mark MIXL1 (figure B) is also influenced in the same manner, the rejection ratio to AFP (internal organ entoderm) in 2%FBS stronger under the 10%FBS condition (figure C).
Figure 25 A-D shows SOX17 in the culture +Fissional displaing micro picture.The SOX17 immunoreactive cell is present in hESC clone's dividing edge (C, D), by proliferating cell nuclear antigen (PCNA) mark (figure B), but is not total to mark (figure C) by OCT4.In addition, at SOX17 +Cell (arrow) and OCT4 +All can see mitotic division figure (D) clearly with DAPI mark nuclear in the cell, undifferentiated hESCs (arrow head).
Figure 26 is the bar graph that is presented at the relative expression's level of CXCR4 among the hESC that is breaking up under the different culture medium condition.
Figure 27 A-D shows that how one group of definitive entoderm mark share the bar graph of closely similar expression pattern with CXCR4 under the identical differentiation that shows as Figure 26 is handled.
Figure 28 A-E is a bar graph, show the mesoderm mark (BRACHYURY, MOX1), the ectoderm mark (SOX1, ZIC1) and internal organ entoderm mark (SOX7) under the same treatment that shows as Figure 26, how to show with CXCR4 and express opposite relation.
Figure 29 A-F is the displaing micro picture that is presented at the relative different of SOX17 immunoreactive cell under three kinds of culture medium condition of showing among Figure 26-28.
Figure 30 A-C is the fluidic cell point diagram, proves CXCR4 +Cell number increases with the increase of the concentration of the activin A that is added into division culture medium.
Figure 31 A-D is a bar graph, shows from high dosage activin A to handle (A100-CX+) isolating CXCR4 +Cell is than the further enrichment definitive entoderm mark of parental generation group (A100).
Figure 32 is a bar graph, shows to use the isolating CXCR4 of fluorescence activated cell sorting (FACS) +And CXCR4 -Genetic expression among gene expression of cells and the parental generation group.This proves CXCR4 +Cell has comprised all CXCR4 genetic expression among each parental cell group, CXCR4 substantially -Cell mass comprises seldom or does not have CXCR4 genetic expression.
Figure 33 A-D is a bar graph, proves from high dosage activin A to handle isolating CXCR4 +Mesoderm in the cell (BRACHYURY, MOX1), (SOX1, ZIC1) and the disappearance of internal organ entoderm (SOX7) genetic expression, the expression of these non-definitive entoderm marks is suppressed ectoderm.
Figure 34 A-M is the bar graph that shows the expression pattern of the marker gene can be used for identifying the definitive entoderm cell.Figure G-L has shown the expression analysis of definitive entoderm mark FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 respectively.Figure A-F shown previously described clone marker gene SOX17, SOX7, SOX17/SOX7, TM, ZIC1 and MOX1 respectively) expression analysis.Figure M has shown the expression analysis of CXCR4.For each figure of figure A-M, the genetic expression from the human embryo stem cell of purifying is shown in the tabulation of mark hESC; 2NF represents that cell through the 2%FBS processing, does not add activin; 0.1A100 the expression cell is handled through 0.1%FBS and 100ng/ml activin A; 1A100 represents that cell is through 1%FBS and 100ng/ml activin A processing; 2A100 represents that cell is through 2%FBS and 100ng/ml activin A processing.
Figure 35 is the chart that is presented under the condition that contains or do not contain activin PDX1 gene relative expression in the hESC culture of cultivating 4 days and 6 days, wherein at the 4th day interpolation vitamin A acid (RA) and fibroblast growth factor (FGF-10).
Figure 36 A-F is the chart that is presented under the condition that contains or do not contain activin marker gene relative expression in the hESCs culture of cultivating 4 days and 6 days, wherein at the 4th day interpolation vitamin A acid (RA) and fibroblast growth factor (FGF-10).Each figure has shown the level relatively that following marker gene is expressed: (A) SOX17; (B) SOX7; (C) AFP; (D) SOX1; (E) ZIC1; (F) NFM.
Figure 37 A-C is the chart that is presented under the condition that contains or do not contain activin marker gene relative expression in the hESC culture of cultivating 4 days and 8 days, wherein in the combination of the 4th day interpolation vitamin A acid (RA), fibroblast growth factor (FGF-10) and fibroblast growth factor (FGF-4).Each figure has shown the level relatively that following marker gene is expressed: (A) PDX1; (B) SOX7 and (C) NFM.
Figure 38 A-G is the chart that is presented at the relative expression of marker gene in the definitive entoderm cell culture that contacts with 50ng/ml FGF-10, wherein adds 1 μ M, 0.2 μ M or 0.04 μ M vitamin A acid (RA) and 50ng/ml FGF-10 at the 4th day and unites use.Each figure has shown the level relatively that following marker gene is expressed: (A) PDX1; (B) HOXA3; (C) HOXC6; (D) HOXA13; (E) CDX1; (F) SOX1 and (G) NFM.
Figure 39 A-E is presented at and contains or do not contain activin, exist vitamin A acid (RA), fibroblast growth factor (FGF-10) and following among a kind of: serum substitute (SR), cultivate the relative expression of marker gene in the hESC culture of 4 days and 8 days under the condition of the combination that calf serum (FBS) or B27 constitute.Each hurdle has shown the level relatively that following marker gene is expressed: (A) PDX1; (B) SOX7; (C) AFP; (D) ZIC1 and (E) NFM.
The relative expression's of the marker gene of pancreas (PDX1, HNF6) and liver (HNF6) chart in hESC culture when Figure 40 A-B is (just before the adding RA) back that shows 6 days and 9 days (after being exposed to RA3 days).Comprise the comparison of different conditions, under the condition of 25ng/ml (A25) or 50ng/ml (A50), added the activin B of 10ng/ml (a10), 25ng/ml (a25) or 50ng/ml (a50).The condition that does not contain any activin A and activin B (NF) is as the negative control that produces definitive entoderm and PDX1-positive entoderm.Each figure has shown the level relatively that following marker gene is expressed: (A) PDX1 and (B) HNF6.
Figure 41 A-C be presented under the condition that contains 100ng/ml (A100), 50ng/ml (A50) or do not contain (NF) activin A when cultivating 5 days (just before adding vitamin A acid) and add RA after the relative expression of marker gene in the hESCs culture of 2,4 and 6 days (being respectively the 7th, 9 and 11 day).The per-cent of direct mark shows the FBS dosage of differentiation during 3 to 5 days under each post figure.Since the 7th day, handle the cell of (R) with RA and in comprising the RPMI substratum of 0.5%FBS, grow.The concentration of RA was 2 μ M at the 7th day, and the 9th day is 1 μ M, and the 11st day is 0.2 μ M.Each figure has shown the level relatively that following marker gene is expressed: (A) PDX1; (B) ZIC1 and (C) SOX7.
Figure 42 A-B demonstration is at first induced definitive entoderm (the 5th day) with activin A processing in low FBS, induce PDX1-to express entoderm with fresh (A25R) substratum that comprises 25ng/ml activin A and RA or various conditioned medium (MEFCM, CM#2, CM#3 and CM#4) and RA then.Detected marker expression at the 5th, 6,7,8 and 9 day.Each figure has shown the level relatively that following marker gene is expressed: (A) PDX1 and (B) CDX1.
Figure 43 has shown at first in low FBS to handle with activin A and has induced definitive entoderm, handles with fresh culture then, and this fresh culture comprises the RA of activin A and vitamin A acid (A25R) or different amounts in the conditioned medium that dilutes with fresh culture.The cumulative volume of substratum all is 5ml.
Figure 44 is the Western trace, shows PDX1 immunoprecipitation of the definitive entoderm cell of RA-processing when 3 days (d8) and 4 days (d9) after adding RA and 50ng/ml activin A.
Figure 45 is a summary chart, the result of the positive anterior intestine endoderm cell's fluorescence activated cell sorting of PDX1-(FACS) of the EGFP reporter gene under the control of PDX1 promotor that shown genetic marker.
Figure 46 is the chart of the relative PDX1 expression level of group after proofreading and correct with respect to house-keeping gene of viable cell (Live), EGFP-negative cells (Neg) and the EGFP-positive cell (GFP+) of demonstration sorting.
Figure 47 is the viable cell (Live) that shows sorting, EGFP-negative cells (Neg), faciation that half has the EGFP-positive cell group of minimum EGFP strength of signal (Lo) and an EGFP-positive cell group that half has the highest EGFP strength of signal (Hi) the relative PDX1 expression level after for the house-keeping gene correction.
Figure 48 A-E shows back 5 kinds of pancreas entoderm marks of the relative expression's level faciation of viable cell (Live), EGFP-negative cells (Neg) and the EGFP-positive cell (GFP+) of sorting is proofreaied and correct to(for) house-keeping gene.Each figure: A-NKX2.2; B-GLUT2; C-HNF3 β; D-KRT19 and E-HNF4 α.
Figure 49 is relative expression's level that the group of viable cell (Live), EGFP-negative cells (Neg) and the EGFP-positive cell (GFP+) of demonstration sorting proofreaies and correct back two kinds of non-pancreas entoderm marks at relative house-keeping gene.Each figure: A-ZIC1 and B-GFAP.
Figure 50 A-D shows the interior differentiation of the body of the definitive endoderm under the kidney peplos of being transplanted to the immunocompromise mouse.Each figure: the A-hematoxylin-eosin staining shows the intestinal tube spline structure; The antibody mediated immunity reactivity of the anti-liver cell specific antigens of B-(liver); The antibody mediated immunity reactivity of the anti-villin of C-(villin) (intestines); Antibody mediated immunity reactivity with the anti-CDX2 of D-(intestines).
Figure 51 A-C is the chart that is presented at the relative expression's level behind the hepatic tissue mark (albumin and PROX1) and lung tissue mark (TITF1) correction in the cell of the 5th to 10 day contact 20ng/ml Wnt3B, 5ng/ml FGF2 or 100ng/ml FGF2.DE appointment type endoderm cell.Each figure: A-albumin, B-PROX1, and C-TITF1.
Figure 52 A-L be presented at the 5th to 10 day contact 20-50ng/ml Wnt3A, 5ng/mlFGF2 or 100ng/ml FGF2 with the 9th with the cell that contact BMP4 in 10 days in the chart of relative expression's level after hepatic tissue mark (AFP, AAT, hHEX, GLUT2, APOA1 and VCAM1) and lung tissue mark (VWF and the CXR4) correction.DE appointment type endoderm cell.Each figure: A-AFP, B-AAT, C-GLUKO, D-hHEX, E-TAT, F-hNF4a, G-CYP7A, H-GLUT2, I-APOA1, J-VCAM1, K-VWF, and L-CXCR4.
Describe in detail
A critical stage of people's early development, namely the term primitive gut forms and occurs in fertilization 2-3 after week. Primitive gut 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 connective 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 the organ of deriving from enteron aisle, such as lung, liver, thymus gland, parathyroid gland and thyroid gland, gall-bladder and pancreas (Grapin-Botton and Melton, 2000; Kimelman and Griffin, 2000; Tremblay et al., 2000; Wells and Melton, 1999; Wells and Melton, 2000). Have very significantly different between definitive entoderm and the clone of separating fully that is called primitive endoderm. Primitive endoderm mainly forms embryo outside organization, mainly is the body wall of placenta yolk bag and the cell epimatrix material of internal organ entoderm part and Reichert ' s film.
In the primitive gut forming process, the definitive entoderm forming process starts from the cell migration event, and wherein, mesendoderm cell (can form mesoderm or endoblastic cellular component) passes a structure that is called former (primitive streak). Definitive entoderm is derived from the cell that passes former front portion and knot (ad hoc structure that is positioned at former line foremost part). When migrating when occuring, definitive entoderm at first forms the foremost part of intestinal tube until finish when forming the intestinal tube rear end.
Definitive entoderm and represented cell-derived important pluripotency starting point from the endoderm cell that it is derived, this cell forms eventually tissue and/or the organ of differentiation derived from the definitive entoderm pedigree. These cells, tissue and/or organ are exceedingly useful in cell therapy. Thus, the method for evaluation differentiation factor described herein is conducive to the progress of cell therapy, and this differentiation factor can make the endoderm cell of definitive entoderm cell and/or expression PDX1 be divided into derived from cytophyletic other cell type of definitive entoderm.
Particularly, embodiments more of the present invention relate to the method for identifying one or more differentiation factors, described differentiation factor can be used for breaking up the cell in the cell mass that comprises PDX1 positive entoderm cell and/or definitive entoderm cell, it can promote the definitive entoderm Cell Differentiation to be the cell of tissue and/or organ precursor, and this tissue and/or organ include but not limited to pancreas, liver, lung, stomach, intestines, thyroid gland, thymus gland, pharynx, gall-bladder and bladder.
This paper has also described the other side that relates to definitive entoderm cell composition, PDX1 positive entoderm cell composition and can be used for preparing the method and composition of these cells.
Definition
Run through some term used in this application and phrase and have following described meaning:
" embryo's " used herein refers to organic a series of stage of development, starts from single embryonated egg, ends at multi-cellular structure, and this multi-cellular structure no longer comprises versatility or totipotent cell except the gamogenesis extracellular of growing. Except the embryo from Gamete Fusion, term " embryo's " also refers to the embryo from SCNT.
" versatility " used herein or " multipotential cell " refer to produce the cell type of other concrete cell type of limited quantity.
" expression " used herein refers to level or the amount of generation and material or the material generation of material or material. Therefore, the expression that detects the special sign thing refer to measure expressed mark relatively or absolute magnitude, or the existence that only detects mark is whether.
" mark " used herein refers to can be observed or any molecule of detecting. For example, mark can include but not limited to nucleic acid such as specific gene transcript, gene polypeptide product, non-genomic polypeptide product, glycoprotein, sugar, glycolipid, lipid, lipoprotein or little molecule (for example, molecular weight is less than the molecule of 10,000amu).
When relating to cell culture and/or cell mass, term " part " expression cell culture or cell mass be non-vanishing amount arbitrarily, and scope is from individual cells to whole cell culture or cell mass.
About the cell in cell culture or the cell mass, term " is substantially free of " and represents that the ratio that cell culture or the specific cell type that cell mass did not contain are occupied is less than 5% in all cells of cell culture or cell mass.
" retinoids (retinoid) " used herein refers to any derivative of retinol, retinene or retinoic acid and these compounds.
" conditioned medium " refers to minimal medium the culture medium that changes occur relatively.
" anterior intestine/middle intestines " used herein duodenum 12 pipe front portion and middle part cell comprises the cell of anterior intestine/middle intestines connecting portion.
Definitive entoderm cell and its correlated process
The embodiment that the present invention describes relates to the new definite method that generates the definitive entoderm cell in culture, and it will be by being divided into such as the multipotential cell of stem cell pluripotency definitive entoderm cell. 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. The tissue that these methods provide effective generation people entoderm to derive is such as the basis of pancreas, liver, lung, stomach, intestines, thyroid gland and thymus gland. For example, the generation of definitive entoderm is that stem cell is to the first step of the β Cell Differentiation of functional generation insulin. For the β cell of the generation insulin that obtains consumption, before reaching pancreas islet/β cell, expect that each differentiation step is efficient differentiation step. Perhaps represent the initial step (as shown in Figure 1) of systematic function pancreas islet/β cell because Stem cell differentiation is the definitive entoderm cell, needing especially this step differentiation efficient.
In view of the needs of effective differentiation multipotential cell to the definitive entoderm cell, some aspects of the present invention relate to in-vitro method to be learned, and the multipotential cell that it will about 50-80% changes the definitive entoderm cell into. Typically, these methods comprise in the mode that clearly reaches temporary transient appointment and use culture and growth factor. The reagent that use can be combined with the definitive entoderm cell-specific with definitive entoderm cell and other cell separation and/or purifying, can obtain the further enrichment of definitive entoderm cell mass from cell mass. Like this, embodiments more of the present invention relate to the definitive entoderm cell and prepare and separate 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 to from culture or cell mass, distinguish the method for this class cell and other cell. Therefore, certain embodiments of the present invention relate to the cell sign thing, and whether it exists and/or relative expression's level is special to definitive entoderm, and detect the method for determining these marker expression.
In some embodiments of the present invention, whether mark exists and/or its expression is determined 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, immunohistochemistry technique is for detection of the albumen of said gene expression. In other embodiments, Q-PCR and immunohistochemistry technique are used for amount or the relative scale of identification and definite these marks.
By using the method for determining one or more suitable marker expression described above, differentiate that in cell culture or cell mass the ratio of definitive entoderm cell and definite definitive entoderm cell is possible. 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 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 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. In embodiments more as herein described, the definitive entoderm cell is not significantly expressed PDX1.
Embodiment described herein also relates to the definitive entoderm cell composition. For example, some embodiments relate to the cell culture that comprises definitive entoderm, however other the cell mass of definitive entoderm cell that related to enrichment. Some preferred 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. Particularly 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 regulated by changing some parameters, these parameters include but not limited to the arrangement of time of Growth of Cells condition, growth factor concentration and incubation step, and 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% multipotential cell. In other preferred embodiment, will be converted into such as the multipotential cell group of stem cell colony substantially pure definitive entoderm cell mass.
Composition of the present invention and method have several useful features. For example, comprise 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 such as the disease of diabetes. For example, because definitive entoderm only is used as the source of the tissue of limited quantity, thereby it can be used for growing pure tissue or cell type.
Prepare definitive entoderm from multipotential cell
Below and in the 11/021st, No. 618 United States Patent (USP) that is entitled as DEFINITIVE ENDODERM (definitive endoderm) of submitting on December 23rd, 2004, described the method that differentiation multipotential cell production comprises the cell mass of the cell culture of definitive endoderm and enrichment. In some such methods, be stem cell as the multipotential cell of parent material. In some method, comprise the cell culture of definitive endoderm and the cell mass of enrichment from embryonic stem cell production. The method of definitive entoderm cell of preferably deriving utilizes human embryo stem cell as the parent material of preparation definitive entoderm. These multipotential cells can be the cells that is derived from mulberry body, embryo's inner cell mass or obtains from embryo's genital ridge. Use art methods, human stem cell can keep the multipotency state and substantially not break up in culture medium. Described method is such as the 5th, 453, and 357,5,670,372,5,690,926,5,843,780,6,200,806 and 6,251, describe to some extent in No. 671 United States Patent (USP)s.
In production definition endoderm cell's certain methods, hESCs remains on trophoderm. In these methods, any trophoderm that can make hESCs remain on the multipotency state all can be used in the method for the present invention. The trophoderm of a cultivation human embryo stem cell commonly used is one deck l cell. Recently, HF's trophoderm has also developed for cultivating hESCs (referring to No. 2002/0072117 disclosed method of U.S. Patent application). Other embodiment of the method for the invention allows not use trophoderm to keep versatility hESCs. These methods have description in No. 2003/0175956 U.S. Patent application.
Human embryo stem cell of the present invention can remain on to have or not to have in the culture medium of serum. In some embodiments, use serum replacement. In other embodiments, use the culture medium technology of serum-free, as described in Application No. 2003/0190748, it is disclosed in this whole introducings, for your guidance.
In culture medium, keep the stem cell of multipotency state to go down to posterity according to routine, until be divided into definitive entoderm. 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 growth factor in stem cell media. TGF beta superfamily growth factor for the preparation of definitive entoderm is selected from Nodal/ activin or BMP subgroup. In some embodiments of differentiation method of the present invention, growth factor is selected from Nodal, activin A, activin B and BMP4. In addition, growth factor Wnt3a and other Wnt family member can be used for preparing the definitive entoderm cell. In some atomization, can use the combination of above-mentioned any growth factor of mentioning.
About the certain methods from multipotent stem cells to the definitive entoderm Cell Differentiation, the above-mentioned growth factor that adds in the cell, its concentration in culture medium is enough to promote at least part of Stem cell differentiation to the definitive entoderm cell. In some embodiments of the present invention, in the culture medium concentration of above-mentioned growth factor 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 5000 ng/ml or greater than 5000ng/ml.
In some method that multipotent stem cells is divided into the definitive entoderm cell, above-mentioned growth factor needs to be removed from cell culture after adding culture medium. For example, the time of removing of growth factor about add rear 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 growth factor is about adding rear 4 days.
The cultivation of definitive entoderm cell can be carried out in the culture medium that contains a small amount of serum or serum-free. Under some condition of culture, the concentration range of serum is that about 0.05%v/v is to about 20%v/v. is for example; ，0.05％ （ v／v ） 、 0.1％ （ v／v ） 、0.2％ （ v／v ） 、0.3％ （ v／v ） 、0.4％ （ v／v ） 、 0.5％ （ v／v ） 、0.6％ （ v／v ） 、0.7％ （ v／v ） 、0.8％ （ v／v ） 、 0.9％ （ v／v ） 、1％ （ v／v ） 、2％ （ v／v ） 、3％ （ v／v ） 、 4％ （ v／v ） 、5％ （ v／v ） 、6％ （ v／v ） 、7％ （ v／v ） 、 8％ （ v／v ） 、9％ （ v／v ） 、10％ （ v／v ） 、15％ （ v／v ） 20％ （ v／v ） 。 In certain methods, the definitive entoderm cell is grown under serum-free or in the presence of serum replacement. 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.
The monitoring multipotential cell is to the differentiation of definitive entoderm
HESC is cultured to the process of definitive entoderm and can monitors by the mark feature representation of determining definitive entoderm. In some embodiments, whether the expression of some marks can exist mark to determine by detecting. 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 a kind of quantitative marker gene produces can be used quantitative PCR (Q-PCR). Carry out the method for Q-PCR in the prior art for known. Other method well known in the prior art also can be used for the expression of quantitative marker gene. For example, the expression of marker gene product can detect by using the antibody for interested specific markers gene outcome. In some embodiments of the present invention, determine the expression of the distinctive marker gene of definitive entoderm, and lacked the expression of hESCs and the distinctive marker gene of 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 cellular expression SOX17 marker gene of the method for the invention preparation produces the SOX17 gene outcome thus. The mark of other definitive entoderm is MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1. Because the level of definitive entoderm cellular expression SOX17 marker gene is higher than the level of SOX7 marker gene, wherein the SOX7 marker gene is original and the endoblastic feature of internal organ (referring to table 1), therefore monitors in some embodiments of the present invention the expression of SOX17 and SOX7. In other embodiments, the expression of monitoring SOX17 marker gene and the expression of OCT4 marker gene, wherein the OCT4 marker gene is the feature of hESCs. In addition, because the level of definitive entoderm cellular expression SOX17 marker gene is higher than the level of AFP, SPARC or thrombomodulin (TM) marker gene, can monitor the expression of these genes.
Other mark of definitive entoderm is the CXCR4 gene. CXCR4 gene code cell surface chemokine receptors, its part is chemoattractant SDF-1. The Main Function that comprises the cell of CXCR4 acceptor among the adult it is believed that as 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.Leukocyte Biol.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.et al.Dev. Biology 213,442-456 (1999)] in the research carried out, chemokine receptors CXCR4 and unique part SDF-1[Kim thereof in adult mice and early development thereof have been described, C., and Broxmyer, H., J.Leukocyte Biol.65,6-15 (1999)] expression. CXCR4/SDF1 is clear in developmental interaction, in transgenic mice, as arbitrary gene disruption [Nagasawa et al. Nature wherein, 382,635-638 (1996)], Ma, Q., et al Immunity, 10,463-471 (1999)] all cause late embryo dead. McGrath etc. use ribonuclease protecting and in-situ hybridization method to confirm that at early stage (E7.5) that form primitive gut, CXCR4 is the abundantest chemokine receptors mRNA that detects. As if at the primitive gut formation stages, the CXCR4/SDF-1 signal is mainly induced the migration of former germinal layer cell, and express on definitive entoderm, mesoderm and the ectoderm that exists at this moment. In the E7.2-7.8 mice embryonic, CXCR4 and α-fetoprotein are mutually exclusive, are presented at the internal organ entoderm and lack expression [McGrath, K.E.et al.Dev.Biology 213,442-456 (1999)].
Owing to the definitive entoderm cellular expression CXCR4 marker gene of multipotential cell preparation, can monitor the expression of CXCR4 to follow the tracks of the generation of definitive entoderm cell. In addition, other definitive entoderm mark of definitive entoderm cellular expression according to the method for the invention preparation includes but not limited to SOX17, MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1. Because the level of definitive entoderm cellular expression CXCR4 marker gene is higher than the level of expressing the SOX7 marker gene, can monitor the expression of CXCR4 and SOX7. In other embodiments, the expression of monitoring CXCR4 marker gene and OCT4 marker gene are expressed. In addition, because the level of definitive entoderm cellular expression CXCR4 marker gene is higher than the level of expressing AFP, SPARC or thrombomodulin (TM) marker gene, can monitor the expression of these genes.
Should be appreciated that the expression of CXCR4 in the endoderm cell do not repel the expression of SOX17. Correspondingly, significantly express SOX17 and CXCR4 according to the definitive entoderm cell of the method for the invention preparation, but significantly do not express AFP, TM, SPARC or PDX1.
Should be appreciated that according to differentiation condition, in the definitive entoderm cell, induce SOX17 and/or the CXCR4 marker expression of varying level scope. Like this, in some embodiments of the present invention, SOX17 mark and/or CXCR4 mark the expression of definitive entoderm cell or cell mass than SOX17 mark and/or CXCR4 mark such as the non-definitive entoderm cell of multipotent stem cells 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 the expression of definitive entoderm cell or cell mass than SOX17 mark and/or CXCR4 mark express such as the non-definitive entoderm cell of multipotent stem cells or cell mass high 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, 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 multipotent stem cells in the expression of definitive entoderm cell or cell mass.
It should also 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.
Should be appreciated that also that in the definitive entoderm cell there are a series of differences in the expression of SOX17 mark and OCT4, SPARC, AFP, TM and/or SOX7 marker expression level. Similarly, in the definitive entoderm cell, also there are a series of differences in the expression of CXCR4 mark and OCT4, SPARC, AFP, TM and/or SOX7 marker expression level. Thereby, in some embodiments of the present invention, the expression of SOX17 mark or CXCR4 mark than the expression height of OCT4, SPARC, 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 of SOX17 mark or CXCR4 mark than the expression height of OCT4, SPARC, 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 significantly expressed at the definitive entoderm cell.
Be to be understood that, in some embodiments of the present invention, in the definitive entoderm cell, compare with the expression of 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 enrichment of definitive entoderm, separation and/or purifying
Definitive entoderm cell with any said method preparation can come enrichment, separation and/or purifying with special affinity tag for these cells. The example of special affinity tag for the definitive entoderm cell is that antibody, part or other are special for the binding reagents (for example polypeptide) such as the marker molecules of polypeptide, this marker molecules is present in the surface of cell definitive entoderm cell, but substantially is not present in other cell type of finding in the cell culture medium for preparing according to method of the present invention. The antibody of being combined with CXCR4 in some embodiments, be used for definitive entoderm enrichment, separate and/or the affinity tag of purifying. In other embodiments, chemotactic factor (CF) SDF-1 or other molecule based on SDF-1 also can be used as affinity tag. These molecules include, but are not limited to SDF-1 segment, SDF-1 fusions or SDF-1 analogies.
Dispersal risk and the method that is used for cell separation thereof are known in the art, and these methods can utilize antibody of the present invention and cell to implement. In one embodiment, be combined with magnetic bead in connection with the antibody of CXCR4, then in cell culture medium with the definitive entoderm Cell binding, after enzyme is processed, reduced iuntercellular and with the adhesion of substrate. Cell/antibody/magnetic bead compound is exposed in the shifting magnetic field separates with unconjugated cell with the definitive entoderm cell with the magnetic bead combination. In case the definitive entoderm cell separates with other cell physics in culture medium, destroy the antibody of combination, cell is placed suitable tissue culture medium (TCM) again.
Also can use enrichment, the definitive entoderm cell culture of isolated or purified or other method of cell mass of obtaining. For example, in some embodiments, CXCR4 antibody is hatched with the cell culture that comprises definitive entoderm, reduced after treatment before this cell culture iuntercellular and with the adhesion of substrate. Then with cell washing, centrifugal and again suspension. The cell suspension and then and SA, if the FITC coupling antibody incubation of being combined with first antibody. And then with cell washing, centrifugal reaching again in the buffer suspension liquid. Subsequent analysis cell suspension 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 cell composition of separation can be further with the affine method that substitutes or with special identical or different mark for definitive entoderm by increase sorting purifying for several times.
In other embodiment of the present invention, use part or other molecule enrichment, separation and/or purifying definitive entoderm in conjunction with CXCR4. In some embodiments, described molecule is SDF-1 or its fragment, fusions or its analogies.
In preferred embodiments, be 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.
Except 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 a series of method enrichment or separation of cultivating again under growth conditions, and this growth conditions is conducive to described definitive entoderm cell selective survival or selective amplification.
Use method of the present invention, can external from pass through at least some differentiation such as the multipotential 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 is directed to and mainly is divided into definitive entoderm. Some preferred enrichments, separation and/or purification process relate to external and prepare definitive entoderm from human embryo stem cell. Use method of the present invention, compare with untreated cell mass or cell culture, the enrichment definitive entoderm is at least about 2 times to about 1000 times in cell mass or the cell culture. In some embodiments, compare with untreated cell mass or cell culture, the definitive entoderm cell enrichment is at least about 5 times to about 500 times. In other embodiments, compare with untreated cell mass or cell culture, but the enrichment of definitive entoderm cell is at least about 10 times to about 200 times. In other embodiments, compare with untreated cell mass or cell culture, but the enrichment of definitive entoderm cell is at least about 20 times to about 100 times. In other embodiments, compare with untreated cell colony or cell culture, the definitive entoderm cell 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 cell enrichment is at least about 2 times to about 20 times.
The composition that comprises definitive entoderm
The cell composition that said method produces comprises the cell culture that comprises definitive entoderm and the cell mass of enrichment definitive entoderm. For example, can produce the cell culture that comprises the definitive entoderm cell, wherein the cell at least about 50-80% is the definitive entoderm cell in the culture. Because the efficient of differentiation method can (these parameters include but not limited to by changing some parameter, the control of time of Growth of Cells condition, growth factor concentration and incubation step) adjust, differentiation method described herein can cause about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or surpass about 95% multipotential cell to the conversion of definitive entoderm. In the method for the separation definitive entoderm cell that adopts, for example, by using the affinity reagent in conjunction with the CXCR4 acceptor, can obtain basically pure definitive entoderm cell mass.
Embodiments more of the present invention relate to and comprising such as the multipotential cell of stem cell and the composition of definitive entoderm cell, for example cell mass and cell culture. For example, can prepare the composition that comprises hESCs and definitive entoderm cell mixture with method of the present invention. In some embodiments, can produce and comprise per approximately 95 multipotential cell correspondences at least about the composition of 5 definitive entoderm cells. In other embodiments, can produce and comprise per approximately 5 multipotential cell correspondences at least about the composition of 95 definitive entoderm cells. In addition, expection can obtain comprising cell culture or the cell mass of multipotential cell and other ratios of definitive entoderm. For example, expection can obtain comprising per approximately 1,000,000 multipotential cell correspondence is at least about 1 definitive entoderm cell, per approximately 100,000 multipotential cell correspondence is at least about 1 definitive entoderm, per approximately 10,000 multipotential cell correspondence is at least about 1 definitive entoderm cell, per approximately 1000 multipotential cell correspondences are at least about 1 definitive entoderm cell, per approximately 500 multipotential cell correspondences are at least about 1 definitive entoderm cell, per approximately 100 multipotential cell correspondences are at least about 1 definitive entoderm cell, per approximately 10 multipotential cell correspondences are at least about 1 definitive entoderm cell, per approximately 5 multipotential cell correspondences are at least about 1 definitive entoderm cell, per approximately 2 multipotential cell correspondences are at least about 1 definitive entoderm cell, per approximately 1 multipotential cell correspondence is at least about 1 definitive entoderm cell, per approximately 1 multipotential cell correspondence is at least about 2 definitive entoderm cells, per approximately 1 multipotential cell correspondence is at least about 5 definitive entoderm cells, per approximately 1 multipotential cell correspondence is at least about 10 definitive entoderm cells, per approximately 1 multipotential cell correspondence is at least about 20 definitive entoderm cells, per approximately 1 multipotential cell correspondence is at least about 50 definitive entoderm cells, per approximately 1 multipotential cell correspondence is at least about 100 definitive entoderm cells, per approximately 1 multipotential cell correspondence is at least about 1000 definitive entoderm cells, per approximately 1 multipotential cell correspondence is at least about 10,000 definitive entoderm cell, about multipotential cell correspondence is at least about 100,000 definitive entoderm cell, per 1 the multipotential cell correspondence of peace treaty is at least about 1, the composition of 000,000 definitive entoderm cell. In some embodiments, this multipotential cell is people's multipotent stem cells. In some embodiment, this is stem cell-derived from mulberry body, embryo's inner cell mass or embryo sexual fold. In some other embodiment, this multipotential cell is derived from growing gonadal tissue or the germinal tissue of having crossed brephic multi-cellular structure.
Embodiments more of the present invention relate to comprise from least about 5% definitive entoderm cell to cell culture or cell mass at least about 95% definitive endoderm. In some embodiments, this cell culture or cell mass comprise mammalian cell. In the preferred embodiment, this cell culture or cell mass comprise people's cell. For example, some particular relates to the cell culture that contains people's cell, wherein from being the definitive entoderm cell at least about 5% to the people's cell at least about 95%. Other embodiment relates to the cell culture that contains 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 more than people's cell of 90%. In some embodiments, cell culture or cell mass comprise people's feeder cells, do not consider the feeder cells in cell culture or the cell mass when calculating above-mentioned percentage.
Further embodiment of the present invention relates to the composition that comprises people's cell (such as people's definitive entoderm cell), for example cell culture or cell mass wherein are higher than the expression of OCT4, SPARC, α-fetoprotein (AFP) is in thrombomodulin (TM) and/or SOX7 mark at least about the expression of SOX17 or CXCR4 mark in people's cell of 5%. other embodiments; 10％、15％、20％、 25％、30％、35％ 、40％、45％、50％ 、55％、60％、 65％、70％、75％、 80％、85％、90％ 、95％95％，SOX17CXCR4 OCT4、SPARC、AFP、TM／SOX7 。 Some cell cultures or cell mass comprise in the embodiment of people's feeder cells, do not consider the feeder cells in cell culture or the cell mass when calculating above-mentioned percentage.
Should be appreciated that embodiments more of the present invention relate to the composition that comprises people's cell (such as people's definitive entoderm cell), for example cell culture or cell mass, wherein from least about 5% to surpassing the expression that is higher than OCT4, SPARC, AFP, TM and/or SOX7 mark at least about one or more expression that are selected from the mark of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 people's cell of 95%. Some cell cultures or cell mass comprise in the embodiment of people's feeder cells, do not consider the feeder cells in cell culture or the cell mass when calculating above-mentioned percentage.
Other embodiment of the present invention relates to the composition that comprises people's cell (such as people's definitive entoderm cell), for example cell culture or cell mass wherein all are higher than the expression of OCT4, SPARC, AFP is in TM and/or SOX7 mark at least about the expression of SOX17 and CXCR4 mark in people's cell of 5%. other embodiments; 10％、15％ 、20％、25％、 30％、35％、40％、 45％、50％、55％ 、60％、65％、70％ 、75％、80％、 85％、90％、95％ 95％，SOX17CXCR4OCT4、 SPARC、AFP、TM／SOX7。 Some cell cultures or cell mass comprise in the embodiment of people's feeder cells, do not consider the feeder cells in cell culture or the cell mass when calculating above-mentioned percentage.
Should be appreciated that embodiments more of the present invention relate to the composition that comprises people's cell (as people's definitive entoderm cell), for example cell culture or cell mass, wherein from least about 5% to surpassing the expression that is higher than OCT4, SPARC, AFP, TM and/or SOX7 mark at least about the expression of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 mark people's cell of 95%.Some cell cultures or cell mass comprise in the embodiment of people's feeder cell, do not consider the feeder cell in cell culture or the cell mass when calculating above-mentioned per-cent.
Other embodiment of the present invention relates to the composition that comprises Mammals endoderm cell (as people endoderm cell), for example cell culture or cell mass wherein are higher than the expression of OCT4, SPARC, AFP, TM and/or SOX7 mark at least about the expression of SOX17 among 5% the endoderm cell or CXCR4 mark.In other embodiments, in 10% endoderm cell, in 15% endoderm cell, in 20% endoderm cell, in 25% endoderm cell, in 30% endoderm cell, in 35% endoderm cell, in 40% endoderm cell, in 45% endoderm cell, in 50% endoderm cell, in 55% endoderm cell, in 60% endoderm cell, in 65% endoderm cell, in 70% endoderm cell, in 75% endoderm cell, in 80% endoderm cell, in 85% endoderm cell, in 90% endoderm cell, among at least 95% the endoderm cell or more than among 95% the endoderm cell, the expression of SOX17 or CXCR4 mark is higher than OCT4, SPARC, AFP, the expression of TM and/or SOX7 mark.
Should be appreciated that embodiments more of the present invention relate to the composition that comprises the Mammals endoderm cell, for example cell culture or cell mass, wherein from least about 5% to surpassing the expression that is higher than OCT4, SPARC, AFP, TM and/or SOX7 mark at least about one or more expression that are selected from the mark of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 95% the endoderm cell.
Other embodiment of the present invention relates to the composition that comprises Mammals endoderm cell (as people endoderm cell), for example cell culture or cell mass wherein all are higher than the expression of OCT4, SPARC, AFP, TM and/or SOX7 mark at least about the expression of SOX17 among 5% the endoderm cell and CXCR4 mark.In other embodiments, in 10% endoderm cell, in 15% endoderm cell, in 20% endoderm cell, in 25% endoderm cell, in 30% endoderm cell, in 35% endoderm cell, in 40% endoderm cell, in 45% endoderm cell, in 50% endoderm cell, in 55% endoderm cell, in 60% endoderm cell, in 65% endoderm cell, in 70% endoderm cell, in 75% endoderm cell, in 80% endoderm cell, in 85% endoderm cell, in 90% endoderm cell, among at least 95% the endoderm cell or more than among 95% the endoderm cell, the expression of SOX17 and CXCR4 mark is higher than OCT4, SPARC, AFP, the expression of TM and/or SOX7 mark.
Should be appreciated that embodiments more of the present invention relate to the composition that comprises the Mammals endoderm cell, for example cell culture or cell mass, wherein from least about 5% to surpassing the expression that is higher than OCT4, SPARC, AFP, TM and/or SOX7 mark at least about the expression of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 95% the endoderm cell.
Use method described herein, can produce the composition that comprises definitive endoderm that is substantially free of other cell types.In embodiments more of the present invention, the definitive endoderm group or the cell culture that produce with method described herein are substantially free of remarkable cell of expressing OCT4, SOX7, AFP, SPARC, TM, ZIC1 and/or BRACH marker gene.
In one embodiment of the invention, according to expression being described below definitive endoderm of marker gene: SOX17 height, MIXL1 height, AFP is low, SPARC is low, thrombomodulin is low, SOX7 is low, CXCR4 is high.
PDX1 genetic expression in the growth course
PDX1 (being also referred to as little IDX-1 of STF and IPF-1) is that pancreas and beak duodenum (rostralduodenum) are grown essential transcription factor.PDX1 at first expresses at the pancreas entoderm, the pancreas entoderm from the rear portion before gut entoderm, produce external secretion and endocrine cell, in mouse, start from E8.5.Thereafter, PDX1 is defined in beta cell and some δ-cells of endocrine pancreas.This expression pattern also keeps after growing up.Grow the also duodenum entoderm expression of the pancreas in contiguous formation of early stage PDX1, in duodenal intestinal cells and enteroendocrine cell, stomach hole and common bile duct, gall-bladder and bile duct, express then.Express when limited at pancreas, this is expressed zone and mainly is limited to the beak duodenum.
PDX1 positive cell and relative step
The embodiment of other differentiation method described herein relates to the new method of determining that produces the PDX1-positive entoderm cell, wherein the PDX1-positive entoderm cell is a multipotential cell, can be divided into cell, tissue or the organ in the anterior intestine/midgut zone (the positive anterior intestine of PDX1-/middle gut entoderm) derived from intestinal tube.Some embodiment preferred relate to the method that produces the positive anterior intestine endoderm cell of PDX1-.In some embodiments, these PDX1 positive entoderm cells are multipotential cells, can be divided into cell, tissue or organ derived from intestinal tube front portion (gut entoderm before the PDX1-positive).Other embodiment preferred relate to the method for the PDX1-positive entoderm cell that produces the intestinal tube rear portion.In some embodiments, these PDX1 positive entoderm cells are multipotential cells, can be divided into cell, tissue or organ derived from the rear portion in the anterior intestine zone of intestinal tube.
The positive anterior intestine endoderm cell of PDX1-, for example those can be used for producing the β cell of the generation Regular Insulin of differentiation fully according to the cell that method described herein produces.In some embodiments, do not express definitive entoderm cell (the negative definitive entoderm cell of PDX1-of PDX1 basically by differentiation; Also specify the shape entoderm herein) the positive anterior intestine endoderm cell of generation PDX1-.Can use method described herein or other any currently known methods differentiation multipotential cells (as embryonic stem cell) to prepare the negative definitive entoderm cell of PDX1-.The title of submitting on December 23rd, 2004 is that the 11/021st, No. 618 U.S. Patent application of " definitive entoderm " described a kind of method from the negative definitive entoderm of the convenient efficient PDX1-of generation of multipotential cell.
The method that produces the positive anterior intestine endoderm cell of PDX1-provides the foundation for efficiently producing pancreatic tissue (as acinous cell, vessel cell and islet cells) from multipotential cell.In some preferred embodiment, the negative definitive entoderm cell of the positive anterior intestine endoderm cell of people PDX1-derived from human PDX1-, and these cells are derived from hESC.The positive anterior intestine endoderm cell of these people PDX1-is used to produce the beta cell of functional generation Regular Insulin then.Be the Regular Insulin β cell of the generation that obtains significant quantity, wish that each differentiation step all has high differentiation efficiency before being divided into pancreas islet/beta cell terminal point.Because the negative definitive entoderm cell of PDX1-especially wishes to have high differentiation efficiency in this step to an early stage step (as shown in Figure 1) of differentiation representative generation functional islets/beta cell of the positive anterior intestine endoderm cell of PDX1-.
Considering needs the effective differentiation of the negative definitive entoderm cell of PDX1-to the positive anterior intestine endoderm cell of PDX1-, aspects more of the present invention relate in vitro method to be learned, and it can cause the negative definitive entoderm cell of PDX1-of about 2%-25% to transform to the positive anterior intestine endoderm cell of PDX1-.Typically, this method comprises with what determine and uses substratum and somatomedin condition with of short duration specific mode.Separate and/or the positive anterior intestine endoderm cell of purifying PDX1-by using in reagent other cells from cell mass with the positive anterior intestine endoderm cell of PDX1-specific combination, further the positive anterior intestine endoderm cell of enrichment PDX1-in cell mass.Perhaps, the positive anterior intestine endoderm cell of PDX1-can use the reporter gene mark such as green fluorescent protein (GFP), expresses so that can detect PDX1.This fluorescently-labeled cell can be used fluorescence activated cell sorting (FACS) purifying.The more deep aspect of the present invention relates to cell culture and comprises the cell mass of the positive anterior intestine endoderm cell's of PDX1-enrichment, and identify gut entoderm before the PDX1-positive and before the PDX1-positive the useful factor the gut entoderm differentiation.
Be to measure the quantity of the positive anterior intestine endoderm cell of PDX1-in cell culture or the cell mass, need a kind of in culture or cell mass the method from this cell type of other cell differentiation.Correspondingly, certain embodiments of the present invention relate to cell sign thing and detection and determine the method for these marker expression, the existence of these marks whether and/or relative expression's level be the positive anterior intestine endoderm cell's of PDX1-indication.
In embodiments more of the present invention, whether and/or expression level the existence of measuring mark with quantitative PCR (Q-PCR).For example, some genetic marker, the amount of the transcription product that produces as PDX1, SOX17, SOX7, SOX1, ZIC1, NFM, α-Jia Taidanbai (AFP), homology frame A13 (HOXA13), homology frame C6 (HOXC6) and/or other marks described herein is determined with Q-PCR.In other embodiments, detect the said gene expressed proteins with the immunohistochemical methods method.In other embodiments, use two kinds of methods of Q-PCR and immunohistochemical methods to identify and detect the amount and the relative proportion of these marks.
Use differentiation described herein and detection method just may in cell culture or cell mass, identify positive anterior intestine endoderm cell of PDX1-and the ratio of determining these cells.For example, in some embodiments of the present invention, positive anterior intestine endoderm cell of the PDX1-of generation or cell mass express the level of PDX1 gene than PDX1-negative cells or cell mass height at least about 2 orders of magnitude.In other embodiments, positive anterior intestine endoderm cell of the PDX1-of generation and cell mass are expressed the level of PDX1 gene than the PDX1-negative cells or high 2 the above orders of magnitude of cell mass.In other embodiments, positive anterior intestine endoderm cell of the PDX1-of generation or cell mass are expressed the level of one or more marks that are selected from PDX1, SOX17, HOXA13 and HOXC6 than the negative definitive entoderm cell of PDX1-high about 2 or 2 above orders of magnitude.
Composition described herein and method have several useful characteristics.For example, the method that comprises the cell culture and the cell mass of PDX1-positive entoderm and produce this cell culture and cell mass helps the commitment that the anthropomorphic dummy grows.In addition, composition described herein and method also can be used for the therapeutic intervention of morbid state (as diabetes).For example, because gut entoderm is as the source of limited quantity tissue before the PDX1-positive, it can be used to grow pure tissue or cell type.
Produce gut entoderm before the PDX1-positive from the negative definitive entoderm of PDX1-
Positive anterior intestine endoderm cell's culture of PDX1-and the cell mass that comprises the positive anterior intestine endoderm cell of PDX1-described herein produced by the negative definitive entoderm of PDX1-, and wherein the negative definitive entoderm of this PDX1-is produced by multipotential cell as mentioned above.A preferable methods utilizes human embryo stem cell as parent material.In one embodiment, hESC at first is converted into the negative definitive entoderm cell of PDX1-, is converted into the positive anterior intestine endoderm cell of PDX1-then.But should be appreciated that the parent material that is used for the positive anterior intestine endoderm cell generation of PDX1-is not limited to the definitive entoderm cell that produces with the multipotential cell differentiation method.And the negative definitive entoderm cell of any PDX1-all can be used for method described herein, and is irrelevant with their source.
In some embodiments of the present invention, the cell culture or the cell mass that comprise the negative definitive entoderm cell of PDX1-can be used for further breaking up to the cell culture that comprises PDX1-positive anterior intestine endoderm cell and/or the cell mass of enrichment.For example, can use cell culture or the cell mass that comprises people PDX1-feminine gender, SOX17-male definitive entoderm cell.In some embodiments, cell culture or cell mass may comprise the differentiation factor that keeps from front differentiation step (promptly breaking up the step that multipotential cell is the definitive entoderm cell), as activin, nodal and/or BMP.In other embodiments, be used for PDX1-feminine gender, the positive definitive entoderm cell of SOX17-before the factor of the positive anterior intestine endoderm cell differentiation of PDX1-, from cell culture or cell mass, remove the factor in the differentiation step of front in interpolation.In other embodiments, the cell mass of using enrichment PDX1-feminine gender, the positive definitive entoderm cell of SOX17-is as the source that produces the positive anterior intestine endoderm cell of PDX1-.
Promote the differentiation factor (anterior intestine differentiation factor) of cell to the positive anterior intestine endoderm cell differentiation of PDX1-by adding in the cell culture that comprises PDX1-feminine gender, the positive definitive entoderm cell of SOX17-, the negative definitive entoderm cell of the PDX1-in the substratum can be divided into the PDX1-positive entoderm cell.In some embodiments of the present invention, the anterior intestine differentiation factor is a retinoid, for example vitamin A acid (RA).In some embodiments, retinoid and fibroblast growth factor (for example FGF-4 and FGF-10) are united use.In other embodiments, retinoid and somatomedin TGF beta superfamily member and/or conditioned medium are united use.
As previously defined, " conditioned medium " is meant with minimum medium the substratum that changes relatively taken place.For example, the conditioning of substratum may cause from the initial level of substratum and add or removal molecule, for example nutrition and/or somatomedin.In some embodiments, in substratum, grow or keep the certain hour section by the cell that makes certain type under certain condition and make culture medium conditionization.For example, make culture medium conditionization by making hESC in the substratum of certain temperature, definite composition, increase, break up or keep the regular hour.It will be recognized by one of ordinary skill in the art that numerous combinations of cell, type of culture medium, time length and envrionment conditions can be used for producing the conditioned medium near infinite arrangement.In embodiments more of the present invention, by comprise about 1% to the substratum of about 20% serum-concentration growth or the multipotential cell of keeping differentiation come the conditioning substratum.In other embodiments, the multipotential cell by making differentiation comprise about 1ng/ml to the substratum of about 1000ng/ml activin A growth or keep the conditioning substratum.In other embodiments, the multipotential cell by making differentiation is grown to the substratum of about 1000ng/ml BMP4 or is kept the conditioning substratum comprising about 1ng/ml.In preferred embodiments, grow in the substratum (as RPMI) that comprises about 25ng/ml activin A and about 2 μ M RA by the hESC that makes differentiation or keep the preparation condition substratum.
In embodiments more of the present invention, the cell that is used for conditioning substratum (these substratum are used to strengthen the negative definitive entoderm of PDX1-to the endoblastic differentiation of the positive anterior intestine of PDX1-) be contain about 0% to the substratum (for example RPMI) of about 20% serum and/or one or more TGF beta superfamily growth/differentiation factors from the cell of multipotential cell (as hESC) differentiation above 5 days.The concentration range of adding differentiation factor (for example activin A and BMP4) is from about 1ng/ml to about 1000ng/ml.In certain embodiments of the present invention, the cell that is used for the conditioning substratum breaks up above 5 days from hESC at low serum RPMI.According to some embodiments, low serum RPMI refers to contain the substratum of low serum, and wherein serum-concentration increases in the certain hour section gradually.For example, in one embodiment, low serum RPMI grows at cell and comprised the foetal calf serum (FBS) of concentration about 0.2% in first day, second day about 0.5%FBS of cell growth, 3 to 5 days about 2%FBS of cell growth regulation.In another embodiment, low serum RPMI comprised about 0%, the second day about 0.2%, the 3-6 of serum-concentration day about 2% in first day.In certain preferred aspects, add one or more differentiation factors, scheme such as activin A and BMP4 among the low serum RPMI.Except preparation is used for the cell of conditioning substratum, low serum RPMI also can be used as the substratum of the negative definitive entoderm cell of PDX1-to the positive anterior intestine endoderm cell differentiation of PDX1-.
Those of ordinary skill in the art should be appreciated that the substratum RPMI just that is used for the preparation condition substratum, and prerequisite is that this substratum does not disturb the positive anterior intestine endoderm cell's of PDX1-growth or keeps.Should also be clear that the cell that is used for the conditioning substratum can be different type.Come in the embodiment of conditioning substratum at the cell that uses fresh differentiation, this cell can break up in being different from the substratum of RPMI, and prerequisite is that this substratum can not suppress the growth of this cell or keeps.In addition, the technician should be appreciated that the time length of the time length of conditioning or the cell that preparation is used for conditioning is not must be respectively 24 hours or 5 days, because other time span also is enough to the effect that obtains to report herein.
Usually, retinoid and fibroblast growth factor (member of somatomedin TGF beta superfamily), conditioned medium or arbitrarily the combinatorial association of these anterior intestine differentiation factors use, can cause than single with the negative definitive entoderm of the stronger PDX1-of retinoid to the endoblastic differentiation of the positive anterior intestine of PDX1-.In preferred embodiments, add RA and FGF-10 in the negative definitive entoderm cell culture of PDX1-.In another preferred embodiment, the negative definitive entoderm cell of PDX1-breaks up in the substratum that contains conditioned medium, activin A, activin B and RA.
With regard to some embodiments of differentiation method described herein, above-mentioned anterior intestine differentiation factor is added in the cell, make the concentration of anterior intestine differentiation factor in cell culture or the cell mass be enough to promote break up to the positive anterior intestine endoderm cell of PDX1-to negative definitive entoderm cell culture of small part PDX1-or cell mass.As previously defined, when relating to cell culture and/or cell mass, term " part " expression cell culture or cell mass be non-vanishing quantity arbitrarily, scope from individual cells to whole cell culture or cell mass.
In embodiments more of the present invention, adding retinoid in the cell of culture makes its concentration at least about 0.01 μ M, at least about 0.02 μ M, at least about 0.04 μ M, at least about 0.08 μ M, at least about 0.1 μ M, at least about 0.2 μ M, at least about 0.3 μ M, at least about 0.4 μ M, at least about 0.5 μ M, at least about 0.6 μ M, at least about 0.7 μ M, at least about 0.8 μ M, at least about 0.9 μ M, at least about 1 μ M, at least about 1.1 μ M, at least about 1.2 μ M, at least about 1.3 μ M, at least about 1.4 μ M, at least about 1.5 μ M,, at least about 1.6 μ M, at least about 1.7 μ M, at least about 1.8 μ M, at least about 1.9 μ M, at least about 2 μ M, at least about 2.1 μ M, at least about 2.2 μ M, at least about 2.3 μ M, at least about 2.4 μ M, at least about 2.5 μ M, at least about 2.6 μ M, at least about 2.7 μ M, at least about 2.8 μ M, at least about 2.9 μ M, at least about 3 μ M, at least about 3.5 μ M, at least about 4 μ M, at least about 4.5 μ M, at least about 5 μ M, at least about 10 μ M, at least about 20 μ M, at least about 30 μ M, at least about 40 μ M or at least about 50 μ M." retinoid " of Shi Yonging refers to any derivative of Vogan-Neu, retinene or vitamin A acid and these compounds herein.In preferred embodiments, retinoid is a vitamin A acid.
In other embodiments of the present invention, there is the differentiation factor of one or more fibroblast growth families in the cell culture.For example, in some embodiments, in the cell culture concentration of FGF-4 at least about 10ng/ml, at least about 25ng/ml, at least about 50ng/ml, at least about 75ng/ml, at least about 100ng/ml, at least about 200ng/ml, at least about 300ng/ml, at least about 400ng/ml, at least about 500ng/ml or at least about 1000ng/ml.In the further embodiment of the present invention, the concentration of FGF-10 is at least about 10ng/ml, at least about 25ng/ml, at least about 50ng/ml, at least about 75ng/ml, at least about 100ng/ml, at least about 200ng/ml, at least about 300ng/ml, at least about 400ng/ml, at least about 500ng/ml or at least about 1000ng/ml in the cell culture.In some embodiments, selecting one from FGF-4 and FGF-10 adds in the cell culture with RA.In the embodiment preferred, RA concentration is 1 μ M in the cell culture, and FGF-10 concentration is 50ng/ml.
In some embodiments of the present invention, in cell culture, there be the somatomedin and/or the conditioned medium of TGF beta superfamily.These differentiation factors can with RA and/or in other anterior intestine differentiation factor (including but not limited to FGF-4 and FGF-10) unite use.For example, in some embodiments, can have activin A and/or activin B in the cell culture, its concentration is at least about 5ng/ml, at least about 10ng/ml, at least about 25ng/ml, at least about 50ng/ml, at least about 75ng/ml, at least about 100ng/ml, at least about 200ng/ml, at least about 300ng/ml, at least about 400ng/ml, at least about 500ng/ml or at least about 1000ng/ml.In another embodiment of the present invention, existence condition substratum in the cell culture, its concentration be total substratum at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100%.In some embodiments, be added with activin A, activin B and conditioned medium and RA in the cell culture.In preferred embodiments, the negative definitive entoderm cytodifferentiation of PDX1-is the positive anterior intestine endoderm cell of PDX1-in the culture that comprises 1 μ M RA, about 25ng/ml activin A and low serum RPMI substratum (about 24 hours of the hESC conditioning that this substratum has been broken up, wherein Fen Hua hESC broke up about 5 days) in comprising the low serum RPMI of 100ng/ml activin A.In another preferred embodiment, also there are activin B and/or FGF-10 in the culture, its concentration is respectively 25ng/ml and 50ng/ml.
In certain embodiments of the present invention, above-mentioned anterior intestine differentiation factor is removed from cell culture after interpolation.For example, the anterior intestine differentiation factor can be removed in about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days or about 10 days after interpolation.
PDX1-is positive, and the anterior intestine endoderm cell can grow in containing the substratum that reduces serum-concentration.The serum-concentration scope can be from about 0.05% (v/v) to about 20% (v/v).In some embodiments, the positive anterior intestine endoderm cell of PDX1-is grown under the condition that contains serum substitute.For example, in certain embodiments, the serum-concentration of substratum can be lower than about 0.05% (v/v), be lower than about 0.1% (v/v), be lower than about 0.2% (v/v), be lower than about 0.3% (v/v), be lower than about 0.4% (v/v), be lower than about 0.5% (v/v), be lower than about 0.6% (v/v), be lower than about 0.7% (v/v), be lower than about 0.8% (v/v), be lower than about 0.9% (v/v), be lower than about 1% (v/v), be lower than about 2% (v/v), be lower than about 3% (v/v), be lower than about 4% (v/v), be lower than about 5% (v/v), be lower than about 6% (v/v), be lower than about 7% (v/v), be lower than about 8% (v/v), be lower than about 9% (v/v), be lower than about 10% (v/v), be lower than about 15% (v/v) or be lower than about 20% (v/v).In some embodiments, the positive anterior intestine endoderm cell of PDX1-can grow under the condition of serum-free.In other embodiments, the positive anterior intestine endoderm cell of PDX1-grows containing under the condition of serum substitute.
In other embodiments, the positive anterior intestine endoderm cell of PDX1-grows in the presence of B27.In these embodiments, B27 can add to the substratum from about 0.1% (v/v) to the concentration range of about 20% (v/v) or greater than the concentration of 20% (v/v).In certain embodiments, about 0.1% (v/v) of the concentration of B27, about 0.2% (v/v), about 0.3% (v/v), about 0.4% (v/v), about 0.5% (v/v), about 0.6% (v/v), about 0.7% (v/v), about 0.8% (v/v), about 0.9% (v/v), about 1% (v/v), about 2% (v/v), about 3% (v/v), about 4% (v/v), about 5% (v/v), about 6% (v/v), about 7% (v/v), about 8% (v/v), about 9% (v/v), about 10% (v/v), about 15% (v/v) or about 20% (v/v) in the substratum.Perhaps, the concentration of the B27 additive of interpolation can be calculated according to the multiple of commercial B27 storage liquid concentration.For example, Invitrogen (Carlsbad, CA) provide 50 * B27 storage liquid.In the growth medium of enough volumes, add this storage liquid of capacity and can produce the substratum that contains aequum B27.For example, in the 90ml growth medium, add 10ml 50 * B27 storage liquid and will obtain having added the growth medium of 5 * B27.In the substratum concentration of B27 additive can be about 0.1 *, about 0.2 *, about 0.3 *, about 0.4 *, about 0.5 *, about 0.6 *, about 0.7 *, about 0.8 *, about 0.9 *, about 1 *, about 1.1 *, about 1.2 *, about 1.3 *, about 1.4 *, about 1.5 *, about 1.6 *, about 1.7 *, about 1.8 *, about 1.9 *, about 2 *, about 2.5 *, about 3 *, about 3.5 *, about 4 *, about 4.5 *, about 5 *, about 6 *, about 7 *, about 8 *, about 9 *, about 10 *, about 11 *, about 12 *, about 13 *, about 14 *, about 15 *, about 16 *, about 17 *, about 18 *, about 19 *, about 20 * and be higher than about 20 *.
The negative definitive entoderm of monitoring PDX1-is to the endoblastic differentiation of the positive anterior intestine of PDX1-
As the situation of multipotential cell to the definitive entoderm cytodifferentiation, the process of PDX1-feminine gender, the gut entoderm differentiation before the PDX1-positive of the positive definitive entoderm of SOX17-can be monitored by the expression of measuring the distinctive mark of these cell types.This monitoring makes and can determine under different condition (for example one or more differentiation factor concentration and envrionment conditionss) to be enough to produce the time of gut entoderm needs before the aequum PDX1-positive.In the embodiment preferred, deciding by the expression that detects PDX1 is enough to produce the required time of gut entoderm before the aequum PDX1-positive.In embodiments more of the present invention, whether measure the expression of some mark by the existence that detects mark.Perhaps, the level that exists by mark in the cell of measuring cell culture or cell mass is measured the expression of some mark.In these embodiments, the measurement of marker expression can be qualitative or quantitative.As mentioned above, preferred detection by quantitative is to use Q-PCR by the method for the marker expression of marker gene generation.In specific embodiments, Q-PCR is used to the disappearance that expression by the distinctive marker gene of gut entoderm before the detection by quantitative PDX1-positive and the distinctive marker gene of other cell types are expressed, and monitors PDX1-feminine gender, the positive definitive entoderm culture of the SOX17-process to the positive anterior intestine endoderm cell differentiation of PDX1-.Also can express with other known method detection by quantitative marker genes of this area.For example, can utilize the expression that the special antibody of interested marker gene product is detected the marker gene product.In some embodiments of the present invention, measured the disappearance of the remarkable expression of the expression of the distinctive marker gene of gut entoderm before the PDX1-positive and the negative definitive entoderm of PDX-1, hESC and other cell types distinctive marker genes.
As further describing of following embodiment, PDX1 be with the PDX1-positive before the relevant marker gene of gut entoderm.Therefore, measured the expression of PDX1 in some embodiments of the present invention.In other embodiments, also measured the expression of other marks that gut entoderm is expressed before the PDX1-positive, these marks include but not limited to SOX17, HOXA13 and/or HOXC6.Because some other cell type (being internal organ entoderm and some neuroderm) are also expressed PDX1, some embodiments of the present invention relate to the disappearance of proof and internal organ entoderm and/or neuroderm gene expression related or lack basically.For example, in some embodiments, the expression of the mark (including but not limited to SOX7, AFP, SOX1, ZIC1 and/or NFM) of expressing at internal organ entoderm and/or neuroderm is determined.
In some embodiments, the positive anterior intestine endoderm cell of the PDX1-culture that produces with method described herein is substantially free of the cell of expressing SOX7, AFP, SOX1, ZIC1 or NFM marker gene.In certain embodiments, the positive anterior intestine endoderm cell of the PDX1-culture that produces with step described herein is substantially free of internal organ entoderm, body wall entoderm and/or neurocyte.
The endoblastic enrichment of the positive anterior intestine of PDX1-, separation and/or purifying
According to other aspects of the invention, the positive anterior intestine endoderm cell of PDX1-can be by enrichment, separation and/or purifying.In embodiments more of the present invention, the positive anterior intestine endoderm cell's of enrichment PDX1-cell mass produces by separate this cell from cell culture.
In embodiments more of the present invention, PDX1-is positive, and the anterior intestine endoderm cell uses fluorescent mark, uses fluorescence-activated cell sorting device (FACS) to separate with unlabeled cells then.In this class embodiment, the Nucleotide mark PDX1-positive cell that can express the fluorescent marker gene with Nucleotide or another coding of encoding green fluorescent protein (GFP).For example, in some embodiments, the coding GFP of at least one copy or the Nucleotide of its biological active fragment are imported into the downstream of multipotential cell (preferably human embryo stem cell) PDX1 promotor, so that the expression of GFP gene product or its biological active fragment is subjected to the control of PDX1 promotor.In some embodiments, the be encoded Nucleotide of GFP or its biological active fragment of the whole coding region of Nucleotide of coding PDX1 is replaced.In other embodiments, the Nucleotide of coding GFP or its biological active fragment carries out (inframe) fusion in the frame with at least a portion of the Nucleotide of coding PDX1, thereby produces fusion rotein.In this class embodiment, fusion rotein keeps the fluorescence ability similar to GFP.
As mentioned above, fluorescently-labeled cell (for example above-mentioned multipotential cell) is divided into definitive entoderm, is gut entoderm before the PDX1-positive then.The anterior intestine endoderm cell expresses the fluorescent marker gene because PDX1-is positive, and the PDX1-negative cells is not expressed, and these two kinds of cell types can be separated.In some embodiments, the cell suspension thing that comprises the mixture of fluorescently-labeled PDX1-positive cell and unlabelled PDX1-negative cells with the FACS sorting.The PDX1 positive cell separates the back from the PDX1-negative cells to be collected, thereby separates this cell type.If desired, can use the identical or different mark increase sorting number of times special, to be further purified the isolated cells composition to gut entoderm before the PDX1-positive.
Except the step of just having described, the positive anterior intestine endoderm cell of the PDX1-also other technologies of available cellular segregation separates.In addition, the positive anterior intestine endoderm cell of PDX1-also can use and carry out go down to posterity cultured method enrichment or separate the definitive entoderm cell of series under the growth conditions that promotes the positive anterior intestine endoderm cell of above-mentioned PDX1-selectivity survival or selective amplification.
Should be appreciated that above-mentioned enrichment, separation and purification step can be used for any differential period of this class culture.
Use method described herein, the positive anterior intestine endoderm cell's of the PDX1-of enrichment, isolating and/or purifying group and/or tissue can obtain from positive definitive entoderm cell culture of PDX1-feminine gender, SOX17-or the cell mass that proceeds to the small part differentiation external.In some embodiments, cell breaks up at random.But in preferred embodiments, the main directed differentiation of cell is the positive anterior intestine endoderm cell of PDX1-.Some preferred enrichments, separation and/or purification process relate to external from the positive anterior intestine endoderm cell of human embryo stem cell generation PDX1-.
Use method described herein, compare with untreated cell mass or cell culture, the positive anterior intestine endoderm cell of the PDX1-in cell mass or cell culture content can be enriched to less about 2 to about 1000 times.In some embodiments, compare with untreated cell mass or cell culture, PDX1-is positive, and the anterior intestine endoderm cell can be enriched to less about 5 to about 500 times.In other embodiments, compare with untreated cell mass or cell culture, PDX1-is positive, and the anterior intestine endoderm cell can be enriched to less about 10 to about 200 times.In other embodiments, compare with untreated cell mass or cell culture, PDX1-is positive, and the anterior intestine endoderm cell can be enriched to less about 20 to about 100 times.In other embodiments, compare with untreated cell mass or cell culture, PDX1-is positive, and the anterior intestine endoderm cell can be enriched to less about 40 to about 80 times.In some embodiment, compare with untreated cell mass or cell culture, PDX1-is positive, and the anterior intestine endoderm cell can be enriched to less about 2 to about 20 times.
Comprise the endoblastic composition of the positive anterior intestine of PDX1-
Embodiments more of the present invention relate to the cell composition that comprises the positive anterior intestine endoderm cell of PDX1-, for example cell culture or cell mass, wherein the positive anterior intestine endoderm cell of PDX1-is multipotential cell (gut entoderm before the PDX1-positive), can be divided into derived from intestinal tube forward cell, tissue or organ.According to some embodiment, gut entoderm is a mammalian cell before the PDX1-positive, and in preferred embodiments, these cells are people's cells.
Other embodiments of the present invention relate to and comprise the composition that one or more are selected from the cell of the negative definitive entoderm cell of hESC, PDX1-, the positive anterior intestine endoderm cell of PDX1-and mesoblastemic cell type, for example cell culture or cell mass.In some embodiments, in the culture hESC be less than about 5%, be less than about 4%, be less than about 3%, be less than about 2% or be less than whole cells of about 1%.In other embodiments, in the culture the negative definitive entoderm cell of PDX1-be less than about 90%, be less than about 85%, be less than about 80%, be less than about 75%, be less than about 70%, be less than about 65%, be less than about 60%, be less than about 55%, be less than about 50%, be less than about 45%, be less than about 40%, be less than about 35%, be less than about 30%, be less than about 25%, be less than about 20%, be less than about 15%, be less than about 12%, be less than about 10%, be less than about 8%, be less than about 6%, be less than about 5%, be less than about 4%, be less than about 3%, be less than about 2% or be less than whole cells of about 1%.In other embodiments, in the culture mesoblastema be less than about 90%, be less than about 85%, be less than about 80%, be less than about 75%, be less than about 70%, be less than about 65%, be less than about 60%, be less than about 55%, be less than about 50%, be less than about 45%, be less than about 40%, be less than about 35%, be less than about 30%, be less than about 25%, be less than about 20%, be less than about 15%, be less than about 12%, be less than about 10%, be less than about 8%, be less than about 6%, be less than about 5%, be less than about 4%, be less than about 3%, be less than about 2% or be less than whole cells of about 1%.
Other embodiments of the present invention relate to the composition that comprises the main cell type of the preceding gut entoderm conduct of the PDX1-positive that produces with method described herein, for example cell culture or cell mass.In some embodiments, produce with method described herein comprise at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 94%, at least about 93%, at least about 92%, at least about 91%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, at least about 54%, at least about 53%, at least about 52% or at least about 51% the positive anterior intestine endoderm cell's of PDX1-cell culture and/or cell mass.In the embodiment preferred, the cell of cell culture or cell mass comprises people's cell.In other embodiments, comprise at least about 50% with method generation described herein, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 24%, at least about 23%, at least about 22%, at least about 21%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2% or at least about the positive anterior intestine endoderm cell's of 1% PDX1-cell culture and/or cell mass.In the preferred embodiment, the cell of cell culture or cell mass comprises people's cell.In some embodiments, do not consider to remain in the feeder cell in the culture when calculating the ratio of the positive anterior intestine endoderm cell of PDX1-in cell culture or the cell mass.
Other embodiments of the present invention relate to the composition of the mixture that comprises positive anterior intestine endoderm cell of PDX1-and the negative definitive entoderm cell of PDX1-, for example cell culture or cell mass.For example, can produce and comprise cell culture or the cell mass of the negative definitive entoderm cell of per approximately 95 PDX1-correspondence at least about the positive anterior intestine endoderm cells of 5 PDX1-.In other embodiments, can produce and comprise cell culture or the cell mass of the negative definitive entoderm cell of per approximately 5 PDX1-correspondence at least about the positive anterior intestine endoderm cells of 95 PDX1-.In addition, expection can obtain comprising the cell culture or the cell mass of positive anterior intestine endoderm cell of PDX1-and negative other ratios of definitive entoderm of PDX1-.For example, expection can obtain comprising per approximately 1,000,000 the negative definitive entoderm cell of PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, per approximately 100,000 the negative definitive entoderm cell of PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, per approximately 10,000 the negative definitive entoderm cell of PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, the negative definitive entoderm cell of per approximately 1000 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, the negative definitive entoderm cell of per approximately 500 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, the negative definitive entoderm cell of per approximately 100 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, the negative definitive entoderm cell of per approximately 10 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, the negative definitive entoderm cell of per approximately 5 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, the negative definitive entoderm cell of per approximately 4 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, the negative definitive entoderm cell of per approximately 2 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, the negative definitive entoderm cell of per approximately 1 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1 PDX1-, the negative definitive entoderm cell of per approximately 1 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 2 PDX1-, the negative definitive entoderm cell of per approximately 1 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 4 PDX1-, the negative definitive entoderm cell of per approximately 1 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 5 PDX1-, the negative definitive entoderm cell of per approximately 1 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 10 PDX1-, the negative definitive entoderm cell of per approximately 1 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 20 PDX1-, the negative definitive entoderm cell of per approximately 1 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 50 PDX1-, the negative definitive entoderm cell of per approximately 1 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 100 PDX1-, the negative definitive entoderm cell of per approximately 1 PDX1-correspondence is at least about the positive anterior intestine endoderm cell of 1000 PDX1-, the negative definitive entoderm cell of per approximately 1 PDX1-correspondence is at least about 10,000 positive anterior intestine endoderm cell of PDX1-, the negative definitive entoderm cell of per approximately 1 PDX1-correspondence is at least about 100,000 positive anterior intestine endoderm cell of PDX1-, the negative definitive entoderm cell of per 1 PDX1-of peace treaty correspondence is at least about 1,000,000 positive anterior intestine endoderm cell's of PDX1-composition.
In some embodiments of the present invention, the negative definitive entoderm of PDX1-that is used to produce the positive anterior intestine endoderm cell of PDX1-is cell-derived from people's multipotential cell, for example people's multipotent stem cells.In some embodiment, people's multipotential cell is derived from morula, embryo's inner cell mass or embryo sexual fold.In some other embodiment, people's multipotential cell is derived from growing the gonadal tissue or the germinal tissue of having crossed brephic multi-cellular structure.
Further embodiment of the present invention relates to and comprises people's cell composition of (comprising the preceding gut entoderm of the people PDX1-positive), for example cell culture or cell mass wherein are higher than the expression of AFP, SOX7, SOX1, ZIC1 and/or NFM mark at least about the expression of PDX1 mark in people's cell of 2%.In other embodiments, in people's cell of at least 5%, in people's cell of at least 10%, in people's cell of at least 15%, in people's cell of at least 20%, in people's cell of at least 25%, in people's cell of at least 30%, in people's cell of at least 35%, in people's cell of at least 40%, in people's cell of at least 45%, in people's cell of at least 50%, in people's cell of at least 55%, in people's cell of at least 60%, in people's cell of at least 65%, in people's cell of at least 70%, in people's cell of at least 75%, in people's cell of at least 80%, in people's cell of at least 85%, in people's cell of at least 90%, in people's cell of at least 95% or in people's cell of at least 98%, the expression of PDX1 mark is higher than AFP, SOX7, SOX1, the expression of ZIC1 and/or NFM mark.In some embodiments, do not consider feeder cell when calculating the ratio of people's cell (wherein the expression of PDX1 is higher than AFP, SOX7, SOX1, the expression of ZIC1 and/or NFM) among cell culture or the group.
Should be appreciated that embodiments more of the present invention relate to the composition that comprises the positive anterior intestine endoderm cell of people PDX1-, for example cell culture or cell mass, wherein from least about 2% to surpassing the expression that one or more expression that are selected from SOX17, HOXA13 or HOXC6 people's cell of about 98% are higher than AFP, SOX7, SOX1, ZIC1 and/or NFM mark.In some embodiments, in people's cell of 5%, in people's cell of 10%, in people's cell of 15%, in people's cell of 20%, in people's cell of 25%, in people's cell of 30%, in people's cell of 35%, in people's cell of 40%, in people's cell of 45%, in people's cell of 50%, in people's cell of 55%, in people's cell of 60%, in people's cell of 65%, in people's cell of 70%, in people's cell of 75%, in people's cell of 80%, in people's cell of 85%, in people's cell of 90%, at least about in people's cell of 95% or at least about in 98% the cell, one or more are selected from SOX17, the expression of HOXA13 or HOXC6 is higher than AFP, SOX7, SOX1, the expression of ZIC1 and/or NFM.In some embodiments, do not consider feeder cell when calculating the ratio of people's cell (one or more expression that are selected from SOX17, HOXA13 or HOXC6 are higher than the expression of AFP, SOX7, SOX1, ZIC1 and/or NFM) among cell culture or the group.
Other embodiments of the present invention relate to the composition that comprises Mammals endoderm cell (for example people endoderm cell), for example cell culture or cell mass wherein are higher than the expression of AFP, SOX7, SOX1, ZIC1 and/or NFM mark at least about the expression of PDX1 mark among 2% the endoderm cell.In other embodiments, in 5% endoderm cell, in 10% endoderm cell, in 15% endoderm cell, in 20% endoderm cell, in 25% endoderm cell, in 30% endoderm cell, in 35% endoderm cell, in 40% endoderm cell, in 45% endoderm cell, in 50% endoderm cell, in 55% endoderm cell, in 60% endoderm cell, in 65% endoderm cell, in 70% endoderm cell, in 75% endoderm cell, in 80% endoderm cell, in 85% endoderm cell, in 90% endoderm cell, at least about among 95% the endoderm cell or at least about among 98% the endoderm cell, the expression of PDX1 mark is higher than AFP, SOX7, SOX1, the expression of ZIC1 and/or NFM mark.
Other embodiments of the present invention relate to the composition that comprises Mammals endoderm cell (for example people endoderm cell), for example cell culture or cell mass wherein are higher than the expression of AFP, SOX7, SOX1, ZIC1 and/or NFM mark at least about one or more expression that are selected from the mark of SOX17, HOXA13 and HOXC6 in 2% the embryonic cell.In other embodiments, in 5% endoderm cell, in 10% endoderm cell, in 15% endoderm cell, in 20% endoderm cell, in 25% endoderm cell, in 30% endoderm cell, in 35% endoderm cell, in 40% endoderm cell, in 45% endoderm cell, in 50% endoderm cell, in 55% endoderm cell, in 60% endoderm cell, in 65% endoderm cell, in 70% endoderm cell, in 75% endoderm cell, in 80% endoderm cell, in 85% endoderm cell, in 90% endoderm cell, at least about among 95% the endoderm cell or at least about among 98% the endoderm cell, one or more are selected from SOX17, the expression of the mark of HOXA13 and HOXC6 is higher than AFP, SOX7, SOX1, the expression of ZIC1 and/or NFM mark.
Use method described herein, can produce the composition that comprises the positive anterior intestine endoderm cell of PDX1-that is substantially free of other cell types.In embodiments more of the present invention, the positive anterior intestine endoderm cell group of PDX1-or the cell culture that produce with method described herein are substantially free of remarkable cell of expressing AFP, SOX7, SOX1, ZIC1 and/or NFM marker gene.
In one embodiment of the invention, according to the expression of marker gene being described below the positive anterior intestine endoderm cell of PDX1-: the PDX1 height, AFP is low, SOX7 is low, SOX1 is low, ZIC1 is low and NFM is low.
Increase the expression of PDX1 in the positive definitive entoderm cell of SOX17-
Some aspects of the method for the invention relate to the method that increases PDX1 gene product expression in the cell culture comprise the positive definitive entoderm cell of SOX17-or the cell mass.In this class embodiment, in the positive definitive entoderm cell of SOX17-, add the differentiation factor that is enough to increase PDX1 gene product expression amount.The SOX17-that contacts with differentiation factor is positive, and the definitive entoderm cell can be the PDX1-feminine gender or the PDX1-positive.In some embodiments, differentiation factor can be a retinoid.Among some embodiment, make the positive definitive entoderm cell of SOX17-and the about 0.01 μ M of concentration range extremely the retinoid of about 50 μ M contact.In preferred embodiments, described retinoid is RA.
In other embodiments of the method for the invention, by the positive definitive entoderm cell of SOX17-is contacted increase the expression of PDX1 gene product in the cell culture that comprises SOX17-positive definitive entoderm cell or the cell mass with the differentiation factor of fibroblast growth family.This class differentiation factor can use separately or unite use with RA.In some embodiments, make the positive definitive entoderm cell of SOX17-and the about 10ng/ml of concentration range extremely the fibroblast growth factor of about 1000ng/ml contact.In the preferred embodiment, the FGF somatomedin is FGF-10.
In embodiments more of the present invention, by the SOX17-positive cell is contacted increase the expression of PDX1 gene product in the cell culture that comprises the positive definitive entoderm cell of SOX17-or the cell mass with B27.This class differentiation factor can use separately or with FGF family differentiation factor and retinoid in one or both unite use.In some embodiments, make the positive definitive entoderm cell of SOX17-and about 0.1% (v/v) of concentration range extremely the B27 of about 20% (v/v) contact.In preferred embodiments, the positive definitive entoderm cell of SOX17-is contacted with B27 with RA, FGF-10.
The method that increases PDX1 gene product expression in the cell culture comprise the positive definitive entoderm cell of SOX17-or the cell mass can be carried out in the growth medium that reduces serum-concentration or serum-free.In some embodiments, the serum-concentration scope is from about 0.05% (v/v) to about 20% (v/v).In some embodiments, the positive definitive entoderm cell of SOX17-is grown containing under the condition of serum substitute.
Can promote the evaluation of the factor of definitive endoderm differentiation
Some screening method as herein described relates at least a method that can promote the factor of definitive endoderm differentiation of identifying.In some embodiments of these methods, obtain to comprise the cell mass of definitive endoderm (as people's definitive endoderm).Provide the candidate differentiation factor to cell mass.At very first time point, promptly before providing candidate's differentiation factor or approximately simultaneously, determine the expression of mark to cell mass.Perhaps, can after providing candidate's differentiation factor, determine the expression of mark to cell mass.At second time point, promptly, determine the expression of same mark at very first time point with after cell mass provides the step of candidate's differentiation factor.Determine in the expression of the very first time point and second time point whether candidate's differentiation factor can promote the differentiation of definitive endoderm by comparing mark.Increase or reduce if mark is compared with its expression at very first time point in the expression of second time point, then candidate's differentiation factor can promote the differentiation of definitive endoderm.
Some embodiments of screening method described herein are used cell mass or the cell culture that comprises people's definitive endoderm.For example, this cell mass can be pure basically people's definitive endoderm group.Perhaps, this cell mass can be people's definitive endoderm group of enrichment, wherein in the cell mass at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97% or be people's definitive endoderm more than people's cell at least about 97%.In other embodiment of the present invention, cell mass comprises people's cell, wherein 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% or be people's definitive endoderm more than people's cell at least about 85%.In some embodiments, cell mass comprises inhuman cell, as inhuman feeder cell.In other embodiment, cell mass comprises people's feeder cell.In these embodiments, except that described feeder cell, 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%, at least about 95% or be people's definitive endoderm more than people's cell at least about 95%.In some embodiments of screening method described herein, cell mass further comprises the PDX1-positive entoderm cell, and it includes but not limited to the positive anterior intestine endoderm cell of PDX1-.
In the embodiment of screening method as herein described, make the cell mass contact or provide candidate's (test) differentiation factor to it.Candidate's differentiation factor can comprise any molecule that may promote the differentiation of people's definitive endoderm.In embodiments more described herein, it is the molecule of the differentiation factor of one or more cell types that candidate's differentiation factor comprises known.In other embodiment, candidate's differentiation factor comprises the molecule that the unknown can promote cytodifferentiation.In the preferred embodiment, candidate's differentiation factor comprises the unknown molecule that can promote the differentiation of people's definitive endoderm.
In some embodiments of screening method as herein described, candidate's differentiation factor comprises small molecules.In the preferred embodiment, small molecules is that molecular weight is about or is lower than 10, the molecule of 000amu.In some embodiments, small molecules comprises retinoid.In some embodiments, small molecules comprises vitamin A acid.
In other embodiment as herein described, candidate's differentiation factor comprises polypeptide.This polypeptide can be any polypeptide that includes but not limited to glycoprotein, lipoprotein, extracellular matrix protein, cytokine, chemokine, peptide hormone, interleukin or somatomedin.Preferred polypeptide comprises somatomedin.In the certain preferred embodiments, candidate's differentiation factor comprises one or more somatomedins that is selected from FGF10, FGF4, FGF2 and Wnt3B.
In some embodiments of screening method described herein, candidate's differentiation factor comprises one or more and is selected from amphiregulin, B-lymphocyte stimulatory factors, IL-16, thymopoietin, TRAIL/Apo-2, PBEF, endothelium differentiation associated factor 1 (EDF1), endothelial mononuclear cell activating polypeptide II, macrophage migration inhibition factor (MIF), natural killer cell enhancement factor (NKEFA), Delicious peptide 2, Delicious peptide 8 (bone morphogenic protein 2), Delicious peptide 6, Delicious peptide 7, Connective Tissue Growth Factor (CTGF), CGI-149 albumen (the NE differentiation factor), cytokine A3 (macrophage inflammatory protein 1 alpha), spongioblast differentiation associated protein (GBDR1), the liver cancer derivative growth factor, neuromedin U-25 precursor, vascular endothelial growth factor (VEGF), vascular endothelial growth factor B (VEGF-B), T cell-specific RANTES precursor, thymus gland dendritic cell derived factor-1, Transferrins,iron complexes, il-1 (IL 1), interleukin-2 (IL 2), interleukin-3 (IL 3), interleukin-4 (IL 4), interleukin-5 (IL 5), interleukin 6 (IL-6), interleukin-7 (IL 7), interleukin-8 (IL 8), interleukin-9 (IL 9), interleukin-10 (IL 10), interleukin-11 (IL 11), il-1 2 (IL 12), interleukin-13 (IL 13), granulocyte colony-stimulating factor (G-CSF), rHuGM-CSF (GM-CSF), macrophage colony stimulating factor (M-CSF), erythropoietin, thrombopoietin, vitamins D 3Urogastron (EGF), neurotrophic factor derived from brain, leukaemia inhibitory factor, Triiodothyronine, Prostatropin (bFGF), aFGF, FGF-4, FGF-6, keratinocyte growth factor (KGF), Thr6 PDGF BB (PDGF), Thr6 PDGF BB BB, beta nerve-growth factor, activin A, transforminggrowthfactor-(TGF-β 1), interferon-' alpha ', interferon-beta, interferon-, tumor necrosis factor-alpha, tumour necrosis factor-β, explosion stimulating activity thing (BPA), erythron stimulating activity thing (EPA), PGE 2Insulin-like growth factor (IGF-1), IGF-II, neurotrophin somatomedin (NGF), neurotrophin-3, neurotrophin 4/5, ciliary neurotrophic factor, colloid source property connection albumen (Glial-derivednexin), dexamethasone (Dexamethasone), beta-mercaptoethanol, vitamin A acid, BHA, 5-azacytidine, amphotericin B, xitix, ascorbate salt, the isobutyl-xanthine, indomethacin, beta-glycerophosphate, niacinamide, DMSO, thiazolidinediones, TWS119, pitocin, beta-hypophamine, melanotropin, corticotropin, lipotropin, thyrotropin, tethelin, prolactin antagonist, lutropin, human chorionic gonadotrophin, follicle stimulating hormone, corticotropin releasing factor(CRF), GnRF, prolactin releasing factor, prolactin-inhibitory factor, somatotropin releasing factor, somatostatin, thyrotrophin releasing factor, calcitonin-gene-related peptide, Rat parathyroid hormone 1-34, glucagon-like peptide, glucose dependent insulotropic poly-peptide, gastrin, secretin, cholecystokinin, motilin, vasoactive intestinal peptide, the P material, pancreatic polypeptide, junket junket peptide, junket neuropeptide (neuropeptide tyrosine, neuropeptide tyrosine), Regular Insulin, hyperglycemic-glycogenolytic factor, choriomammotropin, Relaxin, Angiotensin II, calcitriol, Natriuretic factor, atrial, melatonin, thyroxine, triiodothyronine, thyrocalcitonin, estradiol, oestrone, progesterone, testosterone, hydrocortisone, Kendall compound, aldosterone, suprarenin, norepinephrine, androstene (androstene), calcitriol, collagen protein, dexamethasone, beta-mercaptoethanol, vitamin A acid, BHA, 5-azacytidine, amphotericin B, xitix, ascorbate salt, the isobutyl-xanthine, indomethacin, beta-glycerophosphate, niacinamide, DMSO, the somatomedin of thiazolidinedione and TWS119.
In some embodiments of screening method described herein, provide the candidate differentiation factor to cell mass with one or more concentration.In some embodiments, to cell mass provide concentration range that candidate's differentiation factor makes candidate's differentiation factor in the substratum of cell for from about 0.1ng/ml to about 10ng/ml.In some embodiments, the concentration range that centers on candidate's differentiation factor in the substratum of cell is to about 1mg/ml from about 1ng/ml.In other embodiment, the concentration range that centers on candidate's differentiation factor in the substratum of cell is to about 100 μ g/ml from about 10ng/ml.In other embodiment, the concentration range that centers on candidate's differentiation factor in the substratum of cell is to about 10 μ g/ml from about 100ng/ml.In the preferred embodiment, the concentration that centers on candidate's differentiation factor in the substratum of cell is about 5ng/ml, about 25ng/ml, about 50ng/ml, about 75ng/ml, about 100ng/ml, about 125ng/ml, about 150ng/ml, about 175ng/ml, about 200ng/ml, about 225ng/ml, about 250ng/ml, about 275ng/ml, about 300ng/ml, about 325ng/ml, about 350ng/ml, about 375ng/ml, about 400ng/ml, about 425ng/ml, about 450ng/ml, about 475ng/ml, about 500ng/ml, about 525ng/ml, about 550ng/ml, about 575ng/ml, about 600ng/ml, about 625ng/ml, about 650ng/ml, about 675ng/ml, about 700ng/ml, about 725ng/ml, about 750ng/ml, about 775ng/ml, about 800ng/ml, about 825ng/ml, about 850ng/ml, about 875ng/ml, about 900ng/ml, about 925ng/ml, about 950ng/ml, about 975ng/ml, about 1 μ g/ml, about 2 μ g/ml, about 3 μ g/ml, about 4 μ g/ml, about 5 μ g/ml, about 6 μ g/ml, about 7 μ g/ml, about 8 μ g/ml, about 9 μ g/ml, about 10 μ g/ml, about 11 μ g/ml, about 12 μ g/ml, about 13 μ g/ml, about 14 μ g/ml, about 15 μ g/ml, about 16 μ g/ml, about 17 μ g/ml, about 18 μ g/ml, about 19 μ g/ml, about 20 μ g/ml, about 25 μ g/ml, about 50 μ g/ml, about 75 μ g/ml, about 100 μ g/ml, about 125 μ g/ml, about 150 μ g/ml, about 175 μ g/ml, about 200 μ g/ml, about 250 μ g/ml, about 300 μ g/ml, about 350 μ g/ml, about 400 μ g/ml, about 450 μ g/ml, about 500 μ g/ml, about 550 μ g/ml, about 600 μ g/ml, about 650 μ g/ml, about 700 μ g/ml, about 750 μ g/ml, about 800 μ g/ml, about 850 μ g/ml, about 900 μ g/ml, about 950 μ g/ml, about 1000 μ g/ml or greater than 1000 μ g/ml.
In some embodiment of screening method described herein, provide candidate's differentiation factor of any molecule that comprises except that the anterior intestine differentiation factor to cell mass.For example, in some embodiments, provide the candidate's differentiation factor that comprises any molecule except that member, FGF10 or the FGF4 of retinoid, somatomedin TGF beta superfamily to cell mass.In some embodiments, provide candidate's differentiation factor of any molecule that comprises except that vitamin A acid to cell mass.
In some embodiments, the step of screening method described herein comprises determines the expression of at least one mark at the very first time point and second time point.In some embodiments, this point can be before providing candidate's differentiation factor to cell mass or with it approximately simultaneously very first time.Perhaps, in some embodiments, this very first time point is after providing candidate's differentiation factor to cell mass.In some embodiments, determine of the expression of a plurality of marks at very first time point.
Except that determining the expression of at least one mark at very first time point, some embodiments of screening method described herein consider to determine at second time point expression of at least one mark, it is after very first time point, also after providing candidate's differentiation factor to cell mass.In these embodiments, determine of the expression of same mark at the very first time point and second time point.In some embodiments, determine of the expression of a plurality of marks at the very first time point and second time point.In these embodiments, determine of the expression of identical a plurality of marks at the very first time point and second time point.In some embodiments, determine the expression of mark at a plurality of time points, wherein each is all after very first time point, and each is all after providing candidate's differentiation factor to cell mass.In some embodiment, determine the expression of mark with Q-PCR.In other embodiment, determine the expression of mark with immunocytochemistry.
In some embodiment of screening method described herein, determine that with second time point mark of its expression is to be divided into the relevant mark of specific cells with people's definitive endoderm at very first time point, this specific cells is a composition derived from the precursor of the cell of the tissue of intestinal tube and/or organ.In some embodiments, comprise the cell of differentiation eventually derived from the tissue of intestinal tube and/or organ.In some embodiments, mark indication pancreatic cell or pancreas precursor cell.In the preferred embodiment, mark is pancreas-duodenum homology frame factor-1 (PDX1).In other embodiment, mark is homology frame A13 (HOXA13) or homology frame C6 (HOXC6).In addition, in other embodiment, mark indication liver cell or liver precursor.In some preferred embodiment, mark is albumin, liver cell specific antigens (HSA) and Prospero-relevant homology frame 1 (PROX1).In other embodiment, mark indication lung or lung precursor cell.In the certain preferred embodiments, mark is Tiroidina transcription factor 1 (TITF1).In other embodiment, mark indication intestines or intestines precursor cell.In other preferred embodiment, mark is villin, glucose transporter (GLUT2), aPoA 1 (APOA1), vascular cell adhesion molecule (VACM1), vWF ELISA (VMF), CXC type Chemokine Receptors 4 (CXCR4) or tail type homeobox transcription factor 2 (CDX2).In other embodiment, mark indication stomach or stomach precursor cell.In other preferred embodiment, mark is VACM1, VMF or CXCR4.In other embodiment, mark indication Tiroidina or Tiroidina precursor cell.In these embodiments, mark is TITF1.In other embodiment, mark indication thymus gland or postthymic precursor cell.
In some embodiments of screening method described herein, candidate's chemokine is provided and determines at second time point between the expression of marker through the sufficiently long time to cell mass.This provides candidate's chemokine and determines that at second time point time enough between the expression of marker at interval can be from the shortest about 1 hour to being about most 10 days to cell mass.In some embodiments, after providing candidate's chemokine, cell mass repeatedly determines the expression of at least one mark.In some embodiments, this sufficiently long time is at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 30 hours, at least about 36 hours, at least about 42 hours, at least about 48 hours, at least about 54 hours, at least about 60 hours, at least about 66 hours, at least about 72 hours, at least about 78 hours, at least about 84 hours, at least about 90 hours, at least about 96 hours, at least about 102 hours, at least about 108 hours, at least about 114 hours, at least about 120 hours, at least about 126 hours, at least about 132 hours, at least about 138 hours, at least about 144 hours, at least about 150 hours, at least about 156 hours, at least about 162 hours, at least about 168 hours, at least about 174 hours, at least about 180 hours, at least about 186 hours, at least about 192 hours, at least about 198 hours, at least about 204 hours, at least about 210 hours, at least about 216 hours, at least about 222 hours, at least about 228 hours, at least about 234 hours or at least about 240 hours.
In some embodiments of methods described herein, whether further definite mark is compared in the expression of very first time point with this mark in the expression of second time point increases or reduces.The increase of at least one marker expression or minimizing show that candidate's differentiation factor can promote the differentiation of definitive endoderm.Similarly, if determined a plurality of marker expression, whether further definite these a plurality of marks are compared in the expression of very first time point with these a plurality of marks in the expression of second time point increases or reduces.Can be by quantity, level or active increase or the minimizing of determining marker expression of mark in measurement or the assessment cell mass at first and second time points.Thisly determine it can is to compare with other mark (as the expression of house-keeping gene), or absolute.Wherein mark is in some embodiment that the expression ratio of second time point its expression at very first time point has increased, the quantity of increase be at least about 2 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times or more than at least about 100 times.In some embodiments, the quantity of increase is for being less than 2 times.Mark the expression ratio of second time point its in the embodiment of the expression decreased of very first time point, the quantity of minimizing be at least about 2 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times or more than at least about 100 times.In some embodiments, the quantity of minimizing is for being less than 2 times.
In some embodiments of screening method described herein, after cell mass provided candidate's chemokine, people's definitive endoderm was divided into the cell type of one or more definitive endoderm pedigrees.In some embodiments, after cell mass provided candidate's chemokine, people's definitive endoderm was divided into the cell derived from intestinal tube.These cells include but not limited to, the cell of pancreas, liver, lung, stomach, intestines, Tiroidina, thymus gland, pharynx, gall-bladder and bladder and the precursor of these cells.In addition, these cells can further grow for high-grade structure more as tissue and/or organ.
Should understand the screening method similar to aforesaid method and can be used for identifying one or more differentiation factors, this differentiation factor can promote to comprise the differentiation of the people PDX1-positive entoderm cell in the cell mass of people PDX1-positive entoderm cell.In some embodiment, this people PDX1-positive entoderm cell is the positive anterior intestine of PDX1-/midgut endoderm cell.In the preferred embodiment, this people PDX1-positive entoderm cell is the positive anterior intestine endoderm cell of PDX1-.In other preferred embodiment, this people PDX1-positive entoderm cell is the PDX1 positive entoderm cell at anterior intestine rear portion.In the particularly preferred embodiment, this people PDX1-is positive, and the anterior intestine endoderm cell is the multipotential cell that can be divided into derived from cell, tissue or the organ of intestinal tube fore-end.
Can promote the negative definitive entoderm cell of PDX1-to the positive anterior intestine endoderm cell of PDX1-branch The evaluation of the factor of changing
The aspect of the screening method that the present invention describes relates to identifies that one or more can promote the method for the negative definitive entoderm cell of PDX1-to the differentiation factor of the positive anterior intestine endoderm cell differentiation of PDX1-.In these class methods, obtain to comprise the cell culture or the cell mass of the negative definitive entoderm cell of PDX1-, and measure the expression of PDX1 in cell culture or the cell mass.After measuring the PDX1 expression, the cell of cell culture or cell mass is contacted with candidate's differentiation factor.In some embodiments, when cell is contacted with candidate's differentiation factor or with the expression that detects PDX1 after candidate's differentiation factor contacts soon.One or more time points after making cell and candidate's differentiation factor contacts detect the PDX1 expression then.Compare with the expression of PDX1 before contacting candidate's differentiation factor, if the expression of PDX1 increases behind contact candidate differentiation factor, candidate's differentiation factor can be accredited as and can promote the differentiation of the negative definitive entoderm cell of PDX1-to the positive anterior intestine endoderm cell of PDX1-.
In some embodiments, above-mentioned evaluation can promote the negative definitive entoderm cell of PDX1-also to comprise HOXA13 and/or HOXC6 expression of gene in mensuration cell culture or the cell mass to the method for the differentiation factor of the positive anterior intestine endoderm cell differentiation of PDX1-.In this class embodiment, make cell measure HOXA13 and/or HOXC6 expression of gene before and after contacting with candidate's differentiation factor respectively.With contact differentiation factor before the expression of PDX1 and HOXA13 compare, if behind contact candidate differentiation factor, the expression of PDX1 and HOXA13 increases, and just can identify that candidate's differentiation factor can promote the differentiation of the negative definitive entoderm cell of PDX1-to the positive anterior intestine endoderm cell of PDX1-.Similarly, with contact differentiation factor before relatively, if behind contact candidate differentiation factor, the expression of PDX1 and HOXC6 increases, and just can identify that candidate's differentiation factor can promote the differentiation of the negative definitive entoderm cell of PDX1-to PDX1-positive anterior intestine endoderm cell.In preferred embodiments, identify candidate's differentiation factor that can promote that the negative definitive entoderm cell of PDX1-breaks up to the positive anterior intestine endoderm cell of PDX1-by the expression of measuring PDX1, HOXA13 and HOXC6 before and after contacting with candidate's differentiation factor at the cell that makes cell culture or cell mass respectively.In the preferred embodiment, detect the expression of PDX1, HOXA13 and HOXC6 with Q-PCR.
Should be appreciated that in some embodiments, soon detect among PDX1, HOXA13 and the HOXC6 one or more expression when the cell in making cell culture or cell mass contacts with candidate's differentiation factor or behind contact candidate differentiation factor, rather than before cells contacting candidate differentiation factor, detect.In this class embodiment, will be when cell be contacted with candidate's differentiation factor or behind contact candidate differentiation factor the expression of one or more among PDX1, HOXA13 and the HOXC6 soon compare with PDX1, the HOXA13 of one or more time points behind cells contacting candidate differentiation factor and the one or more expression among the HOXC6.
In some embodiments of aforesaid method, the scope that contacts one or more time points of back detection PDX1 expression at cell with candidate's differentiation factor can be from about 1 hour to about 10 days.For example, can contact about 1 hour of back with candidate's differentiation factor at cell, cell contacts about 2 hours of back with candidate's differentiation factor, cell contacts about 4 hours of back with candidate's differentiation factor, cell contacts about 6 hours of back with candidate's differentiation factor, cell contacts about 8 hours of back with candidate's differentiation factor, cell contacts about 10 hours of back with candidate's differentiation factor, cell contacts about 12 hours of back with candidate's differentiation factor, cell contacts about 16 hours of back with candidate's differentiation factor, cell contacts about 24 hours of back with candidate's differentiation factor, cell contacts about 2 days of back with candidate's differentiation factor, cell contacts about 3 days of back with candidate's differentiation factor, cell contacts about 4 days of back with candidate's differentiation factor, cell contacts about 5 days of back with candidate's differentiation factor, cell contacts about 6 days of back with candidate's differentiation factor, cell contacts about 7 days of back with candidate's differentiation factor, cell contacts about 8 days of back with candidate's differentiation factor, cell contacts about 9 days of back with candidate's differentiation factor, cell contacts about 10 days of back with candidate's differentiation factor or cell contacts the expression that the back surpasses 10 days detection PDX1 with candidate's differentiation factor.
The candidate's differentiation factor that is used for method described herein can be selected from compound, for example polypeptide and small molecules.For example, candidate's polypeptide includes but not limited to somatomedin, cytokine, chemokine, extracellular matrix protein and synthetic peptide.In preferred embodiments, somatomedin is from FGF family, for example FGF-10.Candidate's small molecules includes but not limited to from combinatorial chemistry synthetic compound and natural product, for example steroid, isoprenoid, terpenoid, class phenyl-propane (phenylpropanoid), alkaloids and flavonoid.It will be understood by a person skilled in the art that thousands of kinds of natural and synthetic small molecules can utilize, the small molecules that expection can be used for method described herein is not limited to above kind of giving an example.Typically, micromolecular molecular weight is lower than 10,000amu.In preferred embodiments, small molecules is a retinoid, for example RA. Can promote the evaluation of the factor of the positive anterior intestine endoderm cell differentiation of PDX1-
Other aspects of screening method of the present invention relate to the method for identifying that one or more can promote the differentiation factor of the positive anterior intestine endoderm cell differentiation of PDX1-.In these class methods, obtain to comprise the positive anterior intestine endoderm cell's of PDX1-cell culture or cell mass, and measure the expression of mark in cell culture or the cell mass.After the expression of measuring mark, the cell of cell culture or cell mass is contacted with candidate's differentiation factor.In some embodiments, when cell is contacted with candidate's differentiation factor or behind contact candidate differentiation factor, detect the expression of mark soon.One or more time points after the contact of cell and candidate's differentiation factor detect the expression of identical mark then.With contact differentiation factor before relatively, if behind contact candidate differentiation factor, the expression of mark increases or reduces, and just can identify that candidate's differentiation factor can promote PDX1-positive anterior intestine endoderm cell's differentiation.In preferred embodiments, detect the expression of mark with Q-PCR.
In some embodiments of aforesaid method, the scope that detects one or more time points of marker expression after cell is contacted with candidate's differentiation factor can be from about 1 hour to about 10 days.For example, can make cell contact about 1 hour of back with candidate's differentiation factor, make cell contact about 2 hours of back with candidate's differentiation factor, make cell contact about 4 hours of back with candidate's differentiation factor, make cell contact about 6 hours of back with candidate's differentiation factor, make cell contact about 8 hours of back with candidate's differentiation factor, make cell contact about 10 hours of back with candidate's differentiation factor, make cell contact about 12 hours of back with candidate's differentiation factor, make cell contact about 16 hours of back with candidate's differentiation factor, make cell contact about 24 hours of back with candidate's differentiation factor, make cell contact about 2 days of back with candidate's differentiation factor, make cell contact about 3 days of back with candidate's differentiation factor, make cell contact about 4 days of back with candidate's differentiation factor, make cell contact about 5 days of back with candidate's differentiation factor, make cell contact about 6 days of back with candidate's differentiation factor, make cell contact about 7 days of back with candidate's differentiation factor, make cell contact about 8 days of back with candidate's differentiation factor, make cell contact about 9 days of back with candidate's differentiation factor, make cell contact about 10 days of back with candidate's differentiation factor or make cell contact the back with candidate's differentiation factor to surpass the expression that detected mark in 10 days.
As previously described, the candidate's differentiation factor that is used for method described herein can be selected from for example polypeptide or micromolecular compound.
Although every kind of method disclosed herein is all about the positive anterior intestine endoderm cell of PDX1-, should be appreciated that in certain embodiments this method can be used for producing the composition of the PDX1-positive entoderm cell that comprises the positive anterior intestine of PDX1-described herein/midgut endoderm cell and/or anterior intestine described herein rear portion.In addition, disclosed any PDX1-positive entoderm cell type all can be used in the screening method described herein in this specification sheets.
Above general description the present invention, can be by further understanding the present invention with reference to some specific embodiment provided herein, these embodiment only are in order to illustrate rather than limit the present invention.
Embodiment
Below a lot of embodiment the use of people's multipotential cell has been described.The method of generation people multipotential cell is known in the art, and in many scientific publication things description is arranged all, comprises the 5th, 453,357,5,670,372,5,690,926,6,090,622,6,200,806 and 6,251, No. 671 United States Patent (USP)s and publication number are 2004/0229350 U.S. Patent application.
Embodiment 1
People ES cell
As if grow for the research entoderm, we have adopted the versatility human embryo stem cell, can infinitely divide in substratum and keep normal caryogram simultaneously.Adopt immunology or mechanical means to separate, obtain the ES cell from embryo's inner cell masses of 5 days.Especially, human embryonic stem cell hESCyt-25 obtains from the unnecessary frozen embryo in cycle in vitro fertilization after the patient agrees.After thawing, the blastocyst of hatching is planted on the mouse embryo fibroblasts (MEF) in the ES substratum (DMEM, 20%FBS, nonessential amino acid, beta-mercaptoethanol, ITS additive).The embryo sticks on the culture dish, and after about two weeks, undifferentiated hESC zone is transferred in the new culture dish that contains MEF.Shift to adopt the simple digestion of mechanical shearing and Dispase, remove cell cluster with mechanical force then, clean and repopulating cell again.After obtaining, hESCyt-25 goes down to posterity through a series of about 100 times.We utilize human embryonic stem cell hESCyt-25 as the parent material that produces definitive entoderm.
Described those skilled in the art should be appreciated that stem cell or other multipotential cells also can be used as the parent material of differentiation step described herein.The parent material that for example, can be used as multipotential cell by the isolating cell that derives from embryo sexual fold of method as known in the art.
Embodiment 2
The feature of hESCyt-25
Human embryonic stem cell hESCyt-25 cultivates in substratum and still keeps normal form, caryogram, growth and self character after 18 months.This clone all shows very strong immunoreactivity (these several antigens all are the characteristic antigen that does not break up hESC), shows alkaline phosphatase activities and be similar form to hESC that other has been set up OCT4, SSEA-4 and TRA-1-60 antigen.In addition, human embryonic stem cell hESCyt-25 forms embryoid (EB) easily when suspension culture.HESCyt-25 can be divided into the different cell types of representing three kinds of main germinal layers, versatility that this has shown hESCyt-25.To the Q-PCR of ZIC1 detect and to immunocytochemistry (ICC) assay certificate of nestin and the ripe neural mark of Geng Duo ectodermic generation.Observed the immunocytochemical stain of β-III tubulin in extending cell cluster, this is early stage neuronic feature.Before, we are with the EB in the retinoic acid treatments suspension, with inducing pluripotent stem cells clone differentiation outside internal organ entoderm (VE), embryo.After handling in 54 hours, the mark of treated cell great expression VE: α-Jia Taidanbai (AFP) and SOX7.Immunocytochemical stain shows that cell is expressed in the monolayer of AFP and breaks up in fragmentary piece.To describe below, when no AFP expressed, the real-time quantitative polymerase chain reaction (Q-PCR) of SOX-17 and immunocytochemistry detect proof hESCyt-25 clone can also form definitive entoderm.Different time points detected among the EB of differentiation the Brachyury expression of gene with proof to mesoblastic differentiation.Brachyury expresses gradually and improves in experimentation.In sum, hESCyt-25 clone has the ability that forms the cell of representing three kinds of germinal layers, is versatility therefore.
Embodiment 3
The preparation of SOX17 antibody
The major obstacle of identifying definitive entoderm in the hESC culture lacks proper tools exactly, so we have prepared the proteic antibody of anti-people SOX17.
Definitive entoderm is after the gastrula formation phase produces, and all definitive entoderms are all expressed mark SOX17, and being expressed in the intestinal tube of it continues (although expression level changes along the A-P axle) until organogenetic beginning.SOX17 also expresses in the extraembryonic endoderm cell subsets.In mesoderm or ectoderm, do not find this proteic expression.Find that now SOX17 is the suitable landmarks thing of definitive entoderm clone when uniting use with the mark of getting rid of clone outside the embryo.
As describe in detail herein, it is to produce the different treatment method of the positive definitive entoderm cell of SOX17 and the effect of differentiation step that SOX17 antibody is used for special testing goal.Be used to get rid of the generation of internal organ and body wall entoderm (extraembryonic endoderm) with other antibody of AFP, SPARC and thrombomodulin reaction.
In order to produce the antibody of anti-SOX17, at the GENOVAC (Fu Laibeige of Antibody Preparation company, Germany) according to the step of its exploitation, the groups of people SOX17 cDNA (SEQ ID NO:1) corresponding with SOX17 protein carboxyl terminal amino acid/11 72-414 (SEQ ID NO:2) (Fig. 2) is used to the inherited immunity of rat.The inherited immunity step can be referring to the 5th, 830, and 876,5,817,637,6,165,993 and 6,261, No. 281 United States Patent (USP)s, and publication number is the international patent application of WO00/29442 and WO 99/13915.
Other usability methods of inherited immunity also have description in non-patent literature.For example, people such as Barry are at Biotechniques 16:616-620, have described with the inherited immunity method producing monoclonal antibody in 1994.Below be the specific examples for preparing differential protein antibody with the inherited immunity method: as people such as Costaglia, (1998) (inherited immunity of anti-thyroid stimulating hormone receptor causes thyroiditis to Genetic immunization against the humanthyrotropin receptor causes thyroiditis and allows production ofmonoclonal antibodies recognizing the native receptor, and can produce the monoclonal antibody of discerning natural receptor), J.Immunol.160:1458-1465; People such as Kilpatrick, (1998) Gene gundelivered DNA-based immunizatiohs mediate rapid production of murinemonoclonal antibodies to the Flt-3 receptor (the quick generation that particle gun is carried) based on the mouse monoclonal antibody of the immunization metering needle antagonism Flt-3 acceptor of DNA, Hybridoma 17:569-576; People such as Schmolke (1998) Identification ofhepatitis G virus particles in human serum by E2-specific monoclonalantibodies generated by DNA immunization (hepatitis G virus particle in the E2-specific monoclonal antibody identifier serum that dna immunization produces), J.Virol.72:4541-4545; People such as Krasemann (1999) Generation of monoclonal antibodies againstproteins with an unconventional nucleic acid-based immunizationstrategy (utilizing unconventional immunization strategy to produce) at proteic monoclonal antibody based on Nucleotide, J.Biotechnol.73:119-129; With people (1996) Generation ofa monoclonal antibody to a defined portion of the Heliobacter pylorivacuolating cytotoxin by DNA immunization (producing the monoclonal antibody of anti-Heliobacter pylorivacuolating intracellular toxin specified portions by dna immunization) J.Biotechnol.51:191-194 such as Ulivieri.
Concern shown in the dendrogram that as Fig. 3 SOX7 and SOX18 are the family members nearest with the SOX17 relation in Sox family.We utilize people SOX7 polypeptide special to SOX17 as the SOX17 antibody that negative control proof inherited immunity produces, and not nearest with its relation family member's reaction.Particularly, SOX7 and other albumen are expressed in the human fibroblasts, pass through the cross reactivity of Westem trace and ICC methods analyst and SOX17 antibody then.For example, following method is used for producing SOX17, SOX7 and EGFP expression vector, with carrier transfection human fibroblasts, carries out the Western engram analysis then.The expression vector that is used to produce SOX17, SOX7 and EGFP is respectively pCMV6 (OriGene Technologies, Inc., Rockville, MD), pCMV-SPORT6 (Invitrogen, Carlsbad, CA) and pEGFP-N1 (Clonetech, Palo Alto, CA).Be preparation albumen, super coiled DNA adopts Lipofectamine 2000 (Invitrogen, Carlsbad, CA) the telomerase immortalized MDX human fibroblasts of transient transfection.Transfection after 36 hours at 50mM TRIS-HCl (pH8), 150mM NaCl, 0.1%SDS, 0.5% Septochol, contain mixture (the Roche Diagnostics Corporation of proteinase inhibitor, Indianapolis, IN) the whole lysates of middle collection.Western engram analysis step is as follows: 100 μ g cell proteins are at NuPAGE (4-12% gradient polyacrylamide, Invitrogen, Carlsbad, CA) going up SDS-PAGE separates, electricity forwards PDVF film (Hercules to, CA), rat SOX17 antiserum(antisera) 10mM TRIS-HCl (pH8), 150mM NaCl, 10%BSA, 0.05%Tween-20 (Sigma, St.Louis, MO) 1/1000 dilution is as probe, use mouse IgG (the Jackson ImmunoResearch Laboratories of the alkaline phosphatase link coupled Chinese People's Anti-Japanese Military and Political College then, West Grove PA) is hatched, and uses Vector Black alkaline phosphatase staining (Vector Laboratories at last, Burlingame CA) manifests.Used molecular weight of albumen standard be wide region color-coded thing (Sigma, St.Louis, MO).
Among Fig. 4, from transient transfection human fibroblasts's the protein extract of SOX17, SOX7 or EGFP cDNA carry out the Westem trace with SOX17 antibody and detect.Have only transfection the protein extract of cell of hSOX17 produce a band at～51Kda, very approaching with the proteic molecular weight 46Kda of prediction people SOX17.SOX17 antibody with from transfection the extract of cell of people SOX7 or EGFP do not have reactivity.In addition, SOX17 antibody clearly mark transfection hSOX17 express the human fibroblasts's of member nucleus, but the cell of EGFP that do not had a mark transfection.Equally, ICC detects the specificity that has also shown SOX17 antibody.
Embodiment 4
SOX17 antibody is as the confirmation of definitive entoderm mark
The hESC of part differentiation is specific to SOX17 albumen to show SOX17 antibody, the step mark definitive entoderm of going forward side by side with SOX17 and the same tense marker of AFP antibody.Proved that SOX17, SOX7 (the nearest member of relation of SOX gene family subgroup F) and AFP all have expression in the internal organ entoderm.But, in the definitive entoderm cell, do not find the expression of AFP and SOX7 at the ICC detection level, therefore, they can be used as the negative mark of bonifide definitive entoderm cell.Show that the SOX17 antibody labeling exists as discrete cell cluster form or with AFP positive cell blended cell mass.Particularly, Fig. 5 A shows that minority SOX17 cell is by the common mark of AFP; But, at SOX17 +Also found in the cell scope to be close to and do not had or do not had at all AFP +The zone of cell (Fig. 5 B).Similarly, because it is reported that the body wall entoderm expresses SOX17, can identify with the common mark of SOX-17 antibody with body wall mark SPARC and/or thrombomodulin (TM) to belong to the endoblastic SOX17 of body wall +Cell.Shown in Fig. 6 A-C, thrombomodulin and SOX-17 are total to the at random differentiation generation of the body wall endoderm cell of mark by the hES cell.
To sum up the cell marking experiment is described, by mark feature SOX17 Hi/ AFP Lo/ [TM LoOr SPARC Lo] can set up the method for identifying the definitive entoderm cell.In other words, the expression of SOX17 mark is higher than the expression of AFP mark, and this is the endoblastic features of internal organ, and TM or SPARC mark are the endoblastic features of body wall.Therefore, those SOX17 positives, and AFP cell negative and TM or SPARC feminine gender is the definitive entoderm cell.
Confirm mark feature SOX17 in order to obtain further evidence Hi/ AFP Lo/ TM LoSPARC LoCan predict definitive entoderm, make the cell of the antibody labeling of SOX17 and AFP expression of gene and respective numbers carry out quantitative comparison.Shown in Fig. 7 A, the hESC through vitamin A acid (internal organ entoderm inductor) or activin A (definitive entoderm inductor) processing has 10 times of differences on the SOX17mRNA expression level.This result has reflected 10 times of differences (Fig. 7 B) of SOX17 antibody labeling cell number.In addition, shown in Fig. 8 A, the activated plain A of hESC handles, and with undressed comparison, can suppress 6.8 times of AFP genetic expressions.Shown in Fig. 8 B-C in the culture the remarkable minimizing of the quantity of AFP labeled cell intuitively reflected this point.For further quantitatively, detect (Fig. 9 A-B) through fluidic cell and confirm that it is that AFP antibody labeling cell number reduces about 7 times result that AFP genetic expression reduces about 7 times.This result is extremely important, because the change that it shows the gene expression amount that Q-PCR shows has reflected the variation of cell type, this can observe by antibody staining.
After hatching, hESC and Nodal family member (Nodal, activin A and activin B-NAA) pass the remarkable increase that causes SOX17 antibody labeling cell in time.Can be labeled as SOX17 (Figure 10 A-F) above 50% cell after passing through the continuous 5 days processing of activin.And activated plain to handle after 5 days almost seldom or do not have cell marking be AFP.
In brief, 242 amino acid whose antibody of anti-people SOX17 protein carboxyl terminal of generation have been identified people SOX17 albumen in the Western trace, but nonrecognition SOX17 immediate member SOX7 in Sox family.Subgroup in the hESC culture that the SOX17 antibody recognition is being broken up, they mainly are SOX17 +/ AFP Lo/-(surpassing 95% labeled cell) and small part (＜5%) are total to the cell (internal organ entoderm) of mark SOX17 and AFP.Handle the hESC culture with activin and cause the remarkable rising of SOX17 genetic expression and the obvious increase of SOX17 labeled cell, and significantly suppress the expression of AFP mRNA, reduce the number of AFP antibody labeling cell.
Embodiment 5
The Q-PCR gene expression analysis
In following experiment, real-time quantitative RT-PCR (Q-PCR) is the main analytical procedure of hESC differentiation being carried out detecting after the different treatment effect.Especially, the real-time detection of genetic expression is at the multiple marker gene of different time point analysis with Q-PCR.Characteristic mark to cell type expectation and that do not expect is assessed, so that better understand the overall dynamics of cell mass.The advantage that Q-PCR analyzes is its extreme susceptibility and newly-increased convenient relatively must mark the time, because genome sequence can obtain.In addition, the high susceptibility of Q-PCR can detect fewer relatively purpose gene expression of cells in the very large cell mass.And the ability that detects extremely low-level genetic expression is that " the differentiation deflection " of cell mass provides indication.At cell phenotype significantly before the differentiation, can not detect to the deflection immunocytochemical technique of specific differentiation pathway.For this reason, Q-PCR provides a kind of analytical procedure, and this method is replenishing of immunocytochemical technique concerning being used to screen successful differentiation handling at least, and may more be better than it.In addition, Q-PCR provides a kind of mechanism, and whether this mechanism can utilize half high-throughput quantification assay Analytical Chemical Experiment design successful.
The method of Cai Yonging is for utilizing SYBR Green chemistry and two one step RT-PCR patterns to carry out relative quantification on Rotor Gene 3000 devices (Corbett Research) herein.This method allows to preserve the gene that the cDNA sample is used for analyzing in the future other marks, has avoided the difference of sample room reverse transcription efficient.
According to removing from the experience of the genomic dna amplified production that pollutes, the primer of design should be positioned at exon-exon border, perhaps as being across to the intron of few 800bp.When the marker gene that uses did not contain intron or has pseudogene, RNA sample application DNase I handled.
We detect the genetic expression of the multiple mark of target cell and non-target cell type usually with Q-PCR, thereby produce the gene expression profile in the cell sample.It is right that table 1 has been listed mark and the confirmed available primer relevant with hESC differentiation early stage (particularly ectoderm, mesoderm, definitive entoderm and extraembryonic endoderm).The right human specific of verified these primers, this is extremely important, because hESC grows on the mouse feeder layer usually.The most representative is that every kind of condition is got three samples, independently carries out twice analysis then, with assessment and each relevant biology difference of quantitative assay.
For producing pcr template, separate total RNA and quantitative with RiboGreen (Molecular Probes) with RNeasy (Qiagen).The reverse transcription of the total RNA of 350-500ng uses iScript reversed transcriptive enzyme test kit (BioRad) to carry out, and test kit contains the mixture of oligo-dT and random primer.Per 20 μ L reaction solutions are diluted to cumulative volume 100 μ L, and 3 μ L are used for per 10 μ L Q-PCR reactions (containing 400nM forward and reverse primer and 5 μ L, 2 * SYBR Green mastermix (Qiagen)) then.Two step loop parameters are as follows: 85-94 ℃ of sex change 5 seconds (specifically according to every kind of primer the melting temp of amplicon being selected); 60 ℃ of annealing/extensions 45 seconds.Back 15 seconds collection fluorescence datas at each extended peroid.Each some triplicate produces the typical curve of each runs with 10 times of gradient dilutions series, based on this typical curve cycle threshold (Ct ' s) is converted into quantitative values then.The quantitative values of each sample is proofreaied and correct with the house-keeping gene result, calculates the average and standard deviation of three duplicate samples.When the PCR circulation is finished, carry out the specificity that reaction is determined in the melt curve analysis analysis.Single special product is by the T that is suitable for pcr amplification mThe simple spike at place is represented.In addition,, do not increase as negative control with the reaction that do not contain reversed transcriptive enzyme.
The first step of setting up the Q-PCR method is the suitable house-keeping gene (HG) of conclusive evidence in experimental system.HG through sample RNA add, the correction of RNA integrity and RT efficient, therefore the expression that importantly HG keeps maintenance level in all samples type is like this proofreaied and correct just meaningful.We have detected the expression of cyclophilin G among the hESC that is breaking up (Cyclophilin G), hypoxanthine phosphoribosyltransferase 1 (HPRT), beta-2-microglobulin, hydroxymethylbianesynthase (HMBS), TATA-conjugated protein (TBP) and glucuronidase β (GUS).Our result shows that the expression level of beta-2-microglobulin increases in atomization, so we get rid of this gene and are used for proofreading and correct.Other genes all keep stable at the expression level of the differentiation phase and the phase of processing.We calculate the correction factor of all samples usually with cyclophilin G and GUS.Use multiple HG to reduce the fluctuation of trimming process self simultaneously, thereby improved the reliability of relative genetic expression numerical value.
After acquisition is used for gauged gene, determine the relative gene expression dose of the marker gene of the sample that the process different experiments is handled with Q-PCR.Selecting these marker genes is that what especially pay close attention to is those gene sets at definitive entoderm and extraembryonic endoderm differential expression because of their content height in the cell mass of the early stage germinal layer of representative.These genes and their relative enrichment condition are pointed out in table 1.
Table 1
Germinal layer Gene The expression scope
Entoderm mbryonic ectoderm mesoderm ?SOX17 ?MIXL1 ?GATA4 ?HNF3b ?GSC ?SOX7 ?AFP ?SPARC ?TM ?ZIC1 ?BRACH Setting, internal organ and body wall entoderm entoderm and mesoderm setting and primitive endoderm definitive entoderm and primitive endoderm, mesoderm, neural plate entoderm and mesoderm internal organ entoderm internal organ entoderm, liver body wall entoderm body wall entoderm/trophectoderm neurocele, the newborn mesoderm of neural precursor
Because many genes are expressed on a more than germinal layer, so be necessary the expression level of quantitative comparison several genes in identical experiment.SOX17 expresses in definitive entoderm, expresses in internal organ and body wall entoderm and will hang down.SOX7 and AFP express in growing early stage internal organ entoderm.SPARC and TM express at the body wall entoderm, and Brauchyury mesoderm in early days expresses.
Definitive entoderm be it is predicted and expressed high-caliber SOX17mRNA and low-level AFP and SOX7 (internal organ entoderm), SPARC (body wall entoderm) and Brachyury (mesoderm).In addition, further get rid of early stage ectodermic inducing with ZIC1 herein.At last, GATA4 and HNF3b all express at definitive entoderm and extraembryonic endoderm, therefore with SOX17 at the expression of definitive entoderm be related (table 1).Representational experiment is shown in Figure 11-14, and it has proved how the marker gene that table 1 is described connects each other in different samples, thereby has given prominence to definitive entoderm and extraembryonic endoderm and to the specific differentiation model of mesoderm and neural cell type.
Comprehensive above data can know that the dosage increase that draws activin causes SOX17 genetic expression to improve.And this SOX17 expresses main represent definitive entoderm rather than extraembryonic endoderm.This conclusion is derived from the genetic expression negative correlation of observing SOX17 genetic expression and AFP, SOX7 and SPARC.
Embodiment 6
People ES cell is to the directed differentiation of definitive entoderm
If not cultivating under the condition of effectively keeping the cell undifferentiated state, differentiation at random will take place in people ES cell.This heterogeneous differentiation produces the extraembryonic endoderm cell that comprises body wall and internal organ entoderm (expressing AFP, SPARC and SOX7), and the early stage ectoderm and the mesoderm derivative that are expressed as mark with ZIC1 and Nestin (ectoderm) and Brachyury (mesoderm).Because lack the specific antibody mark in the ES cell culture, the definitive entoderm cellular form does not have detected or describes in detail.Similarly, because the shortage means are not furtherd investigate the generation of early stage definitive entoderm in the ES cell culture.Because there is not the antibody reagent of the suitable anti-definitive entoderm cell of available, the most of evaluation concentrates on ectoderm and extraembryonic endoderm.Generally speaking, in the ES cell culture of differentiation at random, the number of the outer and neuroderm cell type of embryo is much larger than SOX17 HiThe number of definitive entoderm cell.
When undifferentiated hESC is cloned in into when increasing on the fiber feeder layer, the cell at clone edge shows the another kind of form different with cloning inner cell.So these okioplasts are easily distinguished because the expression of heterogeneity, bigger form and OCT4 is higher.The existing description, when the ES cell begins differentiation phase, the undifferentiated relatively ES cell of the expression level of OCT4 rises or descends.If the change that the OCT4 level rises or descends may not show from the transformation of multipotency state to the differentiation starting stage above breaking up threshold value.
When detecting undifferentiated clone, can find the little cell cluster that contains 10-15 SOX17 positive cell at undifferentiated hESC clone's the edge and the random site of intersection sometimes with the SOX17 immunocytochemistry.As mentioned above, the cell dispersion group at these peripheral clone edges may be that clone size this moment increases, becomes denser from first cell of classical ES cellular form differentiation.More early stage, littler complete undifferentiated clone (＜1mm; 4-5 days ages) do not contain the SOX17 positive cell in clone inside or edge, and more late period, bigger clone's (diameter 1-2mm,＞5 day age) have the fragmentary SOX17 positive, AFP negative cells to distribute in the peripheral or above-mentioned inside, edge of typical hESC form that do not show of some clones.Because this is to develop effective SOX17 antibody for the first time, produces the definitive entoderm cell in " not differentiation " ES cell culture in early days and never be proved in the past.
Because Q-PCR determines SOX17 and SPARC genetic expression negative correlation, the most SOX17 positive, AFP negative cells will be to the mark reactions that is negative altogether of the antibody of body wall entoderm mark.This body wall endoderm cell in Expression of TM obtains special proof (Figure 15 A-B).Contact the number that can reduce TM expression and TM positive cell with B with Nodal factor activin A.The culture that activin is handled with SOX17, AFP and TM antibody labeling after, shown in Figure 16 A-D, observe the SOX17 positive cell bunch of AFP and TM feminine gender.This is first at the SOX17 positive definitive entoderm cell of proof on the cell levels in differentiation hESC culture.
We have designed some steps and can effectively hESC be divided into SOX17 with above-mentioned SOX17 antibody and Q-PCR method Hi/ AFP Lo/ SPARC/TM LoThe definitive entoderm cell.We adopt number and the multiplication capacity of different differentiation methods to improve these cells, and this is by measuring SOX17 genetic expression in group's level with Q-PCR and detecting with antibody labeling SOX17 in the individual cells level.
We (analyze and describe from the effect that embryonic stem cell produces the application of definitive entoderm cell in the vitro culture thing as Nodal/ activin/BMP) TGF 'beta ' family somatomedin first.In representativeness experiment, the combination of activin A, activin B, BMP or these factors is added into not among the differentiation of human stem cell line hESCyt-25 with initial atomization.
As shown in figure 19, adding 100ng/ml activin A causes undifferentiated relatively hESC to induce 19 times SOX17 genetic expression when breaking up the 4th day.Add activin B (second member of activin family) simultaneously and cause the relative hESC37 of differentiation times inducing in the time of the 4th day in the plain back of handling of admixture activation with activin A.At last, add the 3rd member BMP4 from the TGF 'beta ' family of Nodal/ activin and BMP subtribe, unite use with activin A and activin B, causing not breaking up hESC relatively increases by induce (Figure 19) of 57 times.When inducing SOX17 and no factor substratum to compare with activin and BMP, 5,10 and 15 times of appearance induces in the time of 4 days.When using activin A, B and the triple processing of BMP after 5 days, the derivative level of SOX17 is higher 70 times than not breaking up hESC.These data show that can increase SOX17 with high dosage and long Nodal/ activin TGF 'beta ' family member processing expresses.
In vivo or external Nodal and associated molecule activin A, B and BMP promotes SOX17 to express and definitive entoderm forms.In addition, adding BMP and can promote the SOX17 abduction delivering, may be because further induced Nodal acceptor Cripto.
We have proved that the use of uniting of BMP4 and activin A and B causes the extra increase of SOX17 and the formation of definitive entoderm.In activin A and B mixture, add the BMP4 long period (＞4 days) and can induce SOX17 at body wall and internal organ entoderm and definitive entoderm.Therefore in the some embodiments of the present invention, handle in 4 days and be necessary to remove BMP4 adding BMP4.
Be the effect of handling in the individual cells level detection TGF β factor, detected the time-histories that the TGF β factor is added with the SOX17 antibody labeling.As shown in Figure 10 A-F, the relative number that prolongs the SOX17 labeled cell in time significantly increases as before.Relative quantification (Figure 20) shows that the SOX17 labeled cell increases above 20 times.This result shows that cell number and SOX17 gene expression dose increased with the TGF β factor treatment time.As shown in figure 21, using Nodal, activin A, activin B and BMP handled after 4 days, and the SOX17 level of inducing reaches 168 times that do not break up hESC.Figure 22 shows that the relative number of SOX17 positive cell is also relevant with dosage.100ng/ml or more dosage can effectively be induced SOX17 genetic expression, increase cell number.
Except TGF 'beta ' family member, the Wnt family molecule works in may and/or keeping in the special differentiation of definitive entoderm.The use of Wnt molecule also helps the differentiation of hESC to definitive entoderm, as shown in figure 23, compares with independent use activin, adds Wnt3a with activin and handles that the SOX17 expression of gene increases in the sample of back.
The tissue culture medium (TCM) of the factor that comprises 10% serum and interpolation is all used in all above-mentioned experiments.Surprisingly, we find that serum-concentration is influential to the expression level of SOX17 under the situation of adding activin.When serum level when 10% drops to 2%, under the condition that contains activin A and B, the expression of SOX17 increases by 3 times.
At last, we prove activin inductive SOX17 shown in Figure 25 A-D +Cell divides in substratum.Arrow shows the cell of the SOX17/PCNA/DAPI mark that is in m period, and this is by the mitotic division plate mode of PCNA/DAPI mark and differs the confirmation of mitotic division feature.
Embodiment 7
It is relevant with the mark of definitive entoderm that Chemokine Receptors 4 (CXCR4) is expressed, and with middle embryo Layer, ectoderm or the endoblastic mark of internal organ are irrelevant
As mentioned above, use the cytokine of TGF 'beta ' family, especially activin/nodal subtribe can induce hESC to be divided into definitive entoderm.In addition, we show that foetal calf serum (FBS) the level affects hESC of division culture medium is divided into the efficient of definitive entoderm.This influence is that activin A is under the given concentration in substratum, and high-caliber FBS can suppress the at utmost differentiation to definitive entoderm.As lack the activin A of external source, and hESC is very low to the differentiation efficiency of definitive entoderm system, and the influence of FBS in the hESCs atomization also reduces.
In these experiments, hESC is containing 0.5%, 2.0% or RPMI substratum (Invitrogen, Carlsbad, the CA of 10%FBS; Cat#61870-036) Growth and Differentiation in is wherein added or is not added 100ng/ml activin A and cultivated 6 days.In addition, preceding 3 days FBS gradient and 100ng/ml activin A with 0.5%-2.0% in differentiation unite use.After 6 days, every kind of culture condition is collected the double sample, with the relative genetic expression of real-time quantitative PCR analysis.Remaining cell is fixed for the proteic immunofluorescence of SOX17 and detects.
According to 7 kinds of culture condition that use, the expression level of CXCR4 changes (Figure 26) very greatly.In general, in the culture (A100) that activin A handles CXCR4 express higher, if do not contain external source activin A (NF) then CXCR4 expresses lower.In addition, in the substratum that A100 handles, CXCR4 expresses the highest when FBS concentration is minimum.The CXCR4 level has remarkable reduction under the 10%FBS condition, so the relative expression meets the condition that does not contain activin A (NF) more.
As mentioned above, SOX17, GSC, MIXL1 are consistent with the feature of definitive entoderm cell with HNF3 β expression of gene.Under 7 kinds of differentiation condition the relative expression of these 4 kinds of genes and CXCR4 consistent (Figure 27 A-D).This proof CXCR4 also is the mark of definitive entoderm.
Expression ectoderm and mesoderm system according to the unlike signal thing can be different from definitive entoderm.Early stage mesoderm is expressed Brachyury and MOX1 gene, and newborn neuroderm is expressed SOX1 and ZIC1.Figure 28 A-d proves in the culture that does not contain external source activin A and is more prone to mesoderm and ectoderm expression of gene, and in the culture that activin A handles, the condition of 10%FBS also increases the level of mesoderm and ectoderm marker expression.These expression patterns are opposite with CXCR4's, show to express not high in this etap differentiation from mesoderm or the ectodermic CXCR4 of hESC.
Grow in early days Mammals, the differentiation of clone outside embryo also takes place.Especially relevant is the endoblastic differentiation of internal organ herein, and it is identical with the many expression of gene of definitive entoderm, as SOX17.For distinguishing the outer internal organ entoderm of definitive entoderm and embryo, should detect marks different between them.It is not the mark of expressing at definitive entoderm at the internal organ entoderm that SOX7 has represented a kind of.Therefore, show the SOX17 gene high expression and do not have the culture condition that SOX7 expresses and to comprise definitive entoderm rather than internal organ entoderm.Shown in Figure 28 E, SOX7 high expression level in the culture that does not contain activin A, even contain activin A when FBS concentration is 10%, SOX7 also shows to express to be increased.This expression pattern with CXCR4 is opposite, and it is not high to show that CXCR4 expresses in the internal organ entoderm.
Also measured to be present in and under above-mentioned every kind of differentiation condition SOX17 immunoreactivity (SOX17 arranged +) the relative number of cell.As hESC differentiation phase under high dosage activin A and FBS lower concentration (0.5%-0.2%) condition, SOX17 +Cell distribution is in whole culture.When using high dosage activin A and 10%FBS (v/v), SOX17 +The cell frequency of occurrences reduces, and generally appears in the isolated cell cluster rather than in whole culture and is evenly distributed (Figure 29 A and C and B and E).SOX17 when no external source activin A +Cell number further reduces.SOX17 under these conditions +Cell also occurs in cell cluster, and these cell clusters are littler still less than handling the cell cluster that obtains with high dosage activin A and lower concentration FBS.These result's proof not only corresponding definitive entoderm genetic expressions of CXCR4 expression pattern under every kind of condition, and the number of corresponding definitive entoderm cell.
Embodiment 8
The differentiation condition of enrichment definitive entoderm increases the ratio of CXCR4 positive cell
The dosage of activin A also influences the efficient of hESC to the definitive entoderm differentiation.This embodiment proves that the dosage that increases activin A can improve CXCR4 in the culture +The ratio of cell.
HESC added 0.5%-2%FBS (being increased to 1.0% to 2.0% from 0.5%) preceding 3 days of differentiation and 0,10 or the RPMI substratum of 100ng/ml activin A break up.Break up after 7 days with containing 2%FBS and 2 mM (EDTA), not containing Ca 2+/ Mg 2+PBS room temperature dissociated cell 5 minutes.Cell filters with 35 μ m nylon mesh, counting and precipitation.Precipitation suspends again with the normal donkey serum of small volume 50% human serum/50%, and ice bath 2 minutes is to seal the non-specific antibody binding site.50 μ l cell suspensions (contain and have an appointment 10 5Cell) (Abcam cat#ab10403-100), carried out mark 45 minutes on ice to add 1 μ l mouse anti CXCR4 antibody in.Contain PBS (damping fluid) washed cell of 2% human serum with 5ml, then precipitation.Carry out washing the second time back with per 10 with the 5ml damping fluid 5Cell suspends in 50 μ l damping fluids again.Add two anti-(the anti-mouse of FITC link coupled donkey with final concentration 5 μ g/ml; JacksonImmunoResearch cat#715-096-151), was hatched 30 minutes, then with above-mentioned damping fluid washing 2 times.Cell is with 5 * 10 6Cell/ml concentration suspends in damping fluid again, and (The Scripps Research Institute) analyzes and sorting with FACS Vantage (BecktonDickenson) at the flow cytometer center.Cell directly uses RLT lysis buffer (Qiagen) to collect, and separates total RNA then, expresses with the real-time quantitative PCR analyzing gene.
When the dosage of activin A in the division culture medium increased, flow cytometer observed CXCR4 +Cell number significantly increases (Figure 30 A-C).CXCR4 +Cell is those cells that fall into R4 door (gate), and this door only uses two anti-to be provided with in contrast, and for this contrast, 0.2% incident is positioned at the R4 door.When activin A dosage improves, CXCR4 +The enhancing (Figure 31 A-D) of the corresponding definitive entoderm genetic expression of remarkable increase of cell number.
Embodiment 9
The CXCR4 positive cell of separation and concentration definitive entoderm genetic expression and removal expression mesoderm, The cell of ectoderm and internal organ entoderm mark
Collect the CXCR4 that identifies among the embodiment 8 +And CXCR4 -Cell is analyzed relative genetic expression, determines parental cell group's genetic expression simultaneously.
Level relatively with the increase CXCR4 genetic expression of activin A dosage significantly increases (Figure 32).This and the CXCR4 of the dosage that relies on activin A +The increase of cell conform to very much (Figure 30 A-C).And from cell mass separation of C XCR4 +Cell is equivalent to separate nearly all CXCR4 genetic expression in the cell mass.This has proved the efficient of FACS method collecting cell.Gene expression analysis shows CXCR4 +Cell not only comprises most of CXCR4 genetic expression, and has comprised the genetic expression of other definitive entoderm marks.Shown in Figure 31 A-D, relative parental generation A100 cell mass CXCR4 +The further enrichment of cell the expression of SOX17, GSC, HNF3B and MIXL1.In addition, CXCR4 -Part contains the genetic expression of these definitive entoderm marks hardly.And CXCR4 +And CXCR4 -Cell mass has shown opposite gene expression pattern to mesoderm, ectoderm and extraembryonic endoderm.Figure 33 A-D shows relative A100 parental cell group, CXCR4 +Cell has been eliminated the genetic expression of Brachyury, MOX1, ZIC1 and SOX7.Compare with low dosage or the condition of not having an activin A, the expression of these marks is lower among the A100 parental cell group.These results are presented under the high dosage activin A differentiation condition from hESCs separation of C XCR4 +Cell has obtained highly enriched, quite pure definitive entoderm cell mass.
Embodiment 10
With definitive entoderm cell in the quantitative cell mass of CXCR4
For confirm as herein noted earlier and submitted on 21 23rd, 2003 the 60/532nd, No. 004 U.S. Provisional Patent Application (title is " definitive entoderm ") is determined the method for definitive entoderm cell proportion in cell culture or the cell mass, FACS is used to analyze the cell of expressing CXCR4 and other definitive entoderm marks.
Use the method in the foregoing description, hESC is broken up to produce definitive entoderm.Particularly, for increasing the output and the purity of the cell culture that is breaking up, serum-concentration is as follows in the control substratum: first day 0.2%FBS, second day 1.0%FBS, 3-6 days 2.0%FBS.FACS utilizes three kinds of cell surface epi-position E-cadherins (ECAD), CXCR4 and thrombomodulin that the culture of differentiation is classified.Analyze the cell mass of classification to detect relative expression's level of setting and extraembryonic endoderm and other cell type marks with Q-PCR then.The cell of the CXCR4 sorting that obtains from best differentiation culture thing can make isolating definitive entoderm cell purity greater than 98%.
Table 2 has shown with the result of method differentiation described herein from the analysis of markers of the definitive entoderm culture of hESC.
The composition of table 2 definitive entoderm culture
Mark The per-cent of culture Definitive entoderm per-cent Extraembryonic endoderm per-cent HES cell per-cent
SOX17 thrombomodulin AFP CXCR4 ECAD other (ECAD feminine genders) 70-80 ＜2 ＜1 70-80 10 10-20 100 0 0 100 0 75 25 0 100
Add up to 100 100 100 100
Particularly, table 2 shows that CXCR4 and SOX17 positive cell (entoderm) account for 70% to 80% of cell culture.Express in the cell of SOX17 at these, be less than 2% Expression of TM (body wall entoderm), be less than 1% and express AFP (internal organ entoderm).Partly remove TM positive and AFP positive cell (chamber wall and internal organ entoderm mixture from the SOX17/CXCR4 positive cell; Amount to 3%) after, the cell culture that can find out about 67%-77% is a definitive entoderm.About 10% cell is the ECAD positive, and this is the mark of hESC, and approximately the cell of 10-20% is other cell types.
We find to compare with above-mentioned low serum step, can improve the purity of definitive entoderm in the noble cells culture that obtains by keep FBS concentration≤0.5% in whole 5-6 days differentiation step before FACS separates.But the definitive entoderm cell number that keeps FBS concentration≤0.5% also to cause producing in whole 5-6 days differentiation step reduces.
The definitive entoderm cell that produces with method described herein was containing maintenance and amplification under the condition of activin, did not also observe differentiation significantly above 50 days.In these examples, SOX17, CXCR4, MIXL1, GATA4 and HNF3 β keep expression in the training period.In addition, do not detect TM, SPARC, OCT4, AFP in these cultures, SOX7, ZIC1 and BRACH.Possible this class cell can be kept in substratum and increase and surpass 50 days and do not observe obvious differentiation.
Embodiment 11
Other marks of definitive entoderm cell
In following experiment, isolation of RNA from the definitive entoderm of purifying and human embryo stem cell group.In the cell mass of every kind of purifying, use the gene chip analyzing and testing genetic expression of RNA then.Further analyze at definitive entoderm and the possible gene of not expressing with Q-PCR again, with mark as definitive entoderm at embryonic stem cell.
Human embryo stem cell (hESC) is maintained in the DMEM/F12 substratum that adds 20%KnockOut serum substitute, the basic fibroblast growth factor of 4ng/ml recombinant human (bFGF), 0.1mM beta-mercaptoethanol, L-glutaminate, non-essential amino acid and penicillin/streptomycin.Be divided into definitive entoderm by hESC after in the RPMI substratum that adds 100ng/ml recombinant human activin A, FBS and penicillin/streptomycin, cultivating 5 days.FBS concentration every day is according to following variation: 0.1% (first day), 0.2% (second day), 2% (3-5 days).
With the hESC and the definitive entoderm cell mass of FACS isolated cell acquisition purifying, to be used for gene expression analysis.With SSEA4 antigen (R﹠amp; D Systems, cat#FAB1435P) immune purifying hESC is with CXCR4 (R﹠amp; D Systems, cat#FAB170P) purifying definitive entoderm.Cell with trypsinase/EDTA (Invitrogen cat#25300-054) dissociates, with phosphate-buffered saline (PBS) washing that contains 2% human serum, then in 100% human serum resuspended 10 minutes of ice bath with the sealing non-specific binding.To containing 5 * 10 6Adding 200 μ l phycoerythrin coupling antibody in the 800 μ l human serums of cell dyeed on ice 30 minutes.Cell is with 8ml PBS washed twice, and is resuspended in 1ml PBS.It is to carry out with FACS Vantage (BD Bioscience) in the core laboratory of The Scripps Research Institute that FACS separates.Cell directly uses RLT lysis buffer (Qiagen) to collect, and uses the RNeasy isolation of RNA according to working instructions (Qiagen) then.
Submit to the double purified RNA give Expression Analysis (Durham, NC) so that adopt Affymetrix platform and U133 Plus 2.0 high-density polynucleotide arrays to produce express spectra.The data that obtain are one group and compare that it identifies the gene of differential expression in hESC and definitive entoderm cell mass.Be chosen on the expression level with hESC in compare and gene that obvious rise changes arranged as the distinctive new candidate markers of tool of definitive entoderm.The gene of selecting detects the changes in gene expression of finding with the confirmation gene chip with above-mentioned Q-PCR, and these expression of gene patterns in the research hESC atomization.
Figure 34 A-M shows the gene expression results of some marks.Shown the cell culture that adds 1,3 and 5 days post analysis of 100ng/ml activin A, the result among the definitive entoderm cell of the expression CXCR4 of 5 days differentiation step (CXDE) end back purifying and the hESC of purifying.Six marker genes of the relatively proof of Figure 34 C and GM, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 have shown expression pattern much at one, and also also similar to the ratio of the expression pattern of CXCR4 and SOX17/SOX7.As previously mentioned, SOX17 all has expression in the extraembryonic endoderm of definitive entoderm and expression SOX7.Because SOX7 does not express at definitive entoderm, can reliably estimate the contribution of definitive entoderm to the expression of observed SOX17 in the whole cell mass according to the ratio of SOX17/SOX7.Figure G-L and figure M show that with the similarity of figure C FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 may be the marks of definitive entoderm, and they express not high in the extraembryonic endoderm cell.
Will be appreciated that Q-PCR result described herein can further be confirmed by ICC.
Embodiment 12
Vitamin A acid and FGF-10 induce PDX1 specifically in the definitive entoderm culture
Experimental results show that below RA and FGF-10 induce the expression of PDX1 in the definitive entoderm cell.
Human embryo stem cell was cultivated 4 days under the condition that contains or do not contain activin.At the 4th day, in substratum, add 1 μ M RA and 50ng/ml FGF-10.After adding RA/FGF-1048 hour, with the quantitative PDX1 marker gene of Q-PCR with to the expression of non-specific other marker genes of preceding gut entoderm.
Cause the remarkable increase (referring to Figure 35) of PDX1 genetic expression with RA processing definitive entoderm cell, but do not increase the expression (Figure 36 A-F) of (SOX1, ZIC1) or neuronic (NFM) gene expression markers of internal organ entoderm (SOX7, AFP), nerve.With observed comparing in the definitive entoderm, be exposed to 1 μ M RA and after 50ng/ml FGF-1048 hour the guarantor, the PDX1 gene expression dose is induced to approximately increases about 500 times.In addition, these results' demonstrations do not use the cell culture of activin to compare with those before RA handles, PDX1 expresses and improves 160 times in the cell culture that activin is handled, and shows that higher PDX1 induces only to be present in the cell culture that is divided into definitive entoderm (SOX17).
Embodiment 13
Compare FGF-10 with independent use RA and promote more high expression level of PDX1
This embodiment demonstration is compared with independent use RA, and RA and FGF-10 unite use and induce higher PDX1 to express.
As among the embodiment in front, hESC was cultivated 4 days under the condition that contains or do not contain activin.At the 4th day, cell was handled with one of following substances: 1 independent μ M RA; 1 μ M RA and FGF-4 or FGF-10 unite use; Or 1 μ M RA and FGF-4 and FGF-10 unite use.RA or RA/FGF handle after 96 hours, with the expression of the quantitative PDX1 of Q-PCR, SOX7 and NFM.
Can induce PDX1 genetic expression to increase by 60 times with retinoic acid treatments hESC culture then with activin earlier.RA adds FGF-4 and can slightly increase PDX1 and express (be approximately use separately RA 3 times) when handling.But, add FGF-10 and vitamin A acid simultaneously, use RA to strengthen 60 times (seeing Figure 37 A) separately to the induction ratio of PDX1.This very strong PDX1 induction ratio is not handled high about 1400 times with activin or RA/FGF.What is interesting is, add the beneficial effect that FGF-4 and FGF-10 have offset FGF-10 simultaneously, the limited PDX1 that has only produced the interpolation that ascribes FGF-4 to strengthens.
Compare with the cell of handling without RA/FGF, unite and use RA/FGF-4 or RA/FGF-10 not to increase the marker gene expression (Figure 37 B-C) irrelevant with preceding gut entoderm.
Embodiment 14
(Anterior-Posterior, A-P) position before and after external vitamin A acid dosage influence
Carry out following experiment to determine whether vitamin A acid dosage influences A-P position in the vitro cell culture.
Human embryo stem cell was cultivated 4 days under the condition that contains or do not contain activin.At the 4th day, add 50ng/ml FGF-10 and 0.04 μ M, 0.2 μ M or 1.0 μ M RA in the substratum.Expression by quantitative PDX1 marker gene of Q-PCR and the non-special mark of other anterior intestine entoderms.
The vitamin A acid and the 50ng/ml FGF-10 that add various dose unite use, induce the differential gene expression pattern relevant with special front and back position pattern.The preferential expression of inducing anterior entoderm mark (HOXA3) of the RA of maximum dose level (1 μ M), and the strongest increase (Figure 38 A-B) that produces PDX1.The expression of gut entoderm mark (CDX1, HOXC6) (seeing Figure 38 C and 41E) during median dose RA (0.2 μ M) induces, and the preferential expression of inducing back gut entoderm mark (HOXA13) (seeing Figure 38 D) of lowest dose level RA (0.04 μ M).RA dosage does not have materially affect (seeing Figure 38 F-G) to the relative expression of neural (SOX1) or neurone (NFM) mark.This embodiment has given prominence to the purposes as morphogen at external use RA, and especially conduct is derived from the morphogen of the entoderm derivative of hESC.
Embodiment 15
Add the expression that B27 strengthens PDX1
By using some factors and cell growth/differentiation condition can influence PDX1 expression in the definitive entoderm.In following experiment, we show that adding B27 can strengthen PDX1 expression in the definitive entoderm cell.
Handled the not differentiation hES cell on the mouse embryo fibroblasts feeder layer, cultivated 4 days with the activin A (100-200ng/ml is dissolved among the 0.5-2%FBS/DMEM/F12) of high dosage, be divided into definitive entoderm with inducing human embryo stem cell.No activin A cultivates impinging upon among the 0.5-2%FBS/DMEM/F12 that does not have activin A.The 4th day, cell culture is handled with following substratum: no activin A (nothing) among the 2%FBS, no activin A (SR) in 2% serum substitute, or the 2%FBS/DMEM/F12 that contains 50ng/ml activin A and 2 μ M RA and 50ng/ml FGF-10 (does not have, + FBS ,+B27) with similar in 2% serum substitute (SR).B27 (Gibco/BRL) through 1/50 the dilution after be added directly to 2%FBS/DMEM/F12 (+B27).Get the double cell sample at every, as above separate total RNA and carry out the Q-PCR analysis.
Figure 39 A-E demonstration is compared with the cell of serum-free culture, and serum-free additive B27 can further induce PDX1 genetic expression, but does not induce the increase of the non-special marker expression of anterior intestine.
Embodiment 16
Using activin B to strengthen PDX1 induces
Present embodiment be presented at cell in vitro cultivate in activin B strengthen of the differentiation of PDX1 negative cells to the PDX1-positive cell.
The not differentiation hES cell of cultivating on the mouse embryo fibroblasts feeder layer with high dosage activin A (50ng/ml) processing in low serum/RPMI 6 days is divided into definitive entoderm with inducing human embryo stem cell.FBS dosage first day was 0%, the second day 0.2%, and 3-6 day 2%.The negative control (NF) that is used for definitive entoderm is cultivated at the 2%FBS/RMPI that does not add activin A.For inducing PDX1 to express, contain the 2%FBS/RPMI of 2 μ M vitamin A acids every kind of culture acceptance in the 6th day.Add the activin A and the activin B of various dose combination in the culture of using activin A to handle in 1-5 days or still keep the independent 50ng/ml of use activin A.Do not add activin A and activin B in the control cultures of no activin A (NF).This RA/ activin is handled and was carried out 3 days, detects the PDX1 genetic expression of double cell sample then with Q-PCR.
Figure 40 A is presented under the condition of 25ng/ml (A25) or 50ng/ml (A50) activin A existence, and the activin B of additive capacity scope 10-50ng/ml (a10, a25 and a50) improves at least 2 times of PDX1 expression than the culture that uses 50ng/ml activin A separately.Increase the increase (Figure 40 B) of not following HNF6 as the PDX1 that adds activin B result, it is this liver and the mark of pancreas that is growing time period.This result shows that with respect to liver the cell proportion that is divided into pancreas increases.
Embodiment 17
Using serum dosage to strengthen PDX1 induces
In whole atomization, the PDX1 that the serum amount of cell culture influences the definitive entoderm cell expresses.Below experiment is presented at hESC serum level in the culture in the atomization of the negative definitive entoderm of PDX1-influences the PDX1 expression of these cells in the further atomization of PDX1-positive entoderm.
The not differentiation hESC that cultivates on the mouse embryo fibroblasts feeder layer with high dosage activin A (100ng/ml) processing in low serum/RPMI 5 days is divided into definitive entoderm with inducing human embryo stem cell.FBS dosage first day was 0.1%, second day 0.5%, 3-5 day 0.5%, 2% or 10%.No activin A contrast (NF) accepts not contain the same FBS/RMPI dosage of activin A.Induced PDX1 to express at the 6th day by adding RA.At 6-7 days, culture was cultivated in the 0.5%FBS/RPMI that contains 2 μ M vitamin A acids, the 8th day 1 μ M, 9-11 days 0.2 μ M.Activin A is reduced to 50ng/ml during retinoic acid treatments, and no activin A contrast (NF) does not still add activin A.
Figure 41 A is presented at the definitive entoderm inductive during 3 days (the 3rd, 4 and 5 day), and FBS dosage has the ability of inducing PDX1 genetic expression during the retinoic acid treatments that continue to change.This does not follow the obvious change of ZIC1 (Figure 41 B) or SOX7 (Figure 41 C) gene expression pattern.
Embodiment 18
The working conditions substratum strengthens PDX1 and induces
We have also studied influences other factors and the growth conditions that definitive entoderm cell PDX1 expresses.Below experiment display condition substratum is to the influence of the negative definitive entoderm cell of PDX1-to the differentiation of PDX1-positive entoderm cell.
The not differentiation hESC that cultivates on the mouse embryo fibroblasts feeder layer with high dosage activin A (100ng/ml) processing in low serum/RPMI 5 days is divided into definitive entoderm with inducing human embryo stem cell.FBS dosage first day was 0.1%, the second day 0.5%, the 3-5 day 2%.
Add RA (being dissolved in the 2%FBS/RPMI that contains 25ng/ml activin A) 4 days in the definitive entoderm culture that after activin A handled 5 days, produces and express endoblastic differentiation to induce to PDX1.A few days ago add 2 μ M RA, the 3rd day 1 μ M, the 4th day 0.5 μ M.Be used for PDX1 inductive minimum medium and be fresh culture (2A25R) or by the substratum of one of four kinds of different cell masses conditioning after 24 hours.The substratum of conditioning (CM) produces by mouse embryo fibroblasts (MEFCM) or with 5 days hESC of one of following three kinds of conditions differentiation: i) 3%FBS/RPMI (CM2), ii) activin A (CM3) or iii) bone morphogenic protein 4 (BMP4) is (CM4).The concentration of adding activin A or BMP4 down at above-mentioned identical FBS dosage (0.2%, 0.5%, 2%) is 100ng/ml.These three kinds different differentiation examples produce three kinds of very different people's cell masses, can carry out the conditioning of PDX1 inducing culture by them.The 3%FBS (NF) that does not add somatomedin produces the foreign cell group who mainly is made up of extraembryonic endoderm, ectoderm and mesoblastema.The culture (A100) that activin A handles produces a high proportion of definitive entoderm, and the culture (B100) that BMP4 handles mainly produces trophectoderm and some extraembryonic endoderms.
Figure 42 A be presented in a few days ago fresh and conditioned medium that RA handles the PDX1 induction phase with.But, to PDX1 expression beginning reduction in fresh culture and the processing of MEF conditioned medium in the 3rd day.The conditioned medium that the hESC of differentiation produces can be kept or further improve PDX1 genetic expression, compares with fresh culture and can improve 3-4 doubly.The hESC-conditioned medium is kept the effect of PDX1 high expression level and is handled further reinforcement in the 4th day at RA, than the high about 6-7 of fresh culture doubly.Figure 42 B display condition substratum is handled and to be caused the CDX1 genetic expression that reduces greatly, and this gene is not partly expressed at the entoderm of expressing PDX1.This shows the conditioned medium processing definitive entoderm of using from the hESC culture of differentiation, can significantly improve PDX1 and express the total purity of entoderm.
Figure 43 shows that PDX1 genetic expression is positive dose response to the amount of the conditioned medium that is used for the definitive entoderm cell.The substratum cumulative volume that adds each culture dish is 5ml, and the conditioned medium of designated volume (as shown in figure 43) is diluted to (A25R) in the fresh culture.It should be noted that only adding the 1ml conditioned medium in the 4ml fresh culture just can induce and keep than single with the higher PDX1 expression level of 5ml fresh culture.This shows that conditioned medium induces PDX1 to express the release of material in conditioned medium that endoblastic beneficial effect depends on certain or some cell, and this or these dosages of substance rely on ground and strengthen PDX1 and express endoblastic generation.
Embodiment 19
Confirmation in conjunction with PDX1 antibody
Help monitoring inducing that PDX1 expresses in the cell mass with PDX1 bonded antibody.This embodiment shows that the rabbit polyclonal IgY antibody of PDX1 can be used for detecting this proteic existence.
In first experiment, the Western engram analysis confirm IgY anti--(antibody of IgY α-PDX1) combines with PDX1 in the cell lysate PDX1.In this analysis, compared the combining of total lysate of IgY α-PDX1 antibody and 50 μ g MDX12 cell after 24 hours from MDX12 human fibroblasts or transfection PDX1 expression vector.Cell lysate separates with SDS-PAGE, and electricity forwards on the film, resists-IgY (Rb α-IgY) two anti-detections with IgY α-PDX1 one anti-antiserum(antisera) and alkaline phosphatase link coupled rabbit subsequently.Different dilution one is anti-and two anti-ly be used for different strip films: A (anti-a 500 * dilution, two anti-10,000 * as to dilute) by following combination, B (2,000 *, 10,000 *), C (500 *, 40,000 *), D (2,000 *, 40,000 *), E (8,000 *, 40,000 *).
In all antibody combinations, the cell of transfection PDX1 expression vector all detects combination.Just detect combination when in the inoblast of untransfected (PDX1-feminine gender), only using one anti-, two anti-(the combination A) of maximum concentration at the same time.This is non-specific combination, because all detect the extra band that a molecular weight is slightly larger than PDX1 in the inoblast of transfection and untransfected.
In second experiment, with immunocytochemistry detect rabbit anti--(polyclonal antibody of Rb α-PDX1) combines with PDX1's PDX1.For producing a kind of PDX1 express cell to this experiment, MS 1-V cell (ATCC#CRL-2460) (is made up with pEGFP-N1, Clontech) by transient transfection PDX 1-EGFP expression vector.Cells transfected is used Rb α-PDX1 and α-EGFP antiserum(antisera) mark then.Can be observed cells transfected with EGFP fluorescence and use Cy5 link coupled two anti-α-EGFP immunocytochemistries.Use α-Rb Cy3-link coupled two to resist and to see the PDX1 immunofluorescence.
The combination of Rb α-PDX1 and α-EGFP antibody is expressed location altogether to GPF.
Embodiment 20
The immunocytochemistry of people's pancreas tissue
This embodiment shows that the PDX1 specific antibody can be used for immunocytochemistry identifier PDX1-positive cell.
In first experiment, section is dyeed detecting Regular Insulin to paraffin-embedded people's pancreas, by (Gp α-Ins) one is anti-, and (D α-Gp) two resists and carries out with the 1/100 anti-cavy of Cy2 link coupled dog of diluting then with the cavy synalbumin of 1/200 dilution earlier.In second experiment,, anti-with the IgY α-PDX1 one of 1/4000 dilution to identical paraffin-embedded people's pancreas section statining by elder generation to detect PDX1, then with the 1/300 AF555 link coupled Rb α-IgY that dilutes, two anti-carrying out.The picture that to collect from first and second experiment is integrated.In the 3rd experiment, also dye with DAPI through the painted cell of IgY α-PDX1.
Analysis to the section of people's pancreas shows that pancreas islet dyeing is darker.Although pancreas islet (the Regular Insulin positive) shows the strongest PDX1 signal, acinar tissue (Regular Insulin feminine gender) also shows faint dyeing.DAPI and PDX1 dye altogether and show that PDX1 mainly but be not to be positioned nuclear fully.
Embodiment 21
PDX1 immunoprecipitation through the cell of retinoic acid treatments
For further confirming in the presence of RA, to exist PDX1 to express in the definitive entoderm cell of differentiation, and the definitive entoderm cell that does not break up under the RA condition do not have PDX1 and expresses, with rabbit anti--PDX1 (the antibody mediated immunity precipitation of Rb α-PDX1) from RA differentiation with PDX1 undifferentiated definitive entoderm cell.Use IgY α-PDX1 antibody to detect the RA of immunoprecipitation by the Western trace.
For obtaining to be used for the definitive entoderm cell that does not break up and break up of immunoprecipitation, hESC handled 5 days with the low serum that contains 100ng/ml activin A, handled 2 days 1 μ M 1 day, 0.2 μ M 1 day (gut entoderm before the PDX1-positive) then with 50ng/ml activin A and 2 μ M alltrans RA.The preparation transfection cell lysate of MS1-V cell (ATCC#CRL-2460) of PDX1 expression vector as positive control.Interpolation Rb α-PDX1 and rabbit specificity two are anti-with immunoprecipitation PDX1 in each lysate.Centrifugal collecting precipitation.Immunoprecipitate is dissolved in the damping fluid that contains SDS, separates through polyacrylamide gel, and the albumen electricity is forwarded on the film, resists with IgY α-PDX1 one link coupled Rb α-IgY two anti-and subsequently and detects.
The collect immunoprecipitate PDX1 albumen of from MS 1-V positive control cell and the 8th day cell of (d8 swimming lane, beginning RA handled back 3 days) and the 9th day (the d9 swimming lane begins RA and handled back 4 days) all shows the positive (Figure 44).Throw out from undifferentiated definitive entoderm cell (activin A handles among cell-Figure 44 of the 5th day and is designated as (A)) and undifferentiated hESC (being designated as (NF) among undressed the 5th day cell-Figure 44) shows negative to PDX1.
Embodiment 22
The generation of PDX1 promotor-EGFP transgenosis hESC system
Be to use PDX1 mark isolated cell, we are with effable reporter gene genetic marker PDX1-positive anterior intestine endoderm cell.Present embodiment is described to make up and is comprised the carrier of reporting box, and this report box comprises the reporter gene that is subjected to the control of PDX1 regulatory region.Present embodiment has also been described the preparation transfection, and the cell of this carrier (for example human embryo stem cell) and this report box are incorporated into genomic cell.
By the GFP reporter gene is placed under the control in PDX1 generegulation district (promotor), made up definitive entoderm clone with the expression PDX1 of reporter gene genetic marker.At first, personnel selection PDX1 control region (Genebank registration number AF192496) is replaced the CMV promotor of pEGFP-N1 (Clontech) carrier, make up EGFP and express by the initial carrier of people PDX1 gene promoter, wherein people PDX1 control region comprises the nucleotide sequence of 85 base pairs (bp) to the downstream from the about 4.4kb in PDX1 transcription initiation site upstream (kilobase to).This zone comprises the characteristic controlling element of PDX1 gene, is enough to show in transgenic mice normal PDX1 expression pattern.In the carrier that makes up, the initial EFGP of PDX1 promotor expresses.In some experiments, this carrier is transfected to advance hESC.
From above carrier excision PDX1 promotor/EGFP box, subclone is gone into one and is selected carrier, and this carrier comprises the neomycin phosphotransferase gene that is subjected to the control of phosphoglyceric kinase-1 promotor.Selecting the box both sides is flp recombinase recognition sites, to remove this box.Make this select the carrier linearizing, dye the back and import hESC with the standard liposomal body method.The G418 screening separated and the undifferentiated transgenosis hESC clone that increases after 10-14 days.
Embodiment 23
The endoblastic separation of the positive anterior intestine of PDX1-
The hESC that following examples proof comprises PDX1 promotor/EGFP box can be divided into the PDX1-positive entoderm cell, uses fluorescence amplifying cell separator (FACS) to separate then.
PDX1 promotor/EGFP transgenosis hESC broke up 5 days in the substratum that contains activin A, broke up 2 days in the substratum that contains activin A and RA then.The cell of differentiation is collected with trysinization, directly is added among RNA lysis buffer or the PBS after Becton Dickinson FACS Diva sorting.Take out single viable cell sample, and not with EGFP (Live) circle choosing (gating), and single viable cell circle selects into the EGFP positive (GFP) and GFP feminine gender (Neg) group.In an experiment, the positive part of EGFP is assigned among the group of two identical sizes according to fluorescence intensity (Hi and Lo).
After the sorting, with Q-PCR and immunocytochemical assay cell mass.Preparing RNA with Qiagen RNeasy post transfers cDNA then to and is used for Q-PCR and analyzes.Q-PCR is undertaken by preceding method.For carrying out immunocytochemical assay, be added to behind the cell sorting among the PBS, in 4% Paraformaldehyde 96, fix 10 minutes, with the centrifugal cell adhesion that makes of cytospin in slide glass.One of cytokeratin 19 (KRT19) resists from Chemicon; One of hepatocyte neclear factor 3 β (HNF3 β) resist from Santa Cruz; Glucose transporter 2 (GLUT2) is from R﹠amp; D system.Use two of suitable coupling FITC (green) or rhodamine (redness) to resist and detect an anti-combination.
The representative FACS sorting of noble cells as shown in figure 45.Isolating PDX1-positive cell ratio is about 7% among this embodiment, changes between 1-20% according to differentiation efficiency.
The cell of sorting is further used for Q-PCR and analyzes.The cell of differentiation shows that EGFP fluorescence is relevant with endogenous PDX1 genetic expression.Compare with no fluorocyte, the PDX1 expression level of EGFP positive cell increases about more than 20 times (Figure 46).Separating of high and low EGFP intensity cell shows EGFP expression level relevant with the PDX1 expression level (Figure 47).Except the PDX1 analysis of markers, in the cell of sorting, utilize Q-PCR to analyze several genes of expressing at the pancreas entoderm.The product of these marker genes (NKX2.2, GLUT2, KRT19, HNF4 α and HNF3 β) obtains enrichment (Figure 48 A-E) at EGFP male portion branch.On the contrary, neural mark ZIC1 and GFAP not enrichment in the EGFP of sorting express cell (Figure 49 A and B).
Use immunocytochemistry to find out, in fact all expressing K RT19 and GLUT2 of the separative PDX1-positive cell of institute.This result indicates that the pancreas entoderm is a cell.Through the many this cells of antibody staining also is HNF3 β male.
Embodiment 24
The subcapsular people's definitive endoderm of mouse kidney is transplanted
To such an extent as to the people's definitive endoderm that produces with methods described herein for explanation can respond the cell of differentiation factor generation derived from intestinal tube, and this people's definitive endoderm is used for differentiation testing program in the body.
People's definitive endoderm is produced in description by previous embodiment.Collect this people's definitive endoderm and it is transplanted under the kidney tunicle of immunocompromise mouse with standard method.After three weeks, put to death this mouse, transplanted tissue is taken out, cuts into slices and be used for histology and immunocytochemical assay.
After Figure 50 A-D had shown transplanting back (post-transplantation) three weeks, people's definitive endoderm was divided into derived from the cell of intestinal tube and cellularstructure.Particularly, Figure 50 A has shown the painted section of h and E, and people's definitive endoderm of transplanting in this section has been divided into the intestinal tube spline structure.Figure 50 B has shown the section through people's definitive endoderm of the painted transplanting of antibody mediated immunity of anti-liver cell specific antigens (HSA).This result shows that people's definitive endoderm can be divided into liver or liver precursor.Figure 50 C and 50D have shown the people's definitive endoderm through the painted transplanting of antibody mediated immunity of anti-villin respectively, and through people's definitive endoderm of the painted transplanting of antibody mediated immunity of anti-tail type homeobox transcription factor 2 (CDX2).These results show that people's definitive endoderm can be divided into intestinal cells or intestinal cells precursor.
Embodiment 25
Determine to promote the differentiation factor of people's definitive endoderm vitro differentiation
For differentiation factor screening method as herein described exemplarily is described, provide multiple candidate's differentiation factor respectively to the people's definitive endoderm group who produces with methods described herein, and determine expression level after the correction of special sign thing gene product at a plurality of time points.
People's definitive endoderm is produced in description by previous embodiment.In brief, the hESC cell was grown 4 days in having the low serum RPMI substratum of 100ng/ml activin A, and wherein calf serum (FBS) concentration was 0% at the 1st day, was 0.2% at the 2nd day, was 2% at 3-4 days.After definitive endoderm forms, started from the 5th day and end at the 10th day, handle cell mass among cultivation contains 0.2%FBS in three independent culture dish the RPMI respectively with 20ng/ml Wnt3B, 5ng/ml FGF2 or 100ng/ml FGF2.Expression with the marker gene product of the quantitative albumin of Q-PCR, PROX1 and TITF1.
Figure 51 A shows response 5ng/ml FGF2, and albumin gene product (liver precursor and hepatocellular mark) is compared remarkable increase with it (before the differentiation factor processing) expression in definitive endoderm in the 4th day with 10 days expression the 9th.Response 20ng/ml Wnt3B, albumin gene product compare also with its expression in untreated definitive endoderm with 10 days expression the 9th have been increased.But increasing amount is not as observed big in 5ng/ml FGF2 handles.Have the observation of remarkable especially meaning to be, for response 100ng/ml FGF2, the albumin gene product was not compared not increase with it the expression in definitive endoderm in the 4th day with the 10th day expression at the 9th day.Shown in Figure 50 B, observe similar results with PROX1 mark (liver precursor and hepatocellular another mark).Figure 51 C has shown in the cell mass that provides 100ng/mlFGF2 that the TITF1 marker gene is compared remarkable increase with it (before the differentiation factor processing) expression in definitive endoderm in the 4th day with 10 days expression the 7th, 9.But 5ng/ml FGF2 handles the almost not influence of expression (comparing with untreated definitive endoderm) to this gene product.In sum, Figure 51 A-C result displayed shows that the concentration of the candidate's differentiation factor that provides to cell mass can influence the differentiation destiny of external definitive endoderm.
Embodiment 26
Be in harmonious proportion on the mark of response candidate differentiation factor and reduce
For differentiation factor screening method as herein described further exemplarily is described, use and the similar method of embodiment 25 described methods, with candidate's differentiation factor screening people definitive endoderm group.
People's definitive endoderm is produced in description by previous embodiment.In brief, the hESC cell was grown 4 days in having the low serum RPMI substratum of 100ng/ml activin A, and wherein calf serum (FBS) concentration was 0% at the 1st day, was 0.2% at the 2nd day, was 2% at 3-4 days.After definitive endoderm forms, started from the 5th day and end at the 10th day, handle cell mass among cultivation contains 0.2%FBS in three independent culture dish the RPMI respectively with 20-50ng/ml Wnt3B, 5ng/ml FGF2 or 100ng/ml FGF2.Form back the 5th day (hESC begin differentiation back the 9th day) at definitive endoderm, adding BMP4 in culture, to make its concentration be 50ng/ml.With the quantitative α-Jia Taidanbai of Q-PCR (AFP), Cytochrome P450 7A (CYP7A), tyrosine aminotransferase (TAT), hepatocyte neclear factor 4a (HNF4a), CXC type Chemokine Receptors 4 (CXCR4), vWF ELISA (VMF), vascular cell adhesion molecule (VACM1), aPoA 1 (APOA1), glucose transporter (GLUT2), α-1-antitrypsin (AAT), glucokinase (GLUKO) and artificial blood are expressed the expression (mRNA) of the marker gene product of homology frame (hHEX).
Figure 52 A-B shows response 5ng/ml FGF2 and 50ng/ml BMP4, and AFP gene product (liver precursor and liver cell mark) is compared remarkable increase with it the expression in definitive endoderm in the 4th day with 10 days expression the 9th with AAT.The expression of AFP and AAT mRNA is significantly increased by the FGF2 of greater concn (100ng/ml), even there is BMP4 to have (Figure 51 A-B the 9th and 10 days).Form contrast with The above results, in the presence of 100ng/ml FGF2 and 50ng/ml BMP4, GLUKO, hHEX compare remarkable rise with it the expression in definitive endoderm in the 4th day with 10 days expression the 9th with TAT mRNA.For GLUKO, no matter be with or without BMP4,5ng/ml Wnt3A and FGF2 can not cause the increase (Figure 52 C) of this marker expression.But 5ng/ml FGF2 causes that the expression of hHEX when BMP exists increases the increase that 100ng/mlFGF2 caused when its degree existed more than or equal to BMP (Figure 52 D).The factor of each test all makes TAT compare increase (Figure 52 E) with it the expression in definitive endoderm in the 4th day with 10 days expression the 9th.In addition, compare with definitive endoderm, with higher horizontal expression, but they do not respond the combination of FGF/BMP to some cell marker when Wnt3A exists.Particularly, response Wnt3A and BMP4 combination, hNF4a mRNA is expressed in the 9th and 10 day and significantly improves (Figure 52 F).In addition, CYP7A response Wnt3A/BMP4 has a small amount of increase (Figure 52 G) the 10th day expression.
More known marks of in the various kinds of cell type, expressing have also been observed.Particularly, mark APOA1, GLUT2, VCAM1, VMF and CXCR4 have been detected.As described below, each in these marks all was associated with specific cell type in the past: mark APOA1 and GLUT2 express at the liver camber, and moderate is expressed in duodenum and small intestine.Mark VCAM1 expresses at the liver camber, and moderate is expressed in stomach, duodenum and small intestine, and is low but express significantly in lung and pancreas.On the contrary, mark VMF and CXCR4 express but only low expression in liver at the lung camber.VMF and CXCR4 also in stomach, pancreas, duodenum and small intestine moderate to highly the expression.
Monitor the expression of each mark described above in the definitive endoderm culture of contact Wnt3A, FGF2 and BMP4 combination.Consistent with The above results, 5ng/ml FGF2 and BMP4 combination are answered in Figure 52 H-J demonstration, and GLUT2, APOA1 have compared increase with 10 days expression with its expression in definitive endoderm the 9th with VCAM1 mRNA.For 100ng/ml FGF2 and BMP4 combination, the mRNA of these marks does not express significantly to be increased.For APOA1 and VCAM1 mark mRNA, mediate it by Wnt3A and BMP4 combination and be expressed in the 9th and 10 day roll up (Figure 52 I-J).
Except that aforementioned, the expression ratio of some mRNA its in the expression decreased of definitive endoderm.For example, compare with its expression at definitive endoderm, no matter whether BMP4 exists, and VMF reduces (Figure 52 K-L) contact Wnt3A or 5ng/ml FGF2 with being expressed in of CXCR4 mRNA after.No matter whether BMP4 exists, and contact 100ng/ml FGF2 has greatly slowed down the speed (Figure 52 K-L) that these two marks reduce.In fact, the expression of CXCR4 keeps substantially, even at the 10th day still so (Figure 52 L).
Method described herein, composition and device are the representatives of present preferred embodiment, play an exemplary role, and are not in order to limit the scope of the invention.In being included in the scope of spirit of the present invention, those skilled in the art can make it and changing or as other purposes, these change and other purposes also in open scope of the present invention.Correspondingly, it will be apparent for a person skilled in the art that do not depart from the scope of the present invention with spirit under, can make different substitutions and modifications to invention disclosed herein.
Be used in phrase in appended claims and the whole disclosure " mainly by ... form " implication be to comprise any key element behind the phrase and be limited to other to disclose the key element that special activity of listed key element or function do not play interference or facilitate effect.Therefore, phrase " mainly by ... form " show that listed key element is essential or compulsory, but other key elements are that optionally being present in of they do not depend on its activity that whether influences listed key element or function.
Reference
Present patent application has been quoted lot of documents and patent.Provided the complete of some documents in the literary composition and quoted, only listed author and age during other reference citations, it is complete quote as follows:
Alexander, J., Rothenberg, M., Henry, G.L., and Stainier, D.Y. (1999) .Casanova plays an early and essential role in endoderm formationin zebrafish (Casanova plays a part early stage and important in the zebra fish entoderm forms) .Dev Biol 215,343-357.
Alexander, J., and Stainier, D.Y (1999) .A molecular pathwayleading to endoderm formation in zebrafish (molecular pathways that causes entoderm to form in the zebra fish) .Curr Biol 9,1147-1157.
Aoki, T.O., Mathieu, J., Saint-Etienne, L., Rebagliati, M.R., Peyrieras, N., and Rosa, F.M. (2002) .Regulation of nodal signalling andmesendoderm formation by TARAM-A, a TGFbeta-related type Ireceptor (regulating the nodal signal and mesendoderm forms) .Dev Biol 241,273-288. with TGF β-correlation type I acceptor TARAM-A
Beck, S., Le Good, J.A., Guzman, M., Ben Haim, N., Roy, K., Beermann, F., and Constam, D.B. (2002) .Extra-embryonic proteasesregulate Nodal signalling during gastrulation (the embryo exoproteinase is regulated the Nodal signal in the gastrula formation phase) .Nat Cell Biol 4,981-985.
Beddington, R.S., Rashbass, P., and Wilson, V. (1992) .Brachyury-agene affecting mouse gastrulation and early organogenesis (one kind of Brachyury-influences the mouse gastrula and forms and organogenetic gene) .Dev Suppl, 157-165.
Bongso, A., Fong, C.Y., Ng, S.C, and Ratnam, S. (1994) .Isolationand culture ofinner cell mass cells from human blastocysts (separating and cultivation inner cell mass cell) .Hum Reprod9,2110-2117. from people's blastocyst
Chang, H., Brown, C.W., and Matzuk, M.M. (2002) .Geneticanalysis of the mammalian transforming growth factor-betasuperfamily (genetic analysis of Mammals transforming growth factor-beta superfamily) .Endocr Rev23,787-823.
Conlon, F.L., Lyons, K.M., Takaesu, N., Barth, K.S., Kispert, A., Herrmann, B., and Robertson, E.J. (1994) .A primary requirement fornodal in the formation and maintenance of the primitive streak in themouse (former of mouse form and keep in mainly need nodal) .Development 120,1919-1928.
Dougan, S.T., Warga, R.M., Kane, D.A., Schier, A.F., and Talbot, W.S. (2003) .The role of the zebrafish nodal-related genes squint andcyclops in patterning of mesendoderm (zebra fish nodal-genes involved squint and the effect of cyclops in mesendoderm forms) .Development 13O, 1837-1851.
Feldman, B., Gates, M.A., Egan, E.S., Dougan, S.T., Rennebeck, G., Sirotkin, H.L, Schier, A.F., and Talbot, W.S. (1998) .Zebrafish organizerdevelopment and germ-layer formation require nodal-related signals (zebra fish tissue development and germinal layer form needs the relevant signal of nodal-) .Nature395,181-185.
Feng, Y., Broder, C.C, Kennedy, P.E., and Berger, E.A. (1996) .HIV-I entry cofactor:functional cDNA cloning of a seven-transmembrane, (HIV-1 enters cofactor to G protein-coupled receptor: stride membrane structure seven times, the functional cDNA clone of g protein coupled receptor) .Science 272,872-877.
Futaki, S., Hayashi, Y., Yamashita, M., Yagi, K., Bono, H., Hayashizaki, Y., Okazaki, Y., and Sekiguchi, (basement membrane components continues the molecular basis of formation to K. (2003) .Molecular basisof constitutive production of basement membrane components:Geneexpression profiles of engelbreth-holm-swarm tumor and F9 embryonalcarcinoma cells: the .J BiolChem. gene expression pattern of engelbreth-holm-swarm tumour and F9 embryo cells)
Grapin-Botton, A., and Melton, D.A. (2000) .Endodermdevelopment:from patterning to organogenesis (entoderm is grown: be formed into tissue from pattern and take place) .Trends Genet16,124-130.
Harris, T.M., and Childs, G. (2002) .Global gene expression patternsduring differentiation of F9 embryonal carcinoma cells into parietalendoderm (F9 embryo cells in body wall entoderm atomization overall gene expression pattern) .Funct Integr Genomics 2,105-119.
Hogan, B.L. (1996) .Bone morphogenetic proteins indevelopment (developmental bone morphogenetic protein) .Curr Opin Genet Dev 6,432-438.
Hogan, B.L. (1997) .Pluripotent embryonic cells and methods ofmaking same (versatility embryonic cell and the method for preparing the versatility embryonic cell) (U.S.A., Vanderbilt University).
Howe, C.C, Overton, G.C, Sawicki, J., Solter, D., Stein, P., andStrickland, S. (1988) .Expression of SPARC/osteonectin transcript inmurine embryos and gonads (expression of SPARC/ osteonectin transcript in mice embryonic and the sexual gland) .Differentiation 37,20-25.
Hudson, C, Clements, D., Friday, R.V., Stott, D., and Woodland, H.R. (1997) .Xsox17alpha and-beta mediate endoderm formation inXenopus (in the African toad Xsox17 α and-β regulates entoderm and form) .Cell91,397-405.
Imada, M., Imada, S., Iwasaki, H., Kume, A., Yamaguchi, H., andMoore, E.E. (1987) .Fetomodulin:marker surface protein of fetaldevelopment which is modulatable by cyclic AMP (fetomodulin: the mark surface protein of fetation is regulated by ring-type AMP) .Dev Biol 122,483-491.
Kanai-Azuma, M., Kanai, Y., Gad, J.M., Tajima, Y., Taya, C, Kurohmaru, M., Sanai, Y., Yonekawa, H., Yazaki, K., Tam, P.P., andHayashi, Y. (2002) .Depletion of definitive gut endoderm in Sox17-nullmutant mice (disappearance of setting gut entoderm in the no Sox17 mutant mice) .Development129,2367-2379.
Katoh, M. (2002) .Expression of human SOX7 in normal tissues andtumors (expression of people SOX7 in healthy tissues and the tumour) .Int J MoI Med9,363-368.
Kikuchi, Y., Agathon, A., Alexander, J., Thisse, C, Waldron, S., Yelon, D., Thisse, B., and Stainier, D.Y. (2001) .casanova encodes a novelSox-related protein necessary and sufficient for early endoderm formationin zebrafish (early stage entoderm forms required albumen in the relevant zebra fish of new Sox of casanova coding) .Genes Dev 15,1493-1505.
Kim, C.H., and Broxmeyer, H.E. (1999) .Chemokines:signallampsfor trafficking of T and B cells for development and effector function (chemokine: grow and the T of effector function and the signal lamp that the B cell transports) .J Leukoc Biol 65,6-15.
Kimelman, D., and Griffin, K.J. (2000) .Vertebrate mesendoderminduction and patteming (inducing and forming of vertebrates mesendoderm) .Curr OpinGenet Dev 1O, 350-356.
Kubo A, Shinozaki K, Shannon JM, Kouskoff V, Kennedy M, Woo S, Fehling HJ, Keller G. (2004) the Development of definitive endoderm fromembryonic stem cells in culture growth of the definitive entoderm of embryonic stem cell (in the culture from) .Development.131,1651-62.
Kumar, A., Novoselov, V., Celeste, A.J., Wolfman, N.M., ten Dijke, P., and Kuehn, M.R. (2001) .Nodal signaling uses activin andtransforming growth factor-beta receptor-regulated Smads (Smad that the Nodal signal uses activin and transforming growth factor to regulate) .J Biol Chem 276,656-661.
Labosky, P.A., Barlow, D.P., and Hogan, B.L. (1994a) .Embryonicgerm cell lines and their derivation from mouse primordial germ cells (embryonic genital cell line and be derived from deriving of mouse archeocyte) .Ciba Found Symp 182,157-168; Discussion 168-178.
Labosky, P.A., Barlow, D.P., and Hogan, B.L. (1994b) .Mouseembryonic germ (EG) cell lines:transmission through the germline anddifferences in the methylation imprint of insulin-like growth factor 2receptor (Igf2r) gene compared with embryonic stem (ES) cell lines (mice embryonic reproduction (EG) clone: by kind be to transmit and rhIGF-1 2 acceptors (Igf2r) gene and embryonic stem cell (ES) are the difference of mark of methylating) .Development 120,3197-3204.
Lickert, H., Kutsch, S., Kanzler, B., Tamai, Y., Taketo, M.M., andKemler, R. (2002) .Formation of multiple hearts in mice followingdeletion of beta-catenin in the embryonic endoderm (the β catenin is removed the formation of the multiple heart in back in the mice embryonic entoderm) .Dev Cell3,171-181.
Lu, C.C, Brennan, J., and Robertson, E.J. (gastrula forms (2001) .From fertilizationto gastrulation:axis formation in the mouse embryo from being fertilized: the formation of mice embryonic axis) .Curr Opin Genet Dev11,384-392.
Ma, Q., Jones, D., and Springer, T.A. (1999) .The chemokinereceptor CXCR4 is required for the retention of B lineage andgranulocytic precursors within the bone marrow microenvironment (B system and keeping of granulocyte precursor need Chemokine Receptors CXCR4 in the bone marrow microenvironment) .Immunity 10,463-471.
McGrath KE, Koniski AD, Maltby KM, McGann JK, Palis J. (1999) Embryonic expression and function of the chemokine SDF-1 and itsreceptor, CXCR4 (embryo of chemokine SDF-1 and its receptor CXCR 4 expresses and function) .Dev Biol.213,442-56.
Miyazono, K., Kusanagi, K., and Inoue, H. (2001) .Divergence andconvergence of TGF-beta/BMP signaling (diversity and the convergence of TGF-β/BMP signal) .J Cell Physiol 187,265-276.
Nagasawa, T., Hirota, S., Tachibana, K., Takakura, N., Nishikawa, S., Kitamura, Y., Yoshida, N., Kikutani, H., and Kishimoto, T. (1996) .Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in micelacking the CXC chemokine PBSF/SDF-1 (the B-cell lymphocyte generates and the Myelopoietic defective of marrow in the mouse of CXC chemokine PBSF/SDF-1 disappearance) .Nature382,635-638.
Niwa, H. (2001) .Molecular mechanism to maintain stem cellrenewal of ES cells (molecule mechanism that keeps the stem cell renewal of ES cell) .Cell StructFunct 26,137-148.
Ogura, H., Aruga, J., and Mikoshiba, K. (2001) .Behavioralabnormalities of Zicl and Zic2 mutant mice:implications as models forhuman neurological disorders (dystropy of Zicl and Zic2 mutant mice :) .Behav Genet 31 as the model of people's nervous dysfunction, 317-324.
Reubinoff.B.E., Pera, M.F., Fong, C.Y., Trounson, A., and Bongso, A. (2000) .Embryonic stem cell lines from human blastocysts:somaticdifferentiation in vitro is (from the embryonic stem cell line of people's blastocyst: external somatocyte differentiation) .Nat Biotechnol 18,399-404.
Rodaway, A., and Patient, R. (2001) .Mesendoderm.an ancient germlayer? (mesendoderm, an ancient germinal layer?) Cell 105,169-172.
Rodaway, A., Takeda, H., Koshida, S., Broadbent, J., Price, B., Smith, J.C, Patient, R., and Holder, N. (1999) .Induction of the mesendoderm inthe zebrafish germ ring by yolk cell-derived TGF-beta family signals anddiscrimination of mesoderm and endoderm by FGF (yolk cell deutero-TGF-beta family signal to zebra fish germ ring mesendoderm induce and with FGF to mesoderm and endoblastic discriminating) .Development 126,3067-3078.
Rohr, K.B., Schulte-Merker, S., and Tautz, D. (1999) .Zebrafish ziclexpression in brain and somites is affected by BMP and hedgehogsignalling (zicl in zebra fish brain and the body segment expresses and is subjected to BMP and hedgehog effect of signals) .Mech Dev 85,147-159.
Schier, A.F. (2003) .Nodal signaling in vertebrate development (the developmental Nodal signal of vertebrates) .Annu Rev Cell Dev Biol19,589-621.
S choenwolf, G.C, and Smith, J.L. (2000) .Gastrulation and earlymesodermal patteming in vertebrates (gastrula forms and early stage mesoderm formation pattern in the vertebrates) .Methods MoI Biol 135,113-125.
Shamblott, M.J., Axelman, J., Wang, S., Bugg, E.M., Littlefield, J.W., Donovan, P.J., Blumenthal, P.D., Huggins, G.R., and Gearhart, J.D. (1998) .Derivation of pluripotent stem cells from cultured humanprimordial germ cells (from the derivation of the multipotent stem cells of people's archeocyte of cultivating) .Proc Natl Acad Sci U S A95,13726-13731.
Shapiro, A.M., Lakey, J.R., Ryan, E.A., Korbutt, G.S., Toth, E., Warnock, G.L., Kneteman, N.M., and Rajotte, R.V. (2000) .Islettransplantation in seven patients with type 1 diabetes mellitus using aglucocorticoid-free immunosuppressive regimen (using the pancreatic islets transplantation do not contain the glucocorticosteroid immunosuppressive therapy among the 7 routine type 1 diabetes patients) .N Engl J Med 343,230-238.
Shapiro, A.M., Ryan, E.A., and Lake y, J.R. (2001a) .Pancreaticislet transplantation in the treatment of diabetes mellitus (pancreatic islets transplantation in treating diabetes) .Best Pract Res Clin Endocrinol Metab15,241-264.
Shapiro, J., Ryan, E., Warnock, G.L., Kneteman, N.M., Lakey, J., Korbutt, G.S., and Rajotte, R.V. (200 1b) .Could fewer islet cells betransplanted in type 1 diabetes? can Insulin independence should be dominantforce in islet transplantation (transplant in the type 1 diabetes by a small amount of islet cells? the Regular Insulin dependent/non-dependent is the principal element in the pancreatic islets transplantation) .Bmj 322,861.
Shiozawa, M., Hiraoka, Y., Komatsu, N., Ogawa, M., Sakai, Y., andAiso, S. (1996) .Cloning and characterization of Xenopus laevis xSox7cDNA (clone and the evaluation of African toad laevis xSox7 cDNA) .Biochim BiophysActa 13O9,13-76.
Smith, J. (1997) .Brachyury and the T-box genes (Brachyury and T-box gene) .Curr Opin Genet Dev 7,474-480.
Smith, J.C, Armes, N.A., Conlon, F.L., Tada, M., Umbhauer, M., and Weston, K.M. (1997) .Upstream and downstream from Brachyury, the agene required for vertebrate mesoderm formation (upstream and downstream of Brachyury, a kind of vertebrates mesoderm forms the gene that needs) .Cold Spring Harb SympQuant Biol 62,337-346.
Takash, W., Canizares, J., Bonneaud, N., Poulat, F., Mattei, M.G., Jay, P., and Berta, P. (2001) .SOX7 transcription factor:sequence, chromosomal localisation, expression, transactivation and interferencewith Wnt signalling (SOX7 transcription factor: sequence, chromosomal localization, express, the commentaries on classics of Wnt signal is lived and is disturbed) .Nucleic Acids Res 29,4274-4283.
Taniguchi, K., Hiraoka, Y., Ogawa, M., Sakai, Y., Kido, S., and Aiso, S. (1999) .Isolation and characterization of a mouse SRY-related cDNA, the mSox7 (cDNA that mouse SRY-is relevant, the separation of mSox7 and evaluation) .BiochimBiophys Acta 1445,225-231.
Technau, U. (2001) .Brachyury, the blastopore and the evolution ofthe mesoderm (Brachyury, blastopore and mesoblastic evolution) .Bioessays 23,788-794.
Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. (1998) .Embryonic stemcell lines derived from human blastocysts (embryonic stem cell line of derived from human blastocyst) .Science 282,1145-1147.
Tremblay, K.D., Hoodless, P.A., Bikoff, E.K., and Robertson, E.J. (2000) .Formation of the definitive endoderm in mouse is a Smad2-dependent process (formation of mouse definitive entoderm is the process that Smad2 relies on) .Development127,3079-3090.
Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., and Speleman, F. (2002) .Accurate normalization ofreal-time quantitative RT-PCR data by geometric averaging of multipleinternal control genes (geometric mean by multiple inherent crt gene is accurately proofreaied and correct real-time quantitative RT-PCR) .Genome Biol 3, RESEARCH0034.
Varlet, I., Collignon, J., and Robertson, E.J. (1997) .nodal expressionin the primitive endoderm is required for specification of the anterior axisduring mouse gastrulation (mouse gastrula form phase front axle specificityization need the expression of nodal in primitive endoderm) .Development 124,1033-1044.
Vincent, S.D., Dunn, N.R., Hayashi, S., Norris, D.P., and Robertson, E.J. (2003) .Cell fate decisions within the mouse organizer are governedby graded Nodal signals (in the mouse organizer cell fate decision be subjected to gradient Nodal signal control) .Genes Dev 17,1646-1662.
Weiler-Guettler, H., Aird, W.C, Rayburn, H., Husain, M., andRosenberg, R.D. (1996) .Developmentally regulated gene expression ofthrombomodulin in postimplantation mouse embryos (transplanting the genetic expression of growing the thrombomodulin of regulating in the mice embryonic of back) .Development 122,2271-2281.
Weiler-Guettler, H., Yu, K., Soff, G., Gudas, L.J., and Rosenberg, R.D. (1992) .Thrombomodulin gene regulation by cAMP and retinoic acid inF9 embryonal carcinoma cells (cAMP and vitamin A acid are regulated the thrombomodulin gene in the F9 embryo cells) .Proceedings Of The National Academy Of SciencesOf The United States Of America 89,2155-2159.
Wells, J.M., and Melton, D.A. (1999) .Vertebrate endodermdevelopment (growth of vertebrates entoderm) .Annu Rev Cell Dev Biol 15,393-410.
Wells, J.M., and Melton, D.A. (2000) .Early mouse endoderm ispatterned by soluble factors from adj acent germ layers (early stage mouse entoderm is by the solvable factor modeization from contiguous germinal layer) .Development 127,1563-1572.
Willison, K. (1990) .The mouse Brachyury gene and mesodermformation (mouse Brachyury gene and mesoderm form) .Trends Genet 6,104-105.
Zhao, G.Q. (2003) .Consequences of knocking out BMP signaling inthe mouse (knocking out the result of BMP signal in the mouse) .Genesis 35,43-56.
Zhou, X., Sasaki, H., Lowe, L., Hogan, B.L., and Kuehn, M.R. (1993) .Nodal is a novel TGF-beta-like gene expressed in the mouse nodeduring gastrulation (nodal is that gastrula forms the new TGF β sample gene that the phase expresses at the mouse joint) .Nature 361,543-547.
Sequence table
＜110〉Novocell Inc. (Novacell, Inc.)
Kevin Alan Da Muer (Kevin Allen D ' Amour)
Alan D A Gunike (Alan D. Agulnick)
Susan Ai Lize (Susan Eliazer)
Rahm Emanuel E Bethe lattice (Emmanuel E.Baetge)
＜120〉evaluation is used for the method for the factor of differentiating definitive endoderm
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<151>2004-07-09
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<151>2004-12-23
<150>US?11/115868
<151>2005-04-26
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<170>FastSEQ?for?Windows?Version?4.0
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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

Claims (43)

1. identify the differentiation factor of the differentiation of people's definitive endoderm in the cell mass can promote to comprise people's cell, said method comprising the steps of:
Acquisition comprises the cell mass of people's definitive endoderm;
Provide the candidate differentiation factor to described cell mass;
Determine the expression of mark in the described cell mass at very first time point;
Determine the expression of identical mark in the described cell mass at second time point, wherein said second time point is after described very first time point, and wherein said second time point is after providing described candidate's differentiation factor to described cell mass; And
With mark described in the described cell mass at the expression ratio of described very first time point, determine whether mark described in the described cell mass increases or reduce in the expression of second time point, and the increase of marker expression or minimizing described in the wherein said cell mass show that described candidate's differentiation factor can promote the differentiation of described people's definitive endoderm.
2. the method for claim 1, wherein said people's definitive endoderm account for people's cell in the described cell mass at least about 10%.
3. the method for claim 1, wherein people's feeder cell are present in the described cell mass, and are definitive endoderms at least about 10% the people's cell except described feeder cell wherein.
4. the method for claim 1, wherein said people's definitive endoderm account for people's cell in the described cell mass at least about 90%.
5. the method for claim 1, wherein said people's feeder cell are present in the described cell mass, and are definitive endoderms at least about 90% the people's cell except described feeder cell wherein.
6. the method for claim 1, wherein said people's definitive endoderm responds described candidate's differentiation factor and is divided into cell, tissue or the organ that is derived from intestinal tube.
7. the method for claim 1, wherein said people's definitive endoderm responds described candidate's differentiation factor and is divided into the pancreas precursor cell.
8. method as claimed in claim 7, wherein said mark are selected from pancreas-duodenum homology frame factor-1 (PDX1), homology frame A13 (HOXA13) and homology frame C6 (HOXC6).
9. the method for claim 1, wherein said people's definitive endoderm responds described candidate's differentiation factor and is divided into liver precursor.
10. method as claimed in claim 9, wherein said mark are selected from albumin, Prospero-relevant homology frame 1 (PROX1) and liver cell specific antigens (HSA).
11. the method for claim 1, wherein said people's definitive endoderm responds described candidate's differentiation factor and is divided into the lung precursor cell.
12. method as claimed in claim 11, wherein said mark are Tiroidina transcription factor 1 (TITF1).
13. the method for claim 1, wherein said people's definitive endoderm responds described candidate's differentiation factor and is divided into the intestines precursor cell.
14. method as claimed in claim 13, wherein said mark are selected from villin and tail type homeobox transcription factor 2 (CDX2).
15. the method for claim 1, wherein said very first time point is before providing described candidate's differentiation factor to described cell mass.
16. the method for claim 1, the wherein said very first time puts and provides described candidate's differentiation factor roughly simultaneously to described cell mass.
17. the method for claim 1, wherein said very first time point is after providing described candidate's differentiation factor to described cell mass.
18. the method for claim 1, the expression of wherein said mark increases.
19. the method for claim 1, the expression decreased of wherein said mark.
20. the method for claim 1, the expression of wherein said mark is determined by quantitative polyase chain reaction (Q-PCR).
21. the method for claim 1, the expression of wherein said mark is determined by immunocytochemistry.
22. the method for claim 1, wherein said mark are selected from pancreas-duodenum homology frame factor-1 (PDX1), homology frame A13 (HOXA13) and homology frame C6 (HOXC6).
23. the method for claim 1, wherein said mark are selected from albumin, Prospero-relevant homology frame 1 (PROX1) and liver cell specific antigens (HSA).
24. the method for claim 1, wherein said mark are selected from villin and tail type homeobox transcription factor 2 (CDX2).
25. the method for claim 1, wherein said mark are Tiroidina transcription factor 1 (TITF1).
26. the method for claim 1, wherein said differentiation factor comprises the anterior intestine differentiation factor.
27. the method for claim 1, wherein said differentiation factor comprises small molecules.
28. the method for claim 1, wherein said differentiation factor comprises retinoid.
29. the method for claim 1, wherein said differentiation factor comprises vitamin A acid.
30. the method for claim 1, wherein said differentiation factor comprises polypeptide.
31. the method for claim 1, wherein said differentiation factor comprises somatomedin.
32. the method for claim 1, wherein said differentiation factor comprises FGF-10.
33. the method for claim 1, wherein said differentiation factor comprises FGF-2.
34. the method for claim 1, wherein said differentiation factor comprises Wnt3B.
35. the method for claim 1, wherein said differentiation factor are not the anterior intestine differentiation factors.
36. the method for claim 1, wherein said differentiation factor are not retinoides.
37. the method for claim 1, wherein said differentiation factor are not vitamin A acids.
38. the method for claim 1, wherein said differentiation factor offers described cell mass with about 0.1ng/ml to the concentration of about 10mg/ml.
39. the method for claim 1, wherein said differentiation factor offers described cell mass with about 1ng/ml to the concentration of about 1mg/ml.
40. the method for claim 1, wherein said differentiation factor offers described cell mass with about 10ng/ml to the concentration of about 100 μ g/ml.
41. the method for claim 1, wherein said differentiation factor offers described cell mass with about 100ng/ml to the concentration of about 10 μ g/ml.
42. the method for claim 1, wherein said differentiation factor offers described cell mass with the concentration of about 1 μ g/ml.
43. the method for claim 1, wherein said differentiation factor offers described cell mass with the concentration of about 100ng/ml.
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CN101684454B (en) * 2008-09-25 2012-08-15 中国科学院上海生命科学研究院 Method for preparing and separating definitive endoderm
CN101760452B (en) * 2008-11-18 2012-12-19 中国科学院上海生命科学研究院 Heterozygous cell line of embryonic stem cell and tumour cell and construction method thereof
CN114214270A (en) * 2021-12-17 2022-03-22 中国农业科学院北京畜牧兽医研究所 Method for regulating and controlling developmental capacity of frozen bovine oocyte and application thereof

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US8623650B2 (en) * 2007-10-19 2014-01-07 Viacyte, Inc. Methods and compositions for feeder-free pluripotent stem cell media containing human serum
CN102822333A (en) * 2010-03-23 2012-12-12 奥林巴斯株式会社 Method for monitoring state of differentiation in stem cells
US9234170B2 (en) * 2010-04-25 2016-01-12 Mount Sinai School Of Medicine Generation of anterior foregut endoderm from pluripotent cells
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Publication number Priority date Publication date Assignee Title
CN101684454B (en) * 2008-09-25 2012-08-15 中国科学院上海生命科学研究院 Method for preparing and separating definitive endoderm
CN101760452B (en) * 2008-11-18 2012-12-19 中国科学院上海生命科学研究院 Heterozygous cell line of embryonic stem cell and tumour cell and construction method thereof
CN114214270A (en) * 2021-12-17 2022-03-22 中国农业科学院北京畜牧兽医研究所 Method for regulating and controlling developmental capacity of frozen bovine oocyte and application thereof
CN114214270B (en) * 2021-12-17 2023-11-24 中国农业科学院北京畜牧兽医研究所 Method for regulating and controlling developmental capacity of frozen bovine oocytes and application thereof

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