CN1946838A - Definitive endoderm - Google Patents

Definitive endoderm Download PDF

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CN1946838A
CN1946838A CNA2004800387201A CN200480038720A CN1946838A CN 1946838 A CN1946838 A CN 1946838A CN A2004800387201 A CNA2004800387201 A CN A2004800387201A CN 200480038720 A CN200480038720 A CN 200480038720A CN 1946838 A CN1946838 A CN 1946838A
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definitive endoderm
cell
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凯文·艾伦·德阿姆尔
艾兰·D·奥戈尔尼克
埃马纽埃尔·E·拜特格
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赛瑟拉公司
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Priority claimed from CN201410120990.2A external-priority patent/CN103898045B/en
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Abstract

本发明公开了包括定形内胚层细胞的细胞培养物及其制备方法。 The present invention discloses a cell comprising a definitive endoderm cell culturing and preparation method thereof. 本发明也公开了基本纯化的定形内胚层细胞的细胞群及从其它细胞类型中分离、富集及纯化定形内胚层细胞的方法。 The present invention also discloses a purified cell population substantially definitive endoderm cells and separated from other cell types, the method for enrichment and purification of definitive endoderm cells.

Description

定形内胚层 Definitive endoderm

相关申请本申请为非临时申请,在35U.SC§119(e)项的规定下享有于2003年12月23日提交的美国临时专利申请号60/532,004、名称为《定形内胚层》的优先权,在35U.SC§119(e)项的规定下还享有于2004年7月9日提交的美国临时专利申请号60/586,566的名称为《用于分离定形内胚层的化学因子细胞表面受体》项的优先权,以及在35U.SC§119(e)项的规定下享有于2004年7月14日提交的美国临时专利申请号60/587,942,名称为《用于分离定形内胚层的化学因子细胞表面受体》的优先权。 RELATED APPLICATION This application is a non-provisional application of US Provisional Patent Application No. 2003, December 23 submitted under the provisions of subparagraph (e) 35U.SC§119 60 / 532,004, the name for the first "definitive endoderm" in right, under the provisions of subparagraph (e) 35U.SC§119 further Related U.S. provisional Patent application number Title 60 / 586,566 in July 9, 2004 entitled "chemical factors for the inner surface of the cell separation by the definitive endoderm body "priority items, as well as the benefit of US provisional patent on July 14, 2004 filed application No. 60 / 587,942 under the provisions of 35U.SC§119 (e) the item, the name" germ layers within a separate setting a cell surface chemokine receptor "priority. 本文将上面列出的各优先权申请的公开全部引入,以供参考。 The priority of each disclosed herein listed above disclosure is fully incorporated by reference.

发明领域本发明涉及医药和细胞生物学领域。 Field of the Invention The present invention relates to the field of medicine and cell biology. 特别是,本发明涉及组合物,包括哺乳动物定形内胚层细胞及制备、分离及使用这些细胞的方法。 In particular, the present invention relates to a composition comprising cells and preparing amorphous endoderm mammal, and methods of using the isolated cells.

发明背景在1994年,首次在不具有成纤维细胞滋养物的培养物中分离了人多能干细胞,例如胚胎干(ES)细胞和胚胎生殖(EG)细胞(Bongso et al.,1994),然后在具有成纤维细胞滋养物的培养物中分离了这些干细胞(Hogan,1997)。 Culture BACKGROUND OF THE INVENTION In 1994, the first time without having fibroblast trophoblast was isolated human pluripotent stem cells, such as embryonic stem (ES) cells and embryonic germ (EG) cells (Bongso et al., 1994), and these isolated stem cells in culture with fibroblasts was nourishment in (Hogan, 1997). 后来,Thomson、Reubinoff和Shamblott使用使有丝分裂失活的小鼠滋养层建立了人ES和EG细胞的连续培养物(Reubinoff et al.,2000;Shamblott et al.,1998;Thomson et al.,1998)。 Later, Thomson, Reubinoff and Shamblott use mitosis trophoblast mice inactivated established human ES and continuous cultures of EG cells (Reubinoff et al, 2000;. Shamblott et al, 1998;.. Thomson et al, 1998) .

人ES和EG细胞(hESCs)为研究人的早期发育和对一些诸如糖尿病和帕金森氏病的疾病治疗干预提供了新的机会。 Human ES and EG cells (hESCs) for the study of early human development and provide new opportunities for some, such as diabetes and Parkinson's disease treatment interventions disease. 例如,使用源于hESCs的产生胰岛素的细胞将对目前的利用供体胰腺细胞的细胞治疗方法提供巨大的改善。 For example, the use of insulin-producing cells derived from hESCs would offer a vast improvement in cell therapy currently using donor pancreatic cells. 然而,目前还不了解怎样从hESCs产生生成胰岛素的β细胞。 However, we do not know how to generate insulin-producing β cells from hESCs. 同样地,目前对糖尿病的利用来自供体胰腺的胰岛细胞的细胞治疗受到了移植所需的高质量胰岛细胞短缺的限制。 Similarly, cell therapy, the current use of diabetes from a donor pancreas islet cells has been a shortage of high quality islet cells needed for transplant restrictions. 对一个I型糖尿病患者的细胞治疗需要移植大约8×108的胰腺胰岛细胞(Shapiro et al.,2000;Shapiro et al.,2001a;Shapiro et al.,2001b)。 A cell therapy for diabetes type I pancreatic islet cell transplantation requires about 8 × 108 of (Shapiro et al, 2000;. Shapiro et al, 2001a;. Shapiro et al, 2001b.). 同样地,就需要至少两个健康供体器官以获得足够的胰岛细胞进行成功的移植。 Similarly, we need at least two healthy donor organs to obtain sufficient islet cells for successful transplantation. HESCs提供了一种起始材料的来源,从该材料发展出基本量的高质量的分化细胞用于人细胞治疗。 HESCs provides a source of starting material, the material is developed from the basic amount of high quality cells for the differentiation of human cell therapy.

使hESCs非常适于细胞治疗应用的两种特性是多能性和在长期的培养中保持这些细胞的能力,不会发生遗传变化的积累。 HESCs are very suitable for the application of cell therapy are two properties and the ability to maintain pluripotency of these cells in long-term culture, the accumulation of genetic changes will not happen. 多能性是指hESCs分化为所有3种原始生殖细胞层(内胚层、中胚层、外胚层)的衍生物的能力,随后这些原始生殖细胞层形成成体的所有体细胞类型和胚外组织(如,胎盘)以及生殖细胞。 Pluripotent refers hESCs differentiate into all three primitive germ cell layers (endoderm, mesoderm, ectoderm) capability derivative, then these primordial germ cell layers form all somatic cell types and extraembryonic tissues into the body (e.g. , placenta) and germ cells. 虽然多能性赋予了hESCs特别的有用性,但这种特性也给研究和操作这些细胞及其衍生物带来了特殊的挑战。 Although pluripotent particularly given the usefulness of hESCs, but this feature is also to study and manipulate these cells and their derivatives poses a particular challenge. 由于在分化的hESC培养物中可能产生大量细胞类型,因此,绝大多数细胞类型产生的效率很低。 Because of the potential large number of cell type differentiation of hESC cultures, and therefore, the efficiency of the vast majority of cell types produce very low. 此外,成功评价任何既定细胞类型的产生关键依赖于确定合适的标志物。 Moreover, the success of any given key generation evaluation cell type depends on the determination of suitable marker. 实现高效率的定向分化对于hESCs的治疗应用非常重要。 High efficiency of directed differentiation is very important for the therapeutic use of hESCs.

为了将hESCs作为起始材料用来产生可用于细胞治疗应用的细胞,克服前述问题是有益的。 To hESCs as a starting material for producing cells used in cell therapy applications, it is useful to overcome the aforementioned problems. 例如,为了达到胰岛细胞移植治疗需要的细胞材料水平,在分化的最早期,将hESCs高效率地定向分化为胰岛/β细胞系是有利的。 For example, in order to achieve the required level of cellular material islet cell transplantation therapy, the most early differentiation of hESCs to efficiently differentiate into insulin / β cell line it is advantageous.

除分化过程的高效定向分化以外,沿着分化途径向胰岛/β细胞系分化的中间细胞类型的分离和定性,并且将这种细胞作为合适的谱系前体用于分化中的其它步骤,也是有益的。 In addition to the differentiation process efficiently directed differentiation, differentiation to islet / β cell differentiation pathway along a line intermediate isolation and characterization of cell types, and other steps such as pre cells suitable for differentiation lineage body, is also beneficial of.

发明概述本发明的一些实施方案涉及包括定形内胚层细胞的细胞培养物,其中所述的定形内胚层细胞为多能细胞,能分化成肠管细胞或衍生于肠管的器官。 SUMMARY Some embodiments of the present invention relates to a cell culture comprising definitive endoderm cells, wherein said cell is definitive endoderm pluripotent cells capable of differentiating into intestinal cells derived from organs or bowel. 根据一些实施方案,定形内胚层细胞为哺乳动物细胞,以及在优选实施方案中,定形内胚层细胞为人细胞。 According to some embodiments, the definitive endoderm cell is a mammalian cell, and in a preferred embodiment, the definitive endoderm cells are human cells. 在本发明的一些实施方案中,定形内胚层细胞表达或不显著表达特定的标志物。 In some embodiments of the present invention, or endoderm cells express significant expression of a particular marker within the setting. 在一些实施方案中,定形内胚层细胞表达一或多种标志物,所述标志物选自SOX17、CXCR4、MIXL1、GATA4、HNF3b、GSC、FGF17、VWF、CALCR、FOXQ1、CMKOR1及CRIP1。 In some embodiments, the definitive endoderm cells express one or more markers, the marker is selected from SOX17, CXCR4, MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1. 在其它实施方案中,定形内胚层细胞不显著表达一或多种标志物,该标志物选自OCT4、α-胎蛋白(AFP)、凝血调节蛋白(TM)、SPARC及SOX7。 In other embodiments, the definitive endoderm cells do not substantially express one or more markers, the marker is selected from OCT4, α- fetoprotein (AFP), thrombomodulin (TM), SPARC and SOX7.

根据本发明的其它实施方案,描述了从多能细胞产生定形内胚层的方法。 According to other embodiments of the present invention, a method of generating definitive endoderm from pluripotent cells. 在一些实施方案中,多能细胞衍生于桑椹胚。 In some embodiments, the pluripotent cells derived from morula. 在一些实施方案中,多能干细胞为干细胞。 In some embodiments, the pluripotent stem cells are stem cells. 用于这些方法的干细胞可包括,但不局限于胚胎干细胞。 Stem cells used in these methods may include, but are not limited to embryonic stem cells. 胚胎干细胞可衍生于胚胎内细胞团或胚胎性腺嵴。 Embryonic stem cells can be derived from the inner cell mass or gonadal ridges embryo. 胚胎干细胞可起源于各种动物种类,这些种类包括,但不局限于各种哺乳动物种类,包括人。 Embryonic stem cells may originate from a variety of animal species, such species include, but are not limited to various mammalian species, including humans. 在优选实施方案中,将人胚胎干细胞用于产生定形内胚层。 In a preferred embodiment, the human embryonic stem cells used to produce definitive endoderm.

在本发明的一些实施方案中,将一种或多种生长因子用于从多能细胞至定形内胚层细胞的分化过程中。 In some embodiments of the present invention, one or more growth factors for differentiation of definitive endoderm cells into pluripotent cells. 这些用于分化过程的一种或多种生长因子可包括来自TGFβ超家族的生长因子。 A method for the differentiation of these processes or more growth factors may include TGFβ superfamily of growth factors from. 在这些实施方案中,所述一种或多种生长因子包括Nodal/活化素和/或生长因子TGFβ超家族的BMP亚群。 In these embodiments, the one or more growth factors including the Nodal / activin and / or TGFβ superfamily growth factor BMP subgroups. 在一些实施方案中,所述一种或多种生长因子选自Nodal、活化素A,活化素B、BMP4、Wnt3a或任何这些生长因子的组合。 In some embodiments, the one or more growth factors selected Nodal, Activin A, Activin B, BMP4, Wnt3a, or any combination of these growth factors.

本发明实施方案还涉及富集于定形内胚层细胞中的细胞群。 Embodiments of the invention further relates to a concentrate in amorphous endoderm cells in a cell population. 在某些实施方案中,将所述定形内胚层细胞分离或基本纯化。 In certain embodiments, the inner definitive endoderm cells isolated or substantially purified. 在一些实施方案中,所述分离或基本纯化的定形内胚层细胞表达SOX17和/或CXRC4标志物,其表达程度高于OCT4、AFP、TM、SPARC和/或SOX7标志物。 In some embodiments, the isolated or substantially purified definitive endoderm cells express SOX17 and / or CXRC4 marker, which is higher than the level of expression OCT4, AFP, TM, SPARC and / or SOX7 marker.

还提供了富集具有定形内胚层的细胞群的方法。 Also it provides a method for enriching a cell population having the endoderm. 在一些实施方案中,可将定形内胚层细胞从混合细胞群体中分离或基本纯化,通过将所述细胞与一种试剂接触,该试剂与一种分子结合,该分子位于定形内胚层细胞表面,而不是混合细胞群体中的其它细胞表面,然后分离与试剂结合的细胞。 , May be separated definitive endoderm cells, in some embodiments from a mixed cell population, or substantially purified, by contacting the cell with an agent, the agent with the binding molecule, the molecule on the cell surface endoderm, and other cell surface is not a mixed cell population, then separated from the cells bound reagent. 在某些实施方案中,位于所述定形内胚层细胞表面的分子为CXCR4。 In certain embodiments, the cell surface molecule located endoderm is CXCR4.

本发明的其它实施方案还涉及CXCR4抗体,SDF-1配体或CXCR4的其它配体,其用于获得富集的、分离的或基本纯化形式的定形内胚层细胞。 Other embodiments of the present invention further relates to an antibody CXCR4, SDF-1 ligand or other ligand of CXCR4, for obtaining the enriched, isolated or substantially purified form definitive endoderm cells. 例如,可将CXCR4抗体、SDF-1配体或CXCR4的另一种配体用作诸如亲和分离或磁分离的方法中的试剂,以富集、分离或基本纯化与所述试剂结合的定形内胚层细胞。 For example, an antibody CXCR4, SDF-1 ligand or another ligand of CXCR4, such methods may be used as an affinity separation or magnetic separation reagent, enriched, isolated or substantially purified binding agent and the setting endodermal cells.

本文所述的本发明的其它实施方案涉及诸如细胞培养物的组合物,其包括多能细胞和定形内胚层细胞。 Other embodiments of the present invention described herein relates to a composition such as a cell culture, comprising pluripotent cells and definitive endoderm cells. 在某些实施方案中,细胞培养物同时包括干细胞和定形内胚层细胞。 In certain embodiments, cell cultures include both stem cells and definitive endoderm cells. 这些培养物中的干细胞数目可大于、等于或小于该培养物中的定形内胚层细胞数目。 Stem cell number in these cultures may be greater than, or equal to the number of the definitive endoderm cell culture less than. 在一些实施方案中,干细胞为人胚胎干细胞。 In some embodiments, the stem cells are human embryonic stem cells. 在某些实施方案中,将hESCs保持在滋养层上。 In certain embodiments, the hESCs maintained on a feeder layer. 在这些实施方案中,所述滋养层细胞可为从人、小鼠或其它合适生物获得的诸如成纤维细胞的细胞。 In these embodiments, the fibroblast trophoblast cells may be cells obtained from human such as a mouse or other suitable organisms.

在本发明的一些实施方案中,包括定形内胚层细胞和hESCs的所述组合物还包括一种或多种生长因子。 In some embodiments of the present invention, the composition comprises cells and definitive endoderm hESCs further comprises one or more growth factors. 这些生长因子可包括来自TGFβ超家族的生长因子。 These growth factors can include growth factors from the TGFβ superfamily. 在这些实施方案中,所述一种或多种生长因子包括Nodal/活化素和/或生长因子TGFβ超家族的BMP亚群。 In these embodiments, the one or more growth factors including the Nodal / activin and / or TGFβ superfamily growth factor BMP subgroups. 在一些实施方案中,所述一种或多种生长因子选自Nodal、活化素A、活化素B、BMP4、Wnt3a或任何这些生长因子的组合。 In some embodiments, the one or more growth factors selected Nodal, Activin A, Activin B, BMP4, Wnt3a, or any combination of these growth factors.

本发明的其它实施方案参考下述编号段落进行描述:1.包括人细胞的细胞培养物,其中至少约10%的所述人细胞为定形内胚层细胞,所述的定形内胚层细胞为能分化成肠管细胞或衍生于肠管的器官的多能细胞。 Other embodiments of the present invention will be described with reference to the following numbered paragraphs: a cell culture comprising human cells, wherein at least about 10% of said human cells are definitive endoderm cells, said definitive endoderm cells capable of differentiating intestinal cells into the intestine of an organ or derived from pluripotent cells.

2.段落1所述的细胞培养物,其中至少约50%的所述人细胞为定形内胚层细胞。 2. The cell culture according to paragraph 1, wherein at least about 50% of said human cell is a definitive endoderm cell.

3.段落1所述的细胞培养物,其中至少约80%的所述人细胞为定形内胚层细胞。 3. The cell culture of paragraph 1, wherein at least about 80% of said human cell is a definitive endoderm cell.

4.段落1所述的细胞培养物,其中所述的定形内胚层细胞表达选自SOX17和CXCR4的标志物。 4. The cell culture according to paragraph 1, wherein said definitive endoderm cells express a marker selected from the group of CXCR4 and SOX17.

5.段落4所述的细胞培养物,其中在所述定形内胚层细胞中,选自SOX17和CXCR4的所述标志物的表达高于选自OCT4、α-胎蛋白(AFP)、凝血调节蛋白(TM)、SPARC及SOX7的标志物的表达。 5. The cell culture of paragraph 4, wherein the definitive endoderm cells, and SOX17 expression of the selected markers above the selected CXCR4 OCT4, α- fetoprotein (AFP), thrombomodulin (TM) expression, SPARC and SOX7 markers.

6.段落4所述的细胞培养物,其中所述定形内胚层细胞不表达选自OCT4、AFP、TM、SPARC和SOX7的标志物。 6. The cell culture of paragraph 4, wherein said definitive endoderm cells do not express selected from OCT4, AFP, TM, SPARC and SOX7 of markers.

7.段落4所述的细胞培养物,其中所述定形内胚层细胞表达选自MIXL1、GATA4和HNF3b的标志物。 7. The cell culture of paragraph 4, wherein said definitive endoderm cells express selected MIXL1, GATA4 and HNF3b marker.

8.段落4所述的细胞培养物,其中所述定形内胚层细胞表达选自FGF17、VWF、CALCR、FOXQ1、CMKOR1和CRIP1的标志物。 8. The cell culture of paragraph 4, wherein said definitive endoderm cells express selected FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 marker.

9.段落1所述的细胞培养物,其中所述定形内胚层细胞表达SOX17和CXCR4。 Paragraph 9. The cell culture of claim 1, wherein said definitive endoderm cells express SOX17 and CXCR4.

10.段落9所述的细胞培养物,其中在所述的定形内胚层细胞中,所述SOX17和CXCR4的表达高于OCT4、AFP、TM、SPARC和SOX7的表达。 10. The cell culture of paragraph 9, wherein in said definitive endoderm cells, the expression of CXCR4 and SOX17 than OCT4, expression of AFP, TM, SPARC and the SOX7.

11.段落9所述的细胞培养物,其中所述定形内胚层细胞不表达OCT4、AFP、TM、SPARC和SOX7。 11. The cell culture of paragraph 9, wherein said definitive endoderm cells not expressing OCT4, AFP, TM, SPARC and SOX7.

12.段落9所述的细胞培养物,其中所述定形内胚层细胞表达MIXL1、GATA4和HNF3b。 12. The cell culture of paragraph 9, wherein said definitive endoderm cells express MIXL1, GATA4 and HNF3b.

13.段落9所述的细胞培养物,其中所述定形内胚层细胞表达选自FGF17、VWF、CALCR、FOXQ1、CMKOR1和CRIP1的标志物。 13. The cell culture of paragraph 9, wherein said definitive endoderm cells express selected FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 marker.

14.段落1所述的细胞培养物,其中在所述的细胞培养物中,对应每一种多能细胞存在至少约2个定形内胚层细胞。 14. The cell culture according to paragraph 1, wherein said cell culture in the presence of a corresponding plurality endoderm cells can be shaped at least about 2 per one cell.

15.段落14所述的细胞培养物,其中所述多能细胞包括胚胎干细胞。 15. The cell culture of paragraph 14, wherein the pluripotent cells include embryonic stem cells.

16.段落15所述的细胞培养物,其中所述的胚胎干细胞衍生于选自自桑椹胚、胚胎的内细胞团(ICM)和胚胎的性腺嵴的组织。 16. The cell culture of paragraph 15, wherein the embryonic stem cells derived from morula embryos inner cell mass (ICM) and gonadal ridge selected from embryo tissue.

17.段落1所述的细胞培养物进一步包括培养基,该培养基包含少于约10%的血清。 17. The cell culture of paragraph 1 further comprising the medium, the medium comprises less than about 10% serum.

18.段落1所述的细胞培养物进一步包括TGFβ超家族的Nodal/活化素亚群的生长因子。 18. The cell culture of paragraph 1 further comprising the TGFβ superfamily Nodal / Activin subgroup growth factor.

19.段落1所述的细胞培养物,进一步包括选自Nodal、活化素A、活化素B及其组合的生长因子。 19. The cell culture according to paragraph 1, further comprising a selected Nodal, Activin A, Activin B growth factors, and combinations thereof.

20.包括细胞的细胞群,其中至少约90%的所述细胞为人定形内胚层细胞,所述的人定形内胚层细胞为多能细胞,该多能细胞能分化成肠管细胞或衍生于肠管的器官。 20. A cell population comprising cells, wherein at least about 90% of said cells are human definitive endoderm cells, said human definitive endoderm cells are pluripotent cells that can differentiate into pluripotent cells derived from intestinal cells or intestinal tract organ.

21.段落20所述的细胞群,其中至少约95%的所述细胞为人定形内胚层细胞。 21. The cell population of paragraph 20, wherein at least about 95% of said cells are human definitive endoderm cells.

22.段落20所述的细胞群,其中至少约98%的所述细胞为人定形内胚层细胞。 22. The cell population of paragraph 20, wherein at least about 98% of the cells are human definitive endoderm cells.

23.段落20所述的细胞群,其中所述人定形内胚层细胞表达选自SOX17和CXCR4的标志物。 23. A cell population according to paragraph 20, wherein said human definitive endoderm cells express a marker selected from the group of CXCR4 and SOX17.

24.段落23所述的细胞群,其中在所述人定形内胚层细胞中,选自SOX17和CXCR4的所述标志物的表达高于选自OCT4、AFP、TM、SPARC和SOX7的标志物的表达。 24. The cell population of paragraph 23, wherein in said human definitive endoderm cells, said CXCR4 and SOX17 expression of selected markers above the selected OCT4, AFP, TM, SPARC and markers of SOX7 expression.

25.段落23所述的细胞群,其中所述人定形内胚层细胞不表达选自OCT4、AFP、TM、SPARC和SOX7的标志物。 25. The cell population of paragraph 23, wherein said human definitive endoderm cells do not express selected from OCT4, AFP, TM, SPARC and SOX7 of markers.

26.段落23所述的细胞群,其中所述人定形内胚层细胞表达选自MIXL1、GATA4和HNF3b的标志物。 26. The cell population of paragraph 23, wherein said human definitive endoderm cells express selected MIXL1, GATA4 and HNF3b marker.

27.段落23所述的细胞群,其中所述定形内胚层细胞表达选自FGF17、VWF、CALCR、FOXQ1、CMKOR1和CRIP1的标志物。 27. The cell population of paragraph 23, wherein said definitive endoderm cells express selected FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 marker.

28.段落20所述的细胞群,其中所述人定形内胚层细胞表达SOX17和CXCR4。 28. The cell population of paragraph 20, wherein said human SOX17 expression of definitive endoderm cells, and CXCR4.

29.段落28所述的细胞群,其中在所述的人定形内胚层细胞中,SOX17和CXCR4的表达高于OCT4、AFP、TM、SPARC和SOX7的表达。 29. The cell population of paragraph 28, wherein said human definitive endoderm cells, the expression of CXCR4 and SOX17 expression than OCT4, AFP, TM, SPARC and the SOX7.

30.段落28所述的细胞群,其中所述人定形内胚层细胞不表达OCT4、AFP、TM、SPARC和SOX7。 30. The cell population of paragraph 28, wherein said human definitive endoderm cells not expressing OCT4, AFP, TM, SPARC and SOX7.

31.段落28所述的细胞群,其中所述的人定形内胚层细胞表达MIXL1、GATA4和HNF3b。 31. The cell population of paragraph 28, wherein said human definitive endoderm cells express MIXL1, GATA4 and HNF3b.

32.段落28所述的细胞群,其中所述定形内胚层细胞表达选自FGF17、VWF、CALCR、FOXQ1、CMKOR1和CRIP1的标志物。 32. The cell population of paragraph 28, wherein said definitive endoderm cells express selected FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 marker.

33.段落20所述的细胞群,其中在所述的细胞群体中,对应每一种多能细胞存在至少约2个定形内胚层细胞。 33. The cell populations of paragraph 20, wherein said cell population in the presence of pluripotent cells corresponding to the at least endoderm cells each shaped approximately two.

34.段落33所述的细胞群,其中所述多能细胞包括胚胎干细胞。 34. The cell population of paragraph 33, wherein the pluripotent cells include embryonic stem cells.

35.段落34所述的细胞群,其中所述的胚胎干细胞衍生于选自桑椹胚、胚胎的ICM和胚胎的性腺嵴的组织。 35. The cell population of paragraph 34, wherein said embryonic stem cell is derived is selected from morula, gonadal ridge ICM embryos and embryonic tissue.

36.产生定形内胚层细胞的方法,所述的方法包括下述步骤:获得包括人多能细胞的细胞群;为所述的细胞群提供至少一种TGFβ超家族生长因子,所述生长因子的量足以促进所述多能细胞分化成定形内胚层细胞,所述的定形内胚层细胞为能分化成肠管细胞或衍生于肠管的器官的多能细胞;以及给予足够的时间来形成定形内胚层细胞,其中所述的形成定形内胚层细胞的足够时间以检测所述细胞群体中定形内胚层细胞的存在来确定。 36. A method of producing definitive endoderm cells, said method comprising the steps of: obtaining a population of pluripotent cells include human cells; providing the cell population to at least one TGFβ superfamily growth factor, said growth factor an amount sufficient to promote the pluripotent cells are differentiated into endoderm cells shaping said definitive endoderm cells capable of differentiating into intestinal cells or derived from organs of the intestine of pluripotent cells; and given sufficient time to form definitive endoderm cells wherein the time sufficient to form the definitive endoderm cells to detect the presence within the population of cells to definitive endoderm cells determined.

37.段落36所述的方法,其中至少约10%的所述多能细胞分化成定形内胚层细胞。 37. The method of paragraph 36, wherein at least about 10% of the pluripotent cells are differentiated into definitive endoderm cells.

38.段落36所述的方法,其中至少约50%的所述多能细胞分化成定形内胚层细胞。 38. The method according to paragraph 36, wherein at least about 50% of the pluripotent cells are differentiated into definitive endoderm cells.

39.段落36所述的方法,其中至少约70%的所述多能细胞分化成定形内胚层细胞。 39. The method of paragraph 36, wherein at least about 70% of the pluripotent cells are differentiated into definitive endoderm cells.

40.段落36所述的方法,其中至少约80%的所述多能细胞分化成定形内胚层细胞。 40. The method according to paragraph 36, wherein at least about 80% of the pluripotent cells are differentiated into definitive endoderm cells.

41.段落36所述的方法,其中检测定形内胚层细胞在所述细胞群体中的存在包括在所述细胞群细胞中检测至少一种选自SOX17和CXCR4的标志物的表达,以及至少一种选自OCT4、AFP、TM、SPARC和SOX7的标志物的表达,其中在所述定形内胚层细胞中,选自SOX17和CXCR4的所述标志物的表达高于选自OCT4、AFP、TM、SPARC和SOX7的所述标志物的表达。 41. The method according to paragraph 36, wherein the detecting the presence of definitive endoderm cells in said cell population of said cell population comprises detecting at least one selected cell expressing markers CXCR4 and SOX17, and at least one is selected from OCT4, expression of AFP, TM, SPARC and markers SOX7, wherein the definitive endoderm cells, said CXCR4 and SOX17 expression of selected markers above the selected OCT4, AFP, TM, SPARC SOX7 and expression of said marker.

42.段落36所述的方法,其中检测定形内胚层细胞在所述的细胞群中的存在包括在所述细胞群细胞中检测至少一种选自SOX17和CXCR4的标志物的表达,以及至少一种选自AFP、TM和SOX7的标志物的表达,其中在所述定形内胚层细胞中,选自SOX17和CXCR4的所述标志物的表达高于选自AFP、TM和SOX7的所述标志物的表达。 42. The method according to paragraph 36, wherein the detecting the presence of definitive endoderm cells in said cell population comprises a cell population detected in cell marker expression of CXCR4 and SOX17 at least one selected from the group, and at least one expression selected from AFP, SOX7 markers (TM) and wherein the definitive endoderm cells, and SOX17 expression of the selected markers above the selected CXCR4 AFP, said markers (TM) and SOX7 expression.

43.段落42所述的方法,其中至少一种所述的标志物的表达以Q-PCR来测定。 43. The method according to paragraph 42, wherein at least the expression of the marker to be measured Q-PCR.

44.段落42所述的方法,其中至少一种所述的标志物的表达以免疫细胞化学来测定。 44. The method according to paragraph 42, wherein at least one of the expression of the marker is determined by immunocytochemistry.

45.段落36所述的方法,其中检测在所述的细胞群体中定形内胚层细胞的存在包括在所述细胞群体细胞中检测至少一种选自VWF、CALCR、FOXQ1、CMKOR1和CRIP1的标志物的表达,以及至少一种选自OCT4、AFP、TM、SPARC和SOX7的标志物的表达,其中在所述的定形内胚层细胞中,选自FGF17、VWF、CALCR、FOXQ1和CRIP1的标志物的表达高于选自OCT4、AFP、TM、SPARC和SOX7的标志物的表达。 45. The method according to paragraph 36, wherein detecting the presence of definitive endoderm cells in said cell population comprises detecting at least one selected VWF, CALCR, FOXQ1 cells in the cell population, and CRIP1 marker CMKORl expression, and at least one selected from OCT4, expression of AFP, TM, SPARC and markers SOX7, wherein in said definitive endoderm cells, selected FGF17, VWF, CALCR, FOXQ1 markers and CRIP1 expression above selected OCT4, AFP, TM, SPARC and SOX7 markers.

46.段落36所述的方法,其中所述的至少一种生长因子为TGFβ超家族的Nodal/活化素亚群。 46. ​​The method according to paragraph 36, wherein said at least one growth factor is Nodal / Activin subgroup TGFβ superfamily.

47.段落46所述的方法,其中所述的至少一种生长因子选自Nodal、活化素A、活化素B及其组合。 47. The method according to paragraph 46, at least one growth factor selected from the group wherein said Nodal, Activin A, Activin B, and combinations thereof.

48.段落47所述的方法,其中所述的至少一种生长因子为Nodal。 48. The method according to paragraph 47, wherein said at least one growth factor is Nodal.

49.段落47所述的方法,其中所述的至少一种生长因子为活化素A。 49. The method according to paragraph 47, wherein said at least one growth factor is activin A.

50.段落47所述的方法,其中所述的至少一种生长因子为活化素B。 50. The method according to paragraph 47, wherein said at least one growth factor is activin B.

51.段落36所述的方法,其中提供了TGFβ超家族的多种生长因子。 51. The method according to paragraph 36, wherein a plurality of the TGFβ superfamily of growth factors.

52.段落51所述的方法,其中所述的多种生长因子包括Nodal、活化素A及活化素B。 52. The method according to paragraph 51, wherein the plurality of growth factors include Nodal, Activin A and Activin B.

53.段落36所述的方法,其中所述至少一种生长因子的浓度为至少约10ng/ml。 53. The method according to paragraph 36, wherein said at least one growth factor in a concentration of at least about 10ng / ml.

54.段落36所述的方法,其中所述至少一种生长因子的浓度为至少约100ng/ml。 54. The method according to paragraph 36, wherein said at least one growth factor in a concentration of at least about 100ng / ml.

55.段落36所述的方法,其中所述至少一种生长因子的浓度为至少约500ng/ml。 55. The method according to paragraph 36, wherein said at least one growth factor in a concentration of at least about 500ng / ml.

56.段落36所述的方法,其中所述至少一种生长因子的浓度为至少约1000ng/ml。 56. The method according to paragraph 36, wherein said at least one growth factor in a concentration of at least about 1000ng / ml.

57.段落36所述的方法,其中所述至少一种生长因子的浓度为至少约5000ng/ml。 57. The method according to paragraph 36, wherein said at least one growth factor in a concentration of at least about 5000ng / ml.

58.段落36所述的方法,其中所述细胞群体生长于包括少于约10%血清的培养基中。 58. The method according to paragraph 36, wherein the population of cells grown in comprising less than about 10% serum medium.

59.段落36所述的方法,其中所述多能细胞包括干细胞。 59. The method of paragraph 36, wherein the pluripotent cells comprise stem cells.

60.段落59所述的方法,其中所述的多能细胞包括胚胎干细胞。 60. The method according to paragraph 59, wherein the pluripotent cells include embryonic stem cells.

61.段落60所述的方法,其中所述的胚胎干细胞衍生于选自桑椹胚、胚胎的ICM和胚胎的性腺嵴的组织。 61. The method according to paragraph 60, wherein said embryonic stem cells are derived from tissue selected morula, ICM embryos and embryonic gonadal ridge.

62.以段落36的方法产生的定形内胚层细胞。 Definitive endoderm cells 62. The method of paragraph 36 to produced.

63.产生富集的定形内胚层细胞的细胞群方法,包括下述步骤:分化多能人细胞群体中的细胞,以产生定形内胚层细胞,所述的定形内胚层细胞为多能细胞,该多能细胞能分化成肠管细胞或衍生于肠管的器官;向所述的细胞群提供试剂,所述试剂与标志物结合,所述标志物表达于所述的定形内胚层细胞中而基本上不表达于所述细胞群中的其它细胞类型中;及将与所述试剂结合的所述定形内胚层细胞与位于所述细胞群中所述其它细胞类型分离,从而产生富集的定形内胚层细胞的细胞群。 63. A method of generating a population of cells of the endoderm cell enrichment, comprising the steps of: pluripotent cells capable cell populations to produce amorphous endoderm cells, said definitive endoderm cells are pluripotent cells that pluripotent cells capable of differentiating into cells or organs derived from intestinal bowel; providing the cell population of the agent, the agent binds with the marker, the marker in the expression of the definitive endoderm cells without substantially expression in other cell types in the population of cells; and the binding of said definitive endoderm cells with the agent with other cell types in said isolated population of said cells to produce a definitive endoderm cell enriched cell populations.

64.段落63所述的方法,其中分化步骤进一步包括,获得包括多能人细胞的细胞群,向所述细胞群体提供所述至少一种TGFβ超家族生长因子,该生长因子的量足以促进将所述多能细胞分化成定形内胚层细胞,所述的定形内胚层细胞为多能细胞,该多能细胞能分化成肠管细胞或衍生于肠管的器官,并给予足够的时间形成定形内胚层细胞,其中所述的形成定形内胚层细胞的足够时间以检测所述细胞群体中定形内胚层细胞的存在来确定。 64. The method according to paragraph 63, wherein the differentiating step further comprises obtaining a cell population comprising pluripotent human cells, said population of cells to provide at least one TGFβ superfamily of growth factors, the growth factor is an amount sufficient to promote the the pluripotent cells are differentiated into endoderm cells shaping said definitive endoderm cells are pluripotent cells, the pluripotent cells can differentiate into intestinal cells or organs derived from the intestine, and given sufficient time to form definitive endoderm cells wherein the time sufficient to form the definitive endoderm cells to detect the presence within the population of cells to definitive endoderm cells determined.

65.段落63所述的方法,其中检测包括,在所述细胞群体的细胞中检测至少一种选自SOX17和CXCR4的标志物的表达,以及至少一种选自OCT4、AFP、TM、SPARC和SOX7的标志物的表达,其中在所述的定形内胚层细胞中,选自SOX17和CXCR4的标志物的表达高于选自OCT4、AFP、TM、SPARC和SOX7的标志物的表达。 65. The method according to paragraph 63, wherein the detecting comprises detecting in a cell of the population of cells expressing at least one selected marker CXCR4 and SOX17, and at least one member selected from OCT4, AFP, TM, SPARC and expression of the marker SOX7, wherein in said definitive endoderm cells, the expression of selected markers CXCR4 and SOX17 higher than the selected OCT4, expression of AFP, TM, SPARC and SOX7 of markers.

66.段落63所述的方法,其中检测包括,在所述细胞群体的细胞中检测至少一种选自SOX17和CXCR4的标志物的表达,以及至少一种选自AFP、TM和SOX7的标志物的表达,其中在所述的定形内胚层细胞中,选自SOX17和CXCR4的标志物的表达高于选自AFP、TM和SOX7的标志物的表达。 66. The method of paragraph 63, wherein the detecting comprises detecting in a cell of the population of cells expressing at least one selected marker CXCR4 and SOX17, and at least one selected from AFP, SOX7 of markers (TM) and expression, wherein in said definitive endoderm cells, selected markers CXCR4 and SOX17 expression than the selected AFP, the expression of markers (TM) and the SOX7.

67.段落63所述的方法,其中检测包括,在所述细胞群体的细胞中检测至少一种选自FGF17、VWF、CALCR、FOXQ1、CMKOR1和CRIP1的标志物的表达,以及至少一种选自OCT4、AFP、TM、SPARC和SOX7的标志物的表达,其中在所述的定形内胚层细胞中,选自FGF17、VWF、CALCR、FOXQ1、CMKOR1和CRIP1的标志物的表达高于选自OCT4、AFP、TM、SPARC和SOX7的标志物的表达。 67. The method according to paragraph 63, wherein detecting comprises detecting at least one selected FGF17, VWF, CALCR, FOXQ1 cells in said cell population, the expression and CRIP1 CMKOR1 markers, and at least one selected from expressing OCT4, AFP, TM, SPARC and markers SOX7, wherein in said definitive endoderm cells, FGF17 is selected, the expression of VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 marker is selected from OCT4 above, expression of AFP, TM, SPARC and SOX7 markers.

68.段落63所述的方法,其中至少约95%的所述细胞为定形内胚层细胞。 68. The method according to paragraph 63, wherein at least about 95% of the cell is definitive endoderm cells.

69.段落63所述的方法,其中至少约98%的所述细胞为定形内胚层细胞。 69. The method according to paragraph 63, wherein at least about 98% of the cell is definitive endoderm cells.

70.段落63所述的方法,其中所述的标志物为CXCR4。 70. The method of paragraph 63, wherein said marker is CXCR4.

71.段落63所述的方法,其中所述的试剂为抗体。 71. The method of paragraph 63, wherein the agent is an antibody.

72.段落71所述方法,其中所述的抗体对CXCR4具有亲和性。 72. The method of paragraph 71, wherein said antibody has an affinity for CXCR4.

73.段落63所述的方法产生的富集定形内胚层细胞的细胞群。 The method of setting the enrichment of the passages 63, 73. The resulting cell population endoderm cells.

74.段落4或9的任一项所述的细胞培养物,其中所述的定形内胚层细胞不显著表达选自OCT4、AFP、TM、SPARC和SOX7的标志物。 74. The cell culture according to any one of paragraphs 4 or claim 9, wherein said definitive endoderm cells do not substantially express selected from OCT4, AFP, TM, SPARC and SOX7 of markers.

75.段落23或28的任一段落的细胞群,其中所述的定形内胚层细胞不显著表达选自OCT4、AFP、TM、SPARC和SOX7的标志物。 A cell population according to any of paragraphs 23 or 75. paragraph 28, wherein said definitive endoderm cells do not substantially express selected from OCT4, AFP, TM, SPARC and SOX7 of markers.

应当了解,上述的方法和组合物涉及体外细胞培养。 It should be appreciated that the above methods and compositions directed to in vitro cell culture. 然而,上述的体外分化细胞组合物可于体内应用。 However, the above compositions may be in vitro differentiated cells in vivo applications.

本发明的其它实施方案还可发现于下述美国临时专利申请中,即2003年12月23日提交的美国临时专利申请第60/532,004号,名为《定形内胚层》;2004年7月9日提交的美国临时专利申请第60/586,566号,名为《用于分离定形内胚层的趋化因子细胞表面受体》;以及2004年7月14日提交的美国临时专利申请第60/587,942号,名为《用于分离定形内胚层的细胞表面受体》,本文将这些公开全部引入,以供参考。 Other embodiments of the present invention may also be found in the following U.S. provisional patent applications, i.e. U.S. Provisional Patent Application filed on December 23, 2003 No. 60 / 532,004, entitled "Definitive endoderm"; July 9, 2004 U.S. provisional Patent application No. filed 60 / 586,566, entitled "chemokine for the separation of definitive endoderm cell surface factor receptor"; and U.S. provisional Patent filed July 14, 2004 application No. 60 / 587,942 , entitled "cell surface receptor for the separation of the definitive endoderm", all incorporated herein will be those disclosed, for reference.

附图简述图1为提出的从hESCs产生β-细胞的分化途径的示意图。 DRAWINGS Figure 1 is a schematic produce differentiation pathway β- cells from hESCs proposed. 该途径中的第一步为将ES细胞转变为定形内胚层细胞系,其代表进一步将ES细胞分化为胰腺内胚层、内分泌腺内胚层或胰岛/β-细胞最早的已知步骤之一。 The first step in the pathway for the ES cells into definitive endoderm cell line, which represents a further differentiation of ES cells into pancreatic endoderm, pancreatic endoderm one of the inner or endocrine / β- cells earliest known procedures. 可用于介导此转变的一些因子为TGFβ家族的成员,这些成员包括,但不局限于活化素、nodals和BMPs。 Some factors may be used to mediate this transition a member of the TGFβ family, these members include, but are not limited to, activin, nodals and BMPs. 确定定形内胚层靶细胞的代表性标志物为SOX17、GATA4、HNF3b、MIX1和CXCR4。 Determining a target cell definitive endoderm markers representative of SOX17, GATA4, HNF3b, MIX1 and CXCR4.

图2为人SOX17 cDNA图解,其显示了保守基序的位置并突出了用于GENOVAC的免疫方法的区域。 Human SOX17 cDNA illustrated in Figure 2, which shows the position of conserved motifs and a projection area for GENOVAC immunization method.

图3为一种相关性树状图,表明SOX17与SOX7的相关性最强,与SOX18的相关性稍弱。 FIG 3 as a correlation tree, SOX7 and SOX17 showed the strongest correlation, and the correlation of weaker SOX18. 在同源物种中的SOX17蛋白质比相同物种中的SOX群F亚族的其它成员的相关性强得多。 SOX17 protein homologous species much stronger correlation than other members of the subfamily SOX group F of the same species.

图4为以大鼠抗SOX17抗体为探针的蛋白质杂交(Western blot)。 FIG 4 is a rat anti-SOX17 antibody to a protein of the hybridization probe (Western blot). 该杂交证明了该抗体对成纤维细胞中过量表达的人SOX17蛋白质的特异性(泳道1),以及对EGFP(泳道2)或最相关的SOX家族成员SOX7(泳道3)缺少免疫反应性。 The hybridization demonstrated that the antibody to human fibroblasts overexpressing SOX17 specific proteins (lane 1), as well as EGFP (lane 2), or the most relevant SOX7 SOX family member (lane 3) the lack of immunoreactivity.

图5A-B为显示SOX17+细胞簇的显微照片,显示大量的共标记的AFP+细胞(A)。 Figures 5A-B show SOX17 + cell clusters of microscopic photograph showing a large number of co-labeled AFP + cells (A). 这与其它SOX17+细胞簇(B)观察到少量AFP+细胞或未观察到AFP+细胞形成鲜明对比。 This was observed with other cell clusters SOX17 + (B) or to a small amount of AFP + cells were AFP + cells was observed contrast.

图6A-C为显示体壁内胚层和SOX17的显微照片。 Figures 6A-C shows a photomicrograph of the inner body and SOX17 in parietal endoderm. 嵌图A显示对人凝血调节蛋白(TM)的免疫细胞化学,该蛋白质位于随机分化的hES细胞培养物中的体壁内胚层细胞的细胞表面。 Panel A shows the insert of human thrombomodulin protein immunocytochemistry (TM) of the protein on the cell surface of the inner wall of the body randomly differentiated hES cell cultures endoderm cells. 嵌图B为TM和SOX17双重标志显示的与嵌图A相同的区域。 Inset B is a double mark (TM) and SOX17 inset shows the same area A. 嵌图C为以DAPI标记核的相同区域的相差图像。 Figure C is fitted to the same region of the nuclear DAPI marked difference image. 注意DAPI标记核与SOX17标记完全相关。 Note DAPI labeling of nuclei labeled with SOX17 perfectly correlated.

图7A-B为显示定量PCR(Q-PCR)的SOX17基因表达和SOX17特异性抗体的抗SOX17阳性细胞的直方图。 Figures 7A-B is an anti-SOX17 positive cells display a histogram quantitative PCR (Q-PCR) and expression of the gene SOX17 SOX17 specific antibodies. 相对于非分化的对照培养基(SR20)而言,嵌图A显示活化素A增加SOX17基因表达,而视黄酸(RA)强烈抑制SOX17表达。 Differentiation with respect to non-control medium (SR20), the embedded Panel A shows activin A increased the expression of gene SOX17, and retinoic acid (RA) strongly inhibited SOX17 expression. 嵌图B表明,相同模式和相似程度的变化反应在SOX17+细胞数目上,表明SOX17基因表达的Q-PCR测试对单细胞水平的变化是非常敏感的。 Inset B showed the same pattern of change of the reaction and the degree of similarity in the number of SOX17 + cells showed Q-PCR test SOX17 gene expression changes in a single cell level is very sensitive.

图8A为直方图,显示在活化素A存在下分化的hESCs培养物保持低水平的AFP基因表达,而在10%的胎牛血清(FBS)中,细胞随机分化,显示AFP严重上调。 FIG 8A is a histogram showing the gene expression of AFP differentiation hESCs cultured under the presence of activin A was kept low, and in 10% fetal bovine serum (FBS), the cells randomly differentiate display upregulation AFP serious. 表达水平上的差异大约为7倍。 Differences in expression level is about 7 times.

图8B-C为两张显微照片图像,显示了活化素A对AFP表达抑制在单细胞水平也很明显,相对于仅用10%的FBS(顶部)而言,在活化素A处理的条件中(底部)观察到的AFP+细胞簇很少而且很小。 FIG. 8B-C of two micrograph shows the inhibition of activin A on AFP expression was also apparent at the single cell level, with respect to only 10% of FBS (top), in activin A treated condition ( bottom) observed AFP + few and small cell clusters.

图9A-B为对照图,显示使用流式细胞仪定量AFP+细胞数。 Figures 9A-B is a comparison view showing the use of quantitative AFP + cells by flow cytometry. 该图表明,在存在(右嵌图)或不存在(左嵌图)活化素A时AFP基因表达变化幅度(图8A)与AFP+细胞数非常一致,进一步表明Q-PCR分析对显示在单细胞水平上出现的变化的实用性。 This figure shows that in the presence (right inset) or absence (left inset) of AFP gene expression when activin A variation width (FIG. 8A) is consistent with the AFP + cells further showed Q-PCR analysis of single cells showed appearing on the practical level change.

图10A-F为显微照片,显示将hESCs暴露于nodal、活化素A和活化素B(NAA),在5天时间产生了细胞数的显著增加(AC)。 FIGS 10A-F is a micrograph showing the exposure of hESCs to nodal, activin A and activin B (NAA), produced a significant increase in the number of cells (AC) for about five days. 通过比较SOX17+细胞与该区域存在的细胞总量的相对量,如DAPI显色核(DF)所示,可以看出约30-50%的所有细胞在以NAA处理5天后对SOX17具有免疫反应性。 SOX17 + cells by comparing the relative amount of the total amount of cells present in the region, such as color DAPI nuclear (DF) as shown, it can be seen about 30-50% of all cells in the immune reactivity to SOX17 NAA for 5 days .

图11为直方图,显示活化素A(0、10、30或100ng/mL)剂量依赖性地增加分化的hESCs的SOX17基因表达。 FIG 11 is a histogram showing activin A (0,10,30 or 100ng / mL) dose-dependently increased differentiation of hESCs SOX17 gene expression. 在对粘附培养物处理3天后,继续再悬浮培养3~5天,表达增加已经很明显。 In the adhesion to the culture for 3 days, the suspension culture was continued for another 3 to 5 days, to increase the expression already evident.

图12A-C为直方图,证实了活化素A对MIXL1(嵌图A)、GATA4(嵌图B)及HNF3b(嵌图C)表达的作用。 Figures 12A-C is a histogram, confirming the role of activin A on MIXLl (inset A), of GATA4 (inset B) and HNF3b (inset C) expression. 在其它三个定形内胚层标志物MIXL1、GATA4及HNF3b中也观察到了对活化素呈剂量依赖性的增加。 In the other three definitive endoderm markers MIXL1, GATA4 and HNF3b also observed to increase in a dose-dependent activin. 对活化素剂量依赖性增加的表达幅度与SOX17的极其相似,强烈显示了活化素A特异作用于共表达所有四个基因(SOX17+,MIXL1+,GATA4+andHNF3b+)的细胞群。 Expression of very similar amplitude activin and dose-dependent increase of SOX17, strongly suggests a specific role of activin A on the expression of all four genes co (+ SOX17, MIXL1 +, GATA4 + andHNF3b +) cell population.

图13A-C为直方图,证实了活化素A对AFP(嵌图A)、SOX7(嵌图B)及SPARC(嵌图C)表达中的作用。 FIGS 13A-C is a histogram confirmed to AFP (inset A), action (inset B) and the SPARC (inset C) the expression of activin A SOX7. 活化素A剂量依赖性地降低内脏内胚层标志物AFP的表达。 Activin A dose-dependently decreased expression of visceral endoderm markers of AFP. 原始内胚层(SOX7)及体壁内胚层(SPARC)标志物仍然不变或仅在某些点显示抑制,表明活化素A并不特异作用于这些胚外内胚层细胞类型。 Primitive endoderm (SOX7) and parietal endoderm (SPARC) markers remained unchanged or only at certain points displayed inhibiting activin A showed no effect on specific cell types from these embryonic endoderm. 这进一步支持SOX17、MIXL1、GATA4及HNF3b的表达增加是由于活化素A引起定形内胚层细胞数量的增加。 This further supports the expression of SOX17, MIXL1, GATA4 and HNF3b the increase is due to increased number of activin A induced definitive endoderm cells of amorphous.

图14A-B为直方图,表明了活化素A对ZIC1(嵌图A)及Brachyury(嵌图B)表达的作用。 Figures 14A-B is a histogram indicates the effect (inset B) the expression of activin A on ZICl (inset A) and Brachyury. 神经标志物ZIC1的持续表达显示活化素A对神经分化无剂量依赖性的效果。 Sustained expression of neural markers ZIC1 display effect of activin A dose-dependent non-neural differentiation. 从brachyury表达降低可以看出,100ng/mL活化素A处理对中胚层的分化有显著的抑制。 As can be seen from the reduced expression brachyury, 100ng / mL activin A treatment significantly inhibit the differentiation of mesoderm. 这可能是来自中内胚层前体的定形内胚层特异性增加的结果。 This may be the amorphous former endoderm body from the mesoderm increased specificity results. 与无处理的空白对照培养物相比,低水平的活化素A处理(10及30ng/mL)在分化后期时间点保持了brachyury表达。 Compared to non-treated vehicle control cultures, low levels of activin A treatment (10 and 30ng / mL) held at a time point late differentiation brachyury expression.

图15A-B为显微照片,活化素处理引起体壁内胚层分化降低。 FIG. 15A-B is a photomicrograph, activin treatment causes parietal endoderm reduced. 在仅以血清分化的培养物(A)内发现TMhi体壁内胚层区域,然而当包括活化素(B)时很少分化为TM+细胞及总免疫反应性的强度较低。 Found within the region TMhi parietal endoderm differentiation in a culture of serum only (A), but when included activin (B) TM + rarely differentiated into cells and a lower total intensity of immunoreactivity.

图16A-D为显微照片,表明活化素A及活化素B处理引起的标志物表达。 FIGS 16A-D is a photomicrograph, shows that the expression of markers activin A and activin B treatment caused. 连续以活化素A及活化素B处理hESCs4天,以SOX17、AFP及TM抗体进行三标记。 Sequentially with activin A and activin B treatment hESCs4 days, SOX17, AFP and TM three labeled antibody. 嵌图A-SOX17;嵌图B-AFP;嵌图C-TM;及嵌图D-Phase/DAPI。 Inset A-SOX17; inset B-AFP; inset C-TM; and the inset D-Phase / DAPI. 注意当完全无AFP(B)及TM(C)免疫反应性时可见大量的SOX17阳性细胞(A)。 Note that when completely AFP (B) and TM (C) immunoreactivity SOX17 visible when a large number of positive cells (A).

图17是显微图片,表明在体外自hESCs出现定形内胚层及内脏内胚层。 FIG 17 is a photomicrograph, shows that visceral endoderm and endoderm in vitro appears from hESCs. 内脏内胚层区域以AFPhi/SOX17lo/-鉴定,然而定形内胚层显示了完全相反的特征,SOX17hi/AFPlo/-。 Visceral endoderm region AFPhi / SOX17lo / - however endoderm show exactly opposite characterization,, SOX17hi / AFPlo / -. 选择该域是由于这两个区域彼此接近,然而,有多次在完全分离的AFPhi细胞区域观察到SOX17hi/AFPlo/-区域,表明部分定形内胚层细胞衍生于内脏内胚层细胞。 This field is due to the choice of these two regions close to each other, however, there are several completely isolated cell region AFPhi observed SOX17hi / AFPlo / - region, inner portion showed definitive endoderm cells derived from visceral endoderm cells.

图18为描述TGFβ家族配体及受体的图表。 FIG 18 is a diagram depicting TGFβ family ligands and receptors. 活化AR-Smads及BR-Smads的因子有利于从人胚胎干细胞产生定形内胚层(参见,J CellPhysiol.187:265-76)。 Activation of AR-Smads and BR-Smads factor facilitates the generation of definitive endoderm (see, J CellPhysiol.187: 265-76) from human embryonic stem cells.

图19为直方图,表明以单一或多种TGF-β因子处理诱导的SOX17随时间表达。 FIG 19 is a histogram shows that a single factor or more TGF-β-induced process SOX17 expression over time.

图20为直方图,表明以多种TGF-β因子处理引起SOX17+细胞数量随时间的增加。 FIG 20 is a histogram to show a variety of factors TGF-β treatment causes SOX17 + increase in cell number over time.

图21为直方图,表明以多种TGF-β因子处理诱导的SOX17随时间表达。 FIG 21 is a histogram to show that a variety of processing factors TGF-β-induced SOX17 expression over time.

图22为直方图,表明活化素A剂量依赖性地增加SOX17+细胞数量。 FIG 22 is a histogram activin A showed a dose-dependent increase in cell number SOX17 +.

图23为直方图,显示Wnt3a加入至活化素A及活化素B处理的培养物中增加SOX17表达,高于活化素A及活化素B单独使用诱导的水平。 FIG 23 is a histogram showing Wnt3a was added to cultures of Activin A and Activin B in treatment increased the expression of SOX17, than activin A and activin B alone induced level.

图24A-C为直方图,表明在低FBS条件下,分化为定形内胚层增加。 FIGS 24A-C is a histogram shows that under conditions of low FBS differentiated into definitive endoderm increase. 在包括2%FBS(2AA)的培养基中以活化素A及B处理hESCs与在10%FBS(10AA)(嵌图A)中同样条件下处理比,SOX17表达水平高2-3倍。 In a medium comprising 2% FBS (2AA) of activin A and B to hESCs treated under the same conditions with the treatment ratio in 10% FBS (10AA) (inset A), SOX17 expression levels 2-3 times higher. 定形内胚层标志物MIXL1(嵌图B)也以相同的方式受到影响,而且2%FBS对AFP(内脏内胚层)(嵌图C)的抑制比在10%FBS条件下高。 Definitive endoderm markers MIXLl (inset B) are also affected in the same manner, but 2% FBS inhibition of AFP (visceral endoderm) (inset C) is higher than in the 10% FBS conditions.

图25A-D为显微照片,显示培养中的SOX17+细胞分裂。 FIGS 25A-D is a micrograph showing SOX17 + cells divide in culture. SOX17免疫反应性细胞处于hESC克隆分化边缘(C,D),以增殖细胞细胞核抗原(PCNA)(嵌图B)标记,而不以OCT4(嵌图C)共标志。 SOX17 immunoreactive cells in hESC differentiation clones edge (C, D), proliferating cell nuclear antigen to (of PCNA) (inset B) tags, and not to of OCT4 (insert panel C) were flag. 此外,以DAPI标记核,在SOX17+细胞(箭头)及OCT4+,未分化的hESCs(箭头)(D)中可清晰看见有丝分裂图片。 Further, with DAPI labeling of nuclei in SOX17 + cells (arrows) and OCT4 +, undifferentiated of hESCs (arrow) (D) can be clearly visible image mitosis.

图26为直方图,显示在各种培养基中,CXCR4在正在分化的hESCs中的相对表达水平。 FIG 26 is a histogram showing the various media, relative expression levels of CXCR4 in differentiating hESCs.

图27A-D为直方图,通过与图26相同处理,显示一系列定形内胚层标志物如何具有非常相似的CXCR4表达模式。 FIGS 27A-D histogram, FIG. 26 by the same process, the display range of the definitive endoderm markers CXCR4 how to have a very similar expression patterns.

图28A-E为直方图,显示中胚层(BRACHYURY,MOX1)、外胚层(SOX1,ZIC1)及内脏内胚层(SOX7)标志物如何通过与图26相同处理具有相反的CXCR4表达。 FIG. 28A-E is a histogram showing how the mesoderm (BRACHYURY, MOX1), ectoderm (SOX1, ZIC1) and visceral endoderm (SOX7) marker having opposite CXCR4 expression by the same processing in FIG. 26.

图29A-F为显微照片,显示在图26-28三种培养基条件下SOX17免疫反应性细胞的相对差异。 FIGS 29A-F is a micrograph showing the relative difference in SOX17 immunoreactive cells at three culture conditions 26-28 in FIG.

图30A-C为流式细胞仪点图,证实随着加入至分化培养基中的活化素A的浓度增加,CXCR4+细胞数量增加。 FIGS 30A-C is a flow cytometry dot plot, it was confirmed with increasing concentrations was added to the differentiation medium activin A, CXCR4 + cells increase in number.

图31A-D为直方图,显示高剂量活化素A处理(A100-CX+)后分离的CXCR4+细胞的定形内胚层标志物数量较其母体细胞群(A100)更为丰富。 FIGS 31A-D is a histogram showing the number of the isolated amorphous high dose activin A treatment (A100-CX +) of CXCR4 + cells endoderm markers is more abundant than the parent cell population (A100).

图32为直方图,显示使用荧光活化细胞分选器(FACS)分离的CXCR4+及CXCR4-细胞及母体细胞群的基因表达。 FIG 32 is a histogram showing the use of fluorescence activated cell sorter (FACS) gene isolated and CXCR4- CXCR4 + cells and maternal cell population. 这证实CXCR4+细胞基本包括所有的在各母体细胞群上存在的CXCR4基因表达,CXCR4-则包括很少或不包括CXCR4基因表达。 This demonstrates CXCR4 + cells comprises substantially all of the CXCR4 gene expression in the presence of each of the parent cell population, CXCR4- comprising little or no comprising the gene expression of CXCR4.

图33A-D为直方图,证实高剂量活化素A处理的CXCR4+细胞的中胚层(BRACHYURY,MOX1)、外胚层(SOX1,ZIC1)及内脏内胚层(SOX7)基因表达缺失,这些非定形内胚层标志物的表达被抑制。 FIGS 33A-D is a histogram confirm CXCR4 + mesoderm cells (BRACHYURY, MOX1), ectoderm (SOX1, ZIC1) and visceral endoderm high dose of activin A treatment (SOX7) gene absence, these non-definitive endoderm expression of a marker is suppressed.

图34A-M为直方图,表明标志物基因表达模式,其可用于鉴定定形内胚层细胞。 FIGS 34A-M is a histogram markers indicated that gene expression patterns, which can be used to identify definitive endoderm cells. 定形内胚层标志物FGF17、VWF、CALCR、FOXQ1、CMKOR1及CRIP1的表达分析分别如嵌图GL所示。 Definitive endoderm markers FGF17, expression analysis VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 are fitted as shown in FIG GL. 前述谱系标志基因SOX17、SOX7、SOX17/SOX7、TM、ZIC1及MOX1分别如嵌图AF所示。 The lineage marker gene SOX17, SOX7, SOX17 / SOX7, TM, ZIC1 MOX1 and AF respectively insert as shown in FIG. 嵌图M显示CXCR4的表达分析。 Analysis of expression of CXCR4 M inset display. 关于嵌图AM,标记hESC一栏显示纯化的人胚胎干细胞基因表达;2NF显示以2%FBS处理的细胞,不加活化素;0.1A100显示以0.1%FBS、100ng/ml活化素A处理的细胞;1A100显示以1%FBS、100ng/ml活化素A处理的细胞;2A100显示以2%FBS、100ng/ml活化素A处理的细胞。 About inset AM, labeled hESC column shows purified human embryonic stem cell gene expression; 2NF displayed in 2% FBS treated cells without activin; 0.1A100 display cells in 0.1% FBS, 100ng / ml activin A treatment ; 1A100 displayed in 1% FBS, a treated 100ng / ml activin cell; 2A100 display cells 2% FBS, 100ng / ml activin a treatment.

发明详述人早期发育的一个关键阶段,即术语原肠胚形成发生在受精2-3周后。 Detailed Description of a critical phase of early human development of the invention, i.e., the term & gastrulation occurs 2-3 weeks after fertilization. 原肠胚形成极端重要,因为在此期三个原始胚首先专一化和有序化(Lu et al.,2001;Schoenwolf and Smith,2000)。 Gastrulation extremely important, because in this period three primary germ first single-minded and ordering (Lu et al, 2001;. Schoenwolf and Smith, 2000). 外胚层负责身体外层及全部神经系统的形成,然而,心脏、血液、骨骼、骨骼肌及其它结缔组织均衍生于中胚层。 Ectoderm responsible for forming the outer layer of the body and all of the nervous system, however, heart, blood, bone, skeletal muscle and other connective tissues are derived from mesoderm. 定形内胚层定义为负责全部肠管形成的胚层,该全部的肠管包括食道、胃、小肠及大肠,以及从肠道衍生的器官,如肺、肝、胸腺、甲状旁腺及甲状腺、胆囊及胰(Grapin-Botton and Melton,2000;Kimelman and Griffin,2000;Tremblay et al.,2000;Wells and Melton,1999;Wells and Melton,2000)。 Is defined as the endoderm germ layer is responsible for all of the amorphous form of the bowel, the esophagus include all of the intestine, stomach, small intestine and large intestine, and derived from the intestinal organs, such as lungs, liver, thymus, parathyroid, thyroid, 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). 定形内胚层与称为原始内胚层的完全分离的细胞系之间有着非常明显的不同。 And referred to definitive endoderm has a very clear difference between the fully isolated primitive endoderm cell lines. 原始内胚层主要形成胚外组织,主要是胎盘卵黄囊的体壁及内脏内胚层部分及Reichert′s膜的细胞外基质材料。 Primitive endoderm mainly formed extraembryonic tissues, mainly extracellular parietal yolk sac and placenta visceral endoderm Reichert's membrane and the portion of the matrix material.

在原肠胚形成过程中,定形内胚层形成过程始于细胞迁徙事件,其中,中内胚层细胞(能够形成中胚层或内胚层的细胞组分)穿过一个称做原线的结构。 In the process of gastrulation, the formation of definitive endoderm cell migration event begins, wherein the mesodermal cells (mesoderm cell components or can be formed endoderm) through a structure called the original line. 定形内胚层衍生于穿过原线前部及结(一个位于原线最前面部分的特定结构)的细胞。 Endoderm derived from the original line through the junction and the front portion (a portion located in front of the specific structure of the original line) cells. 当迁徙发生时,定形内胚层首先形成肠管的最前面部分直至形成肠管后端时结束。 When migration occurs, definitive endoderm foremost part of the intestine is first formed until the end of the rear end of the bowel is formed.

定形内胚层形成的体内分析,如Conlon et al.,1994;Feldman et al.,1998;Zhou et al.,1993;Aoki et al.,2002;Dougan et al.,2003;Tremblay etal.,2000;Vincent et al.,2003;Alexander et al.,1999;Alexander and Stainier,1999;Kikuchi et al.,2001;Hudson et al.,1997及小鼠Kanai-Azuma et al.,2002进行的斑马鱼及非洲蟾蜍的研究,这些研究为如何在培养皿中使用人胚胎干细胞完成特定胚层细胞类型的发育奠定了基础。 Vivo definitive endoderm formation analysis, as Conlon et al, 1994;. Feldman et al, 1998;. Zhou et al, 1993;. Aoki et al, 2002;. Dougan et al, 2003;. Tremblay etal, 2000.; Vincent et al, 2003;. Alexander et al, 1999;. Alexander and Stainier, 1999; Kikuchi et al, 2001;. Hudson et al, 1997 and mouse Kanai-Azuma et al, 2002 conducted in zebrafish and Africa. toad research, studies on how to use human embryonic stem cells in a petri dish complete cell type specific mesoderm basis. 与体外ESC培养相关的两个方面构成了在培养皿中重启发育的主要瓶颈。 ESC culture in vitro with two related aspects constitute a major bottleneck in the development restart dish. 首先,不会产生有序的胚层或器官结构。 First of all, no orderly mesodermal organ or structure. 在正在分化的hESC培养系统中,大部分胚层及器官的特异遗传标志物会以异源的形式来表达。 In differentiating hESC culture system, most organs and mesoderm specific genetic marker can be in the form of a heterologous expression. 因此,由于缺乏器官特异性界限,很难估计特异性组织或细胞类型的形成。 Therefore, due to the lack of organ-specific limits, it is difficult to estimate specific tissue or cell type formed. 在一个具体胚层的细胞类型或组织类型中表达的几乎所有基因也在其它胚层的细胞类型或组织类型中表达。 Is also expressed in other cell types or tissue types endoderm almost all genes expressed in a cell type or tissue type specific endoderm. 没有特异的界限,很难以1-3个基因样本赋予基因表达的特异性。 No specific limits, it is difficult to samples 1-3 genes confer specific gene expression. 因而,必须仔细检测相当多的基因,有些基因应当也可在不感兴趣器官或组织的具体细胞类型上表达。 Therefore, must be carefully monitored a considerable number of genes, some genes may also be interested in not expressed on specific cell types of the organ or tissue. 其次,基因表达类型的时机对沿特定通道发育的活动至关重要。 Secondly, the expression patterns of genes critical to the timing of development activities in a particular channel.

至于更复杂的事件,应当指出,体外干细胞分化是相当不同步的且可能比体内要显著得多。 For a more complex event, it should be noted that the in vitro differentiation of stem cells is quite out of sync and possibly much more significant than the body. 这样,一组细胞可能在表达与原肠胚形成有关的基因时,而另一组可能正在开始最终的分化。 In this way, a group of cells may be formed in the expression of genes associated with gastrulation, while the other set may be beginning the final differentiation. 而且,在具有或不具有外源因素参与时,处理hESC单层或胚状体(EBs)可能导致全部基因表达模式或分化状态的显著差异。 Further, when with or without exogenous factors are involved, a single layer or processing hESC embryoid bodies (the EBs) can result in significant differences in gene expression patterns of all or differentiation state. 因而,为了有效地沿特定的分化通道前进,根据异质细胞混合物的基因表达模式,外源因子的使用必须进行时间控制。 Accordingly, in order to effectively advanced along a particular path of differentiation, gene expression pattern according to a heterogeneous mixture of cells, exogenous factors must be time controlled. 在培养容器里考虑这些细胞的形态学关系也是有益的。 Consider the relationship between the morphology of these cells in the culture container is also beneficial. 与在培养基容器中成长或分化为单层和/或hESC克隆时能力相比,均一影响形成所谓胚状体时的hESCs的能力远非最理想。 And growth or differentiation in a culture vessel capacity when compared to a single layer and / or cloning hESC, homogeneity affect the ability of hESCs when a so-called embryoid body formation is far from ideal.

作为处理上述异质和不同步的一个有效方法,本发明的一些实施方案考虑将分化细胞的方法与富集、分离和/或纯化分化通道中的中间细胞类型的方法联合。 As the heterogeneous process and not an effective method for synchronizing, some embodiments of the present invention contemplates a method and enrichment of differentiated cells, and / or purification of the intermediate passage differentiation of cell types combined separation methods.

本发明实施方案涉及在培养物中生成定形内胚层细胞的新的确定方法,其通过将诸如干细胞的多能细胞分化为多能定形内胚层细胞。 Embodiments of the invention relates to a new method of determining definitive endoderm cells generated in culture, by cells such as pluripotent stem cells to differentiate into definitive endoderm pluripotent cells. 本文使用的“多能”或“多能细胞”指能产生有限数量的其它具体细胞类型的细胞类型。 As used herein, "pluripotent" or "pluripotent cell" refers to a cell capable of producing other types of specific cell types in a limited number. 如上所述,定形内胚层细胞并不分化为源于外胚层或中胚层的组织,然而,可分化为肠管及源于肠管的器官。 As described above, definitive endoderm cells do not differentiate into tissues derived from ectoderm or mesoderm, however, can be divided into intestinal and bowel derived organs. 在一些优选的实施方案中,定形内胚层细胞衍生于hESCs。 In some preferred embodiments, the definitive endoderm cells derived from hESCs. 这些方法提供了有效生成人内胚层衍生的组织,如胰、肝、肺、胃、肠及甲状腺的基础。 These methods provide the effective production of human endoderm-derived tissues, such as basic pancreas, liver, lung, stomach, intestine and thyroid. 例如,定形内胚层的生成第一步可分化干细胞为功能性的产生胰岛素的β细胞。 For example, amorphous generated endoderm differentiation of stem cells may be the first step in the functional insulin-producing β cells. 为了获得有用量的产生胰岛素的β细胞,在达到胰岛/β细胞前,期望每个分化步骤都是高效的分化步骤。 To obtain an amount of the insulin-producing beta] cells, before reaching the islet / β cells, is desirable for each step of differentiation efficient differentiation step. 因为干细胞分化为定形内胚层细胞也许代表着生成功能性胰岛/β细胞的最初步骤(如图1所示),特别需要此步分化高效。 Because stem cells into definitive endoderm cells may represent the initial step of generating functional islet / β cells (Figure 1), this step requires special differentiation efficiency.

鉴于分化多能细胞至定形内胚层细胞的需要,本发明一些方面涉及体外方法学,将约50-80%的多能细胞转变为定形内胚层细胞。 In view of the need for differentiation of pluripotent cells into definitive endoderm cells, some aspects of the present invention is directed to in vitro methodology, about 50-80% of the pluripotent cells into definitive endoderm cells. 典型地,这些方法包括已定义及暂时指定的方式使用培养物及生长因子。 Typically, these methods include a defined manner and using the temporary designated culture and growth factors. 使用可与定形内胚层细胞特异结合的试剂,从细胞群中将定形内胚层细胞与其它细胞分离和/或纯化,可获得定形内胚层细胞群的更多富集。 Be used in combination with the cell-specific reagent endoderm, mesoderm cells and other cells isolated and / or purified, more enriched cell populations can be obtained from the endoderm in the inner cell mass setting. 这样,本发明涉及定形内胚层细胞及制备分离和/或纯化这些细胞的方法。 Thus, the present invention relates to a definitive endoderm cell separation method and the preparation and / or purification of these cells.

为了确定细胞培养物或细胞群内定形内胚层细胞的数量,需要从培养物或细胞群中区分这类细胞与其它细胞的方法。 To determine the number of inner cell culture or cell population of definitive endoderm cells, such cells and methods need to be distinguished from other cells in culture or cell population. 因此,本发明实施方案涉及细胞标志物,其存在、缺失和/或相对表达水平对定形内胚层是特异的,以及检测确定这些标志物表达的方法。 Thus, embodiments of the present invention relates to a cellular marker whose presence, absence and / or the relative expression levels of definitive endoderm is specific, and the expression of these markers is determined. 此处使用的“表达”指材料或物质的产生,或者材料或物质产生的水平或含量。 "Expression" as used herein refers to the production of a material or substance, or the level or amount of materials or substances. 因而,确定特异标志物的表达指检测表达的标志物的相对或绝对含量,或仅检测标志物是否存在。 Thus, specific markers to determine the relative or absolute expression refers to quantitative expression of the detectable marker, or only detectable marker is present. 此处使用的“标志物”指能够被观察或检测到的任何分子。 As used herein, "marker" refers to any molecule capable of being observed or detected. 例如,标志物可包括但不局限于核酸,如特异基因的转录本、基因的多肽产物、非基因多肽产物、糖蛋白、糖、糖脂、脂质、脂蛋白或小分子。 For example, the marker may include, but are not limited to nucleic acids, such as specific gene transcript, the polypeptide product of a gene, a non-polypeptide gene products, glycoproteins, glycopeptides, glycolipids, lipids, lipoproteins, or small molecules.

在本发明的一些实施方案,标志物表达的存在与否和/或其水平由定量PCR(Q-PCR)确定。 In some embodiments of the present invention, the expression of marker presence or absence thereof and / or a level determined by quantitative PCR (Q-PCR). 例如,一些遗传标志物产生的转录本量,例如SOX17、CXCR4、OCT4、AFP、TM、SPARC、SOX7、MIXL1、GATA4、HNF3b、GSC、FGF17、VWF、CALCR、FOXQ1、CMKOR1、CRIP1及本文所述的其它标志物,由定量Q-PCR确定。 For example, the amount of transcript produced in a number of genetic markers, e.g. SOX17, CXCR4, OCT4, AFP, TM, SPARC, SOX7, MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1, CRIP1 and described herein other markers, determined by quantitative Q-PCR. 在其它实施方案中,Q-PCR及免疫组化技术用于识别及确定这些标志物的量或相对比例。 In other embodiments, Q-PCR and immunohistochemical techniques for identifying and determining the amount or relative proportions of these markers.

通过使用如上述确定的一种或多种合适的标志物表达的方法,在细胞培养物或细胞群内识别定形内胚层细胞以及确定定形内胚层细胞的比例是可能的。 By using such as one or more of the above-identified expression of a suitable marker, identifying cells in a definitive endoderm cell culture or cell population and determining the proportion of definitive endoderm cells it is possible. 例如,在本发明的一些实施方案中,产生的定形内胚层细胞或细胞群表达SOX17和/或CXCR4基因的水平比非定形内胚层细胞类型或细胞群高约2个数量级。 For example, in some embodiments of the present invention, the resulting definitive endoderm cells or cell populations SOX17 expression and / or levels of CXCR4 gene than non-definitive endoderm cell type or cell population of from about 2 orders of magnitude. 在其它实施方案中,产生的定形内胚层细胞或细胞群表达SOX17和/或CXCR4基因的水平比非定形内胚层细胞类型或细胞群高2个以上数量级。 In other embodiments, the resulting definitive endoderm cells or cell populations of two or more SOX17 expression and / or levels of CXCR4 gene than non-definitive endoderm cell types or cell populations of magnitude. 在另一些其它实施方案中,产生的定形内胚层细胞或细胞群表达一种或多种选自SOX17、CXCR4、GSC、FGF17、VWF、CALCR、FOXQ1、CMKOR1及CRIP1的标志物,比非定形内胚层细胞类型或细胞群的表达高约2个或2个以上的数量级。 In still other embodiments, the resulting definitive endoderm cells or cell populations expressing one or more selected SOX17, CXCR4, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 marker, than the non-amorphous high expression of mesodermal cell types or cell populations of two or more, or about two orders of magnitude.

本发明涉及细胞培养物,包括具有大量定形内胚层及包含富集的定形内胚层细胞的细胞群。 The present invention relates to a cell culture, comprising a large number of the definitive endoderm population of cells and definitive endoderm cells comprising enriched. 由此,一些实施方案涉及包括定形内胚层细胞的细胞培养物,其中所述培养物中至少约50-80%的细胞为定形内胚层细胞。 Accordingly, some embodiments relate to cell cultures comprising definitive endoderm cells, wherein said culture of at least about 50-80% of the cells to definitive endoderm cells. 一个优选实施方案涉及包括人细胞的细胞培养物,其中培养物中至少约50-80%人细胞为定形内胚层细胞。 A preferred embodiment relates to a cell culture comprising human cells, wherein the culture is at least about 50-80% of the human cells to definitive endoderm cells. 由于分化方法的效果可以通过改变一些参数调节,包括但不限于细胞生长条件、生长因子浓度及培养步骤的时间,本发明所述的分化方法可使约5%、约10%、约15%、约20%、约25%、约30%、约约40%、约45%、约50%、约55%、约60%、约65%、约70%、约75%、约80%、约85%、约90%、约95%、或大于95%的多能细胞转化为定形内胚层。 Due to the effect of the differentiation process can be adjusted by varying several parameters, including, but not limited to, cell growth conditions, the growth factor concentrations and the time of the culturing step, the differentiation method of the present invention enables to about 5%, about 10%, about 15% about 20%, about 25%, about 30%, from about to 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 greater than 95% pluripotent cells are transformed into definitive endoderm. 在本发明其它实施方案中,将诸如干细胞群体的多能细胞群转化为基本纯的定形内胚层细胞。 In other embodiments of the present invention, such as a pluripotent stem cell population into a population of cells substantially pure definitive endoderm cells.

本发明所述的组合物及方法具有几个有用的特征。 Compositions and methods of the present invention has several useful features. 例如,包括定形内胚层的细胞培养物及细胞群,以及制备这些细胞培养物及细胞群的方法可用于模建人发育的早期阶段。 For example, including the definitive endoderm population of cells and cell cultures, and cell culture methods and populations of cells can be used to prepare these modeling the early stages of human development. 此外,本发明所述的组合物及方法也可用于如糖尿病的疾病的干预治疗。 Furthermore, the compositions and methods of the present invention may also be used for therapeutic intervention in diseases such as diabetes. 例如,由于定形内胚层仅为有限数量的组织来源,因而其可用于发育纯的组织或细胞类型。 For example, since the definitive endoderm only a limited number of tissue sources, and thus it can be used for pure tissue or cell type development.

从多能细胞制备定形内胚层包括本文所述的定形内胚层细胞的定形内胚层细胞培养物及组合物可自如胚胎干细胞的多能细胞中制备。 Prepared from amorphous endodermal multipotent cells within the cell culture preparation comprising amorphous shaped inner herein endoderm endoderm cells can be freely and compositions thereof embryonic stem cells are pluripotent cells. 本文使用的“胚胎”指有机体发育阶段范围,其自单个的受精卵开始至除发育的配子细胞以外不再包含多能或全能细胞的多细胞结构结束。 As used herein, "embryo" refers to a range of developmental stages of an organism, which is to start from a single fertilized egg cell development other than gametes end can no longer contain multiple multicellular structure or totipotent cells. 除衍生于配偶子融合的胚胎,术语“胚胎”指衍生于体细胞核转移的胚胎。 In addition to the gametes derived embryos fused, the term "embryo" means derived from somatic cell nuclear transfer embryos. 优选的衍生定形内胚层细胞的方法利用人胚胎干细胞作为制备定形内胚层的起始材料。 The preferred method of the definitive endoderm cells derived from human embryonic stem cells using the preparation as a starting material of definitive endoderm. 该方法使用的胚胎干细胞可以是衍生于桑椹胚、胚胎内细胞团或从胚胎性腺嵴获得的细胞。 The method using embryonic stem cells may be derived from morula, the inner cell mass or cells obtained from embryonic gonadal ridges. 使用现有技术方法,人干细胞可在培养基中保持多能状态而基本不分化。 Using the prior art method, human stem cells can be maintained in a pluripotent state in culture without substantial differentiation. 所述的方法,例如,美国专利第5,670,5,690,9265,843,6,200,806及6,251,671号中公开的方法,本文全部引入,以供参考。 The method according to, e.g., U.S. Patent Publication No. 6,251,671 and the 5,670,5,690,9265,843,6,200,806, fully incorporated herein by reference.

在本发明所述方法的一些实施方案中,hESCs保持在滋养层。 In some embodiments of the method according to the present invention, hESCs maintained in the feeder layer. 在这些实施方案中,任何能够使hESCs保持在多能状态的滋养层可用于本发明所述的方法中。 In these embodiments, any method capable of maintaining hESCs in a pluripotent state feeder layer may be used in the present invention. 一个常用的培养人胚胎干细胞的滋养层为一层小鼠成纤维细胞。 A common culture human embryonic stem cells to trophoblast layer of mouse fibroblasts. 最近,人成纤维细胞滋养层也已开发出来用于培养hESCs(参见美国专利申请号2002/0072117中公开的方法,本文全部引入,以供参考)。 Recently, human fibroblast feeder layer have also been developed for the culture of hESCs (see, U.S. Patent Application No. 2002/0072117 disclosed a method, fully incorporated herein by reference). 本发明所述方法的其它实施方案允许不使用滋养层保持多能hESCs。 Other embodiments of the method of the present invention permit dispensing with feeder layer to maintain pluripotent hESCs. 这些方法如美国专利申请号2003/0175956所述,其公开在此全部引入,以供参考。 The method as described in U.S. Patent Application No. 2003/0175956, the entire disclosure of which is incorporated herein by reference.

本发明所述的人胚胎干细胞可以保持在具有或不具有血清的培养基中。 The present invention may be human embryonic stem cells maintained in medium with or without serum. 在一些实施方案中,使用血清替代品。 In some embodiments, a serum replacement. 在其它实施方案,无血清的培养基技术,如美国专利申请号2003/0190748所述,其公开在此全部引入,以供参考。 In other embodiments, serum-free medium technology, as described in US Patent Application No. 2003/0190748, the entire disclosure of which is incorporated herein by reference.

在培养基中保持多能状态的干细胞按照常规传代,直到分化为定形内胚层。 Maintaining a pluripotent state in culture medium of stem cells according to routine passage until differentiate into definitive endoderm. 在一些实施方案中,分化至定形内胚层的完成是通过向干细胞培养基中加入TGFβ超家族生长因子,其加入的量足以促进分化至定形内胚层。 In some embodiments, the differentiation to definitive endoderm is completed by addition of the TGFβ superfamily of growth factors stem cell culture medium, which was added an amount sufficient to promote differentiation to definitive endoderm. 用于制备定形内胚层的TGFβ超家族生长因子选自Nodal/活化素或BMP亚群。 TGFβ superfamily definitive endoderm prepared for growth factor selected Nodal / Activin or BMP subgroups. 在本发明所述分化方法的一些实施方案中,生长因子选自Nodal、活化素A、活化素B及BMP4。 In some embodiments of the present invention of the differentiation process, the growth factor is selected from Nodal, activin A, activin B and BMP4. 此外,生长因子Wnt3a及其它Wnt家族成员可用于制备定形内胚层细胞。 In addition, other growth factors Wnt3a and Wnt family members may be used for the preparation of the definitive endoderm cells. 在本发明的一些实施方案中,可以使用上述提及的任何生长因子。 In some embodiments of the present invention, any growth factors mentioned above.

在本发明所述分化方法的一些实施方案中,向细胞中加入的上述生长因子,其在培养基中的浓度足以促进至少部分干细胞分化至定形内胚。 In some embodiments of the present invention of the differentiation process, the cells were added to the growth factors in the medium at a concentration sufficient to promote differentiation of stem cells at least partially into definitive endoderm. 在本发明的一些实施方案中,培养基中上述生长因子的浓度至少约5ng/ml、至少约10ng/ml、至少约25ng/ml、至少约50ng/ml、至少约75ng/ml、至少约100ng/ml、至少约200ng/ml、至少约300ng/ml、至少约400ng/ml、至少约500ng/ml、至少约1000ng/ml、至少约2000ng/ml、至少约3000ng/ml、至少约4000ng/ml、至少约5000ng/ml或大于5000ng/ml。 In some embodiments of the present invention, the above-mentioned growth factor concentration in the medium 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 200 ng of / ml, at least about 300ng / ml, at least about 400ng / ml, at least about 500ng / ml, at least about lOOOng / ml, at least about 2000ng / ml, at least about 3000 ng / ml, at least about 4000ng / ml , at least about 5000ng / ml or more than 5000ng / ml.

在本发明一些实施方案中,上述生长因子加入培养基后需要从细胞培养物除去。 In some embodiments of the present invention, the growth factor needs to be removed from the cell culture medium after the addition. 例如,生长因子的除去时间约在加入后1天、约2天、约3天、约4、约5天、约6天、约7天、约8天、约9天或约10天。 For example, time for removing the growth factor for about 1 day after the addition, 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. 在一个优选实施方案中,生长因子的除去时间约在加入后4天。 In a preferred embodiment, the time for removing the growth factor for about 4 days after addition of.

定形内胚层细胞的培养可在包含少量血清或无血清的培养基中。 Endoderm cells cultured in serum-shaped or may include a small amount of serum-free medium. 在本发明的一些实施方案中,血清的浓度范围为约0.05%v/v至约20%v/v。 In some embodiments of the present invention, the serum concentration in the range of about 0.05% v / v to about 20% v / v. 例如,在一些实施方案中,培养基中血清的浓度可以低于约0.05%(v/v)、低于约0.1%(v/v)、低于约0.2%(v/v)、低于约0.3%(v/v)、低于约0.4%(v/v)、低于约0.5%(v/v)、低于约0.6%(v/v)、低于约0.7%(v/v)、低于约0.8%(v/v)、低于约0.9%(v/v)、低于约1%(v/v)、低于约2%(v/v)、低于约3%(v/v)、低于约4%(v/v)、低于约5%(v/v)、低于约6%(v/v)、低于约7%(v/v)、低于约8%(v/v)、低于约9%(v/v)、低于约10%(v/v)、低于约15%(v/v)或低于约20%(v/v)。 For example, in some embodiments, the serum concentration in the medium may be less than about 0.05% (v / v), less than about 0.1% (v / v), less than about 0.2% (v / v), below about 0.3% (v / v), less than about 0.4% (v / v), less than about 0.5% (v / v), less than about 0.6% (v / v), less than about 0.7% (v / v), less than about 0.8% (v / v), less than about 0.9% (v / v), less than about 1% (v / v), less than about 2% (v / v), less than about 3% (v / v), less than about 4% (v / v), less than about 5% (v / v), less than about 6% (v / v), less than about 7% (v / v ), less than about 8% (v / v), less than about 9% (v / v), less than about 10% (v / v), less than about 15% (v / v) or less than about 20 % (v / v). 在一些实施方案中,定形内胚层细胞在无血清下生长。 In some embodiments, definitive endoderm cells were grown in serum free. 在其它实施方案中,定形内胚层细胞在血清替代品存在下生长。 In other embodiments, definitive endoderm cells were grown in the presence of serum replacement. 在其它实施方案中,定形内胚层细胞在B27存在下生长。 In other embodiments, definitive endoderm cells were grown in the presence of B27. 在这些实施方案中,B27添加物的浓度范围为约0.2%至约20%v/v。 In these embodiments, the concentration of B27 supplement is from about 0.2% to about 20% v / v.

hESC培养至定形内胚层可以通过确定定形内胚层的标志物特征表达监测。 hESC cultures to definitive endoderm can be monitored by determining the expression of the features of the marker of definitive endoderm. 在一些实施方案中,一些标志物的表达可通过是否存在标志物来确定。 In some embodiments, the expression of some markers can be determined whether the presence of the marker. 可选择地,一些标志物的表达可通过测定标志物在细胞培养物或细胞群细胞中的水平来确定。 Expression Alternatively, some of the markers may be determined in cell culture cells or population of cells by measuring levels of markers. 在这些实施方案中,可以定性或定量测定标志物的表达。 In these embodiments, expression of a marker may be qualitative or quantitative determination. 定量标志物基因产生的表达标志物的方法可以使用定量PCR(Q-PCR)。 The method of expression of a marker gene produced a quantitative marker may be used in quantitative PCR (Q-PCR). 开展Q-PCR的方法为现有技术。 Q-PCR method is carried out prior art. 现有技术的其它方法也可用于定量标志物基因的表达。 Other methods of the prior art can also be used to express the marker gene quantification. 例如,标志物基因产物的表达可以通过使用针对感兴趣的特异标志物基因产物的抗体来检测。 For example, expression of a marker gene product may be detected by using antibodies specific for the gene product of the marker of interest. 在本发明的一些实施方案中,确定了具有定形内胚层的标志物特征基因的表达,及缺乏显著表达的hESCs及其它细胞类型的标志物特征基因的表达。 In some embodiments of the present invention, determining the gene expression of a marker characteristic of the definitive endoderm with, and the lack of expression of marker genes characteristic of hESCs and other cell types significantly expressed.

如下述实施例的进一步所述,定形内胚层的一个可靠标志物为SOX17基因。 As further described in the following embodiment, a reliable marker of definitive endoderm object of SOX17 gene. 这样,本发明所述方法制备的定形内胚层细胞表达SOX17标志物基因,由此产生SOX17基因产物。 Thus, definitive endoderm cells prepared according to the present invention, the method of expression of the marker gene SOX17, SOX17 gene product produced thereby. 其它定形内胚层的标志物为MIXL1、GATA4、HNF3b、GSC、FGF17、VWF、CALCR、FOXQ1、CMKOR1和CRIP1。 The other markers of definitive endoderm MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1. 在本发明的一些实施方案中,定形内胚层细胞表达SOX17标志物基因的水平高于SOX7标志物基因的水平,其具有原始及内脏内胚层特征(参见表1)。 In some embodiments of the present invention, the definitive endoderm cells express the marker gene SOX17 level higher than the level of gene markers SOX7, having original characteristics and visceral endoderm (see Table 1). 此外,在一些实施方案中,SOX17标志物基因表达高于OCT4标志物基因的表达,其具有hESCs的特征。 Further, in some embodiments, expression of marker genes than SOXl 7 expression of OCT4 marker gene, which has a characteristic of hESCs. 在本发明其它实施方案中,定形内胚层细胞表达SOX17标志物基因的水平高于AFP、SPARC或凝血调节蛋白(TM)标志物基因的水平。 In other embodiments of the present invention, the level of expression of definitive endoderm cells marker gene SOX17 than AFP, the coagulation level of the marker gene or regulatory protein SPARC (TM). 在本发明的一些实施方案中,根据本发明所述方法表达SOX17的定形内胚层细胞不表达显著水平或量的PDX1(PDX1-阴性)。 In some embodiments of the present invention, the expression of mesodermal cells do not express significant levels or amounts of PDX1 (PDX1- negative) within SOX17 shaped in accordance with the method of the present invention.

定形内胚层的其它标志物为CXCR4基因。 Other markers of definitive endoderm is the CXCR4 gene. CXCR4基因编码细胞表面趋化因子受体,其配体为趋化物SDF-1。 CXCR4 gene encodes a cell surface chemokine receptor, which is a chemoattractant ligand is SDF-1. 成年人中包含CXCR4受体的细胞的主要作用据信为迁移造血细胞至骨髓、运载淋巴细胞、分化各种B细胞及巨噬血细胞系[Kim,C.,and Broxmeyer,HJLeukocyteBiol.65,6-15(1999)]。 The main role of adult cells containing the CXCR4 receptor is believed to migrate to the bone marrow hematopoietic cells, lymphocytes carrier, B cells and differentiation of various blood macrophage cell line [Kim, C., and Broxmeyer, HJLeukocyteBiol.65,6- 15 (1999)]. CXCR4受体也起着HIV-1进入T细胞中的共受体作用[Feng,Y.,et al.Science,272,872-877(1996)]。 CXCR4 receptor also plays into the HIV-1 co-receptor of T cells [Feng, Y., et al.Science, 272,872-877 (1996)]. 在一系列[McGrath,KEetal.Dev.Biology213,442-456(1999)]开展的研究中,描述了在成年小鼠及其早期发育中趋化因子受体CXCR4及其独特配体SDF-1的表达[Kim,C.,andBroxmyer,H.,J.Leukocyte Biol.65,6-15(1999)]。 In a series of [McGrath, KEetal.Dev.Biology213,442-456 (1999)] studies conducted, it is described in the early development of adult mice and chemokine receptor CXCR4 and its ligand SDF-1 unique to expression [Kim, C., andBroxmyer, H., J.Leukocyte Biol.65,6-15 (1999)]. CXCR4/SDF1在发育中的相互作用已经清楚,在转基因小鼠中,当其中任一基因破坏[Nagasawa et al.Nature,382,635-638(1996)],Ma,Q.,et alImmunity,10,463-471(1999)]都导致晚期胚胎死亡。 CXCR4 / SDF1 interactions in development has been clearly in transgenic mice, which either gene destroyed when [Nagasawa et al.Nature, 382,635-638 (1996)], Ma, Q., et alImmunity, 10 , 463-471 (1999)] have led to the late embryonic death. McGrath等使用核糖核酸酶保护及原位杂交方法证实,在形成原肠胚的早期(E7.5),CXCR4为最丰富的趋化因子受体信使RNA。 McGrath et using ribonuclease protection and in situ hybridization confirmed, early gastrula (E7.5) formed, CXCR4 is the most abundant chemokine receptor messenger RNA. 处于原肠胚阶段,CXCR4/SDF-1信号似乎主要诱导原线细胞的迁移,并表达在此时存在的定形内胚层、中胚层及外胚层上。 In gastrula stage, CXCR4 / SDF-1 induced migration of primary signal appears to be primarily cell line and expression in the endoderm in the present case, mesoderm and ectoderm. 在E7.2-7.8小鼠胚胎中,CXCR4及α胎蛋白互相排斥,显示在内脏内胚层缺乏表达[McGrath,KEet al.Dev.Biology 213,442-456(1999)]。 E7.2-7.8 in mouse embryos, CXCR4 and α fetoprotein mutually exclusive, displayed a lack of expression in the endoderm organs [McGrath, KEet al.Dev.Biology 213,442-456 (1999)].

在本发明的一些实施方案中,根据本发明所述方法制备的定形内胚层细胞表达CXCR4标志物基因。 In some embodiments of the present invention, according to definitive endoderm cells prepared according to the present invention, methods of expressing CXCR4 marker gene. 其它实施方案中,根据本发明所述方法制备的定形内胚层细胞表达CXCR4标志物基因及其它定形内胚层标志物,包括但不限于SOX17、MIXL1、GATA4、HNF3b、GSC、FGF17、VWF、CALCR、FOXQ1、CMKOR1及CRIP1。 Other embodiments, the expression of CXCR4 marker gene and the other definitive endoderm markers according to definitive endoderm cells prepared according to the present invention method, including but not limited to, SOX17, MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1. 在本发明一些实施方案中,定形内胚层细胞表达CXCR4标志物基因的水平高于SOX7标志物基因。 In some embodiments of the present invention, definitive endoderm cells express the CXCR4 marker gene is higher than SOX7 marker gene. 此外,在一些实施方案中,CXCR4标志物基因表达高于OCT4标志物基因表达的水平。 Further, in some embodiments, the gene expression levels of CXCR4 above markers OCT4 marker gene expression. 在本发明其它实施方案中,定形内胚层细胞表达CXCR4标志物基因的水平高于AFP、SPARC或凝血调节蛋白(TM)标志物基因的水平。 In other embodiments of the present invention, definitive endoderm cells express the CXCR4 marker gene is higher than the AFP, the coagulation level of the marker gene or regulatory protein SPARC (TM). 在本发明一些实施方案中,根据本发明所述的方法制备表达CXCR4的定形内胚层细胞不表达显著水平或量的PDX1(PDX1-阴性)。 In some embodiments of the present invention, the method according to the present invention prepared expressing endoderm cells do not express significant levels or amounts of PDX1 (PDX1- negative) of amorphous CXCR4.

应当理解,在内胚层细胞中CXCR4的表达并不排斥SOX17的表达。 It should be understood endoderm cells express the CXCR4 does not exclude the expression of SOX17. 相应地,在本发明一些实施方案中,定形内胚层细胞表达SOX17和CXCR4标志物基因的水平均高于SOX7标志物基因的水平。 Accordingly, in some embodiments of the invention, the definitive endoderm cells express CXCR4 and SOX17 water marker gene is higher than the average level SOX7 marker gene. 此外,在一些实施方案中,SOX17及CXCR4标志物基因的表达均高于OCT4标志物基因的表达。 Further, in some embodiments, and SOXl 7 expression of CXCR4 marker gene expression are higher than OCT4 marker gene. 在本发明的其它实施方案中,定形内胚层细胞表达SOX17及CXCR4标志物基因的水平高于AFP、SPARC或凝血调节蛋白(TM)标志物基因的水平。 In other embodiments of the present invention, the definitive endoderm cells express CXCR4 and SOX17 levels of marker genes than AFP, SPARC protein levels or thrombomodulin (TM) marker gene. 在本发明一些实施方案中,根据本发明所述的方法制备表达SOX17/CXCR4的定形内胚层细胞不表达显著水平或量的PDX1(PDX1-阴性)。 In some embodiments of the present invention, the method according to the present invention prepared expressing endoderm cells do not express significant levels or amounts of PDX1 (PDX1- negative) of amorphous SOX17 / CXCR4 in.

应当理解,根据分化条件,在定形内胚层细胞内诱导不同水平范围的SOX17和/或CXCR4标志物表达。 It should be appreciated that, in accordance with the conditions of differentiation, induction of SOX17 and / or different marker expression levels in the range of CXCR4 or definitive endoderm cells. 这样,在本发明一些实施方案中,SOX17标志物和/或CXCR4标志物在定形内胚层细胞或细胞群的表达比SOX17标志物和/或CXCR4标志物在诸如多能干细胞的非定形内胚层细胞或细胞群中的表达至少高约2倍至至少约10,000倍。 Thus, in some embodiments of the present invention, SOX17 markers and / or CXCR4 marker in the amorphous expressing endoderm cells or cell populations than SOX17 markers and / or CXCR4 marker non-definitive endoderm cells, such as pluripotent stem cells or population of cells expressing at least about 2-fold higher to at least about 10,000 fold. 在本发明其它实施方案中,SOX17标志物和/或CXCR4标志物在定形内胚层细胞或细胞群的表达比SOX17标志物和/或CXCR4标志物在诸如多能干细胞的非定形内胚层细胞或细胞群表达高至少约4倍、至少约6倍、至少约8倍、至少约10倍、至少约15倍、至少约20倍、至少约40倍、至少约80倍、至少约100倍、至少约150倍、至少约200倍、至少约500倍、至少约750倍、至少约1000倍、至少约2500倍、至少约5000倍、至少约7500倍或至少约10,000倍。 In other embodiments of the present invention, SOX17 markers and / or CXCR4 marker in the amorphous expressing endoderm cells or cell populations than SOX17 markers and / or CXCR4 marker non-definitive endoderm cells or cells such as pluripotent stem cells population expresses at least about 4-fold, at least about 6-fold, at least about 8 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, at least about 40 fold, at least about 80 fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 500-fold, at least about 750-fold, at least about 1000-fold, at least about 2500-fold, at least about 5000-fold, at least about 7500-fold, or at least about 10,000 fold. 在一些实施方案中,SOX17标志物和/或CXCR4标志物在定形内胚层细胞或细胞群的表达无限制地高于SOX17标志物和/或CXCR4标志物在诸如多能干细胞的非定形内胚层细胞或细胞群表达。 In some embodiments, SOX17 markers and / or CXCR4 marker in the amorphous expressing endoderm cells or cell populations without limitation than SOX17 markers and / or CXCR4 marker non-definitive endoderm cells, such as pluripotent stem cells or expressing cell populations.

应当理解,在本发明一些实施方案中,与在非定形内胚层细胞或细胞群中的GATA4、MIXL1、HNF3b、GSC、FGF17、VWF、CALCR、FOXQ1、CMKOR1及CRIP1标志物的表达相比,在定形内胚层细胞或细胞群体中的GATA4、MIXL1、HNF3b、GSC、FGF17、VWF、CALCR、FOXQ1、CMKOR1及CRIP1标志物的表达增加。 It should be appreciated that in some embodiments of the present invention as compared to the expression of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 markers in a non-definitive endoderm cells or cell population, the endoderm cells or cell populations within the amorphous GATA4, MIXL1, HNF3b, GSC, FGF17, increased expression of VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 markers.

也应当理解,在定形内胚层细胞内,SOX17标志物的表达水平与OCT4、SPARC、AFP、TM和/或SOX7标志物表达水平存在差异范围。 It should also be appreciated that, in the definitive endoderm cells, and OCT4 SOX17 expression levels of the marker, the presence of SPARC, AFP, TM and / or difference in expression level marker SOX7 range. 类似地,在定形内胚层细胞内,CXCR4标志物的表达水平与OCT4、SPARC、AFP、TM和/或SOX7标志物表达水平存在差异范围。 Similarly, in the definitive endoderm cells, and OCT4 CXCR4 expression level of the marker, there is a difference range SPARC, AFP, TM and / or SOX7 marker expression levels. 类似地,在本发明一些实施方案中,SOX17标志物或CXCR4标志物的表达比OCT4、SPARC、AFP、TM和/或SOX7标志物的表达高至少约2倍至至少约10,000倍。 Similarly, in some embodiments of the present invention, SOXl 7 expression marker or markers than CXCR4 OCT4, high expression of SPARC, AFP, TM and / or markers SOX7 least about 2-fold to at least about 10,000 fold. 在本发明其它实施方案中,SOX17标志物或CXCR4标志物的表达比OCT4、SPARC、AFP、TM和/或SOX7标志物的表达高至少约4倍、至少约6倍、至少约8倍、至少约10倍、至少约15倍、至少约20倍、至少约40倍、至少约80倍、至少约100倍、至少约150倍、至少约200倍、至少约500倍、至少约750倍、至少约1000倍、至少约2500倍、至少约5000倍、至少约7500倍或至少约10,000倍。 In other embodiments of the present invention, SOXl 7 marker expression or CXCR4 marker than OCT4, expression of SPARC, AFP, TM and / or SOX7 marker is at least about 4-fold, at least about 6-fold, at least about 8-fold, at least about 10-fold, at least about 15 fold, at least about 20 fold, at least about 40 fold, at least about 80 fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 500-fold, at least about 750-fold, at least about 1000-fold, at least about 2500-fold, at least about 5000-fold, at least about 7500-fold, or at least about 10,000 fold. 在一些实施方案中,OCT4、SPARC、AFP、TM和/或SOX7标志物在定形内胚层细胞表达不显著。 In some embodiments, OCT4, SPARC, AFP, TM and / or SOX7 markers of definitive endoderm cells were not significantly decreased.

应当理解,在本发明一些实施方案中,在定形内胚层细胞内,与OCT4、SPARC、AFP、TM和/或SOX7相比,选自GATA4、MIXL1、HNF3b、GSC、FGF17、VWF、CALCR、FOXQ1、CMKOR1及CRIP1的标志物的表达增加。 It should be appreciated that in some embodiments of the present invention, in the definitive endoderm cells, compared with OCT4, SPARC, AFP, TM and / or SOX7, selected GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1 increased expression CMKOR1 and CRIP1 markers.

包括定形内胚层的组合物本发明一些方面涉及诸如细胞群体及细胞培养物的组合物,其包括诸如干细胞的多能细胞及定形内胚层细胞。 The composition comprising the definitive endoderm of the present invention is directed to several aspects of the groups, and combinations thereof, such as cell cultures of cells, including pluripotent cells and definitive endoderm cells, such as stem cells. 例如,使用本发明所述的方法,可以制备包括hESCs混合物及定形内胚层细胞的组合物。 For example, using the method of the present invention can be prepared and a composition comprising a mixture of hESCs definitive endoderm cells. 在一些实施方案中,制备的组合物中每包括95个多能细胞就包括至少约5个定形内胚层细胞。 In some embodiments, the composition prepared in each cell can include more than 95 including at least about 5 definitive endoderm cells. 在其它实施方案中,每包括5个多能细胞就包括至少约95个定形内胚层细胞。 In other embodiments, each cell can include more than five can comprise at least about 95 definitive endoderm cells. 此外,组合物包括其它比例的定形内胚层细胞与多能细胞。 Furthermore, the compositions comprising other ratios definitive endoderm cells pluripotent cells. 例如,在组合物中,每包括1,000,000个多能细胞就包括至少约1个定形内胚层细胞、每包括100,000个多能细胞就包括至少约1个定形内胚层细胞、每包括100,000个多能细胞就包括至少约1个定形内胚层细胞、每包括10,000个多能细胞就包括至少约1个定形内胚层细胞、每包括1,000个多能细胞就包括至少约1个定形内胚层细胞、每包括500个多能细胞就包括至少约1个定形内胚层细胞、每包括100个多能细胞就包括至少约1个定形内胚层细胞、每包括10个多能细胞就包括至少约1个定形内胚层细胞、每包括5个多能细胞就包括至少约1个定形内胚层细胞、每包括2个多能细胞就包括至少约1个定形内胚层细胞、每包括1个多能细胞就包括至少约2个定形内胚层细胞、每包括1个多能细胞就包括至少约5个定形内胚层细胞、每包括1个多能细胞就包括至少约10个定形内胚层细胞、每包括1个 For example, in the composition, each comprising a 1,000,000 pluripotent cells can comprise at least about 1 definitive endoderm cells, each comprising a 100,000 pluripotent cells including at least endoderm cells amorphous about 1, each comprising a 100,000 pluripotent cells including at least about 1 definitive endoderm cells, each comprising 10,000 pluripotent cells including at least endoderm cells amorphous about 1, each comprising 1,000 pluripotent cells including at least endoderm cells amorphous about 1, each comprising 500 pluripotent cells can comprise at least about 1 definitive endoderm cells, each including 100 pluripotent cells including at least endoderm cells amorphous about 1, each including 10 pluripotent cells can comprise at least about 1 definitive endoderm cells , each including five pluripotent cells including at least endoderm cells amorphous about 1, each comprising two pluripotent cells including at least endoderm cells amorphous about 1, each comprising a pluripotent cell including at least about 2 endoderm cells, each comprising a pluripotent cell including at least endoderm cells amorphous about 5, each comprising a pluripotent cell including amorphous endoderm cells at least about 10, each comprising a 能细胞就包括至少约20个定形内胚层细胞、每包括1个多能细胞就包括至少约50个定形内胚层细胞、每包括1个多能细胞就包括至少约100个定形内胚层细胞、每包括1个多能细胞就包括至少约1,000个定形内胚层细胞、每包括1个多能细胞就包括至少约10,000个定形内胚层细胞、每包括1个多能细胞就包括至少约100,000个定形内胚层细胞、每包括1个多能细胞就包括至少约1,000,000个定形内胚层细胞。 Pluripotent cells including at least about 20 definitive endoderm cells, each comprising 1 pluripotent cells including at least endoderm cells amorphous to about 50, each including one pluripotent cell including at least endoderm cells amorphous about 100, per comprising 1 pluripotent cells can comprise at least about 1,000 definitive endoderm cells, each comprising 1 pluripotent cells can comprise at least about 10,000 definitive endoderm cells, each comprising 1 pluripotent cells can comprise at least about 100,000 within amorphous endoderm cells, each comprising more than one cell can comprise at least about 1,000,000 to definitive endoderm cells. 在本发明一些实施方案中,多能细胞为人多能干细胞。 In some embodiments of the present invention, the pluripotent cell is a human pluripotent stem cells. 在一些实施方案中,干细胞衍生于桑椹胚、胚胎内细胞团或胚胎的性腺嵴。 In some embodiments, stem cells derived from morula, the gonadal ridge of embryos or embryonic cell mass. 在一些其它实施方案中,多能细胞衍生于已经过胚胎阶段发育的多细胞结构的性腺或生殖组织。 In some other embodiments, the pluripotent cells derived from the gonads, or reproductive tissues of the multicellular structure have been embryonic stages of development.

本发明一些方面涉及细胞培养物或细胞群,包括至少约5%定形内胚层细胞至至少约95%定形内胚层细胞。 Some aspects of the invention relates to a cell culture or population of cells, comprising at least about 5% of the definitive endoderm cells to at least about 95% of the definitive endoderm cells. 在一些实施方案中,细胞培养物或细胞群包括哺乳动物细胞。 In some embodiments, the cell culture or cell population comprising a mammalian cell. 在优选实施方案中,细胞培养物或细胞群包括人细胞。 In a preferred embodiment, the cell culture or cell population comprising human cells. 例如,一些具体实施方案中涉及细胞培养物,包括人细胞,其中至少约5%至至少约95%人细胞为定形内胚层细胞。 For example, some embodiments relate to cell cultures, including human cells, wherein at least about 5% to at least about 95% of the human cells to definitive endoderm cells. 本发明其它实施方案涉及细胞培养物,包括人细胞,其中至少约5%、至少约10%、至少约15%、至少约20%、至少约25%、至少约30%、至少约35%、至少约40%、至少约45%、至少约50%、至少约55%、至少约60%、至少约65%、至少约70%、至少约75%、至少约80%、至少约85%、至少约90%或大于90%的人细胞为定形内胚层细胞。 Other embodiments of this invention relate to cell cultures, including human cells, 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 greater than 90% of the human cells to definitive endoderm cells.

本发明其它实施方案涉及诸如细胞培养物或细胞群体的组合物,其包括诸如人定形内胚层细胞的人细胞,其中在至少在约5%的人细胞中SOX17或CXCR4标志物的表达均大于OCT4、SPARC、α胎蛋白(AFP)、凝血调节蛋白(TM)和/或SOX7标志物的表达。 Other embodiments of the present invention relates to compositions such as culture or cell population of cells comprising such as human definitive endoderm cells, human cells, wherein at least about 5% of the human cells expressing SOX17 or the CXCR4 marker is greater than OCT4 expression, SPARC, α-fetoprotein (AFP), thrombomodulin (TM) and / or SOX7 marker. 在其它实施方案中,其中在至少在约10%的人细胞中、至少约15%的人细胞中、至少约20%的人细胞中、至少约25%的人细胞中、至少约30%的人细胞中、至少约35%的人细胞中、至少约40%的人细胞中、至少约45%的人细胞中、至少约50%的人细胞中、至少约55%的人细胞中、至少约60%的人细胞中、至少约65%的人细胞中、至少约70%的人细胞中、至少约75%的人细胞中、至少约80%的人细胞中、至少约85%的人细胞中、至少约90%的人细胞中、至少约95%人细胞中或大于95%人细胞中,SOX17或CXCR4标志物的表达均大于OCT4、SPARC、AFP、TM和/或SOX7标志物的表达。 In other embodiments, wherein at least about 10% of the human cells, at least about 15% of the human cells, at least about 20% of the human cells, at least about 25% of the human cells, at least about 30% human cells, at least about 35% of the human cells, at least about 40% of the human cells, at least about 45% of the human cells, at least about 50% of the human cells, at least about 55% of the human cells, at least people about 60% of the human cells, at least about 65% of the human cells, at least about 70% of the human cells, at least about 75% of the human cells, at least about 80% of the human cells, at least about 85% cells, at least about 90% of the human cells, at least about 95% of the human cells or greater than 95% human cells, expression of SOX17 or the CXCR4 marker is greater than OCT4, SPARC, AFP, TM and / or SOX7 markers expression.

应当理解,本发明一些实施方案涉及诸如细胞培养物或细胞群的组合物,其包括如人定形内胚层细胞的人细胞,其中在至少约5%至大于至少约95%人细胞中,其中选自GATA4、MIXL1、HNF3b、GSC、FGF17、VWF、CALCR、FOXQ1、CMKOR1及CRIP1的一种或多种标志物的表达高于OCT4、SPARC、AFP、TM和/或SOX7标志物的表达。 It should be understood that the present invention Some embodiments relate to a culture or a population of cells a composition such as a cell, which comprises as the definitive endoderm cells human human cells, wherein at least about 5% greater than at least about 95% of human cells, which is selected from since GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 CRIP1 and expression of one or more of the above markers OCT4, expression of SPARC, AFP, TM and / or SOX7 marker.

本发明其它实施方案涉及如细胞培养物或细胞群的组合物,其包括如人定形内胚层细胞的人细胞,其中在至少约5%的人细胞中SOX17及CXCR4标志物的表达均高于OCT4、SPARC、AFP、TM和/或SOX7标志物表达。 Other embodiments of the present invention relates to compositions, such as cell culture or cell population comprising definitive endoderm cells, such as human human cell, wherein at least about 5% of the human cells expressing SOX17 and CXCR4 markers were higher than OCT4 , SPARC, AFP, TM and / or SOX7 marker expression. 在其它实施方案中,在至少约10%人细胞中、至少约15%的人细胞中、在至少约20%人细胞中、在至少约25%人细胞中、在至少约30%人细胞中、在至少约40%人细胞中、在至少约50%人细胞中、在至少约55%人细胞中、在至少约60%人细胞中、在至少约65%人细胞中、在至少约70%人细胞中、在至少约75%人细胞中、在至少约80%人细胞中、在至少约85%人细胞中、在至少约90%人细胞中、在至少约95%人细胞中或大于95%人细胞中,SOX17及CXCR4标志物的表达均高于OCT4、SPARC、AFP、TM和/或SOX7标志物的表达。 In other embodiments, at least about 10% of the human cells, at least about 15% of the human cells, at least about 20% of the human cells, at least about 25% of the human cells, at least about 30% of the human cells , at least about 40% of the human cells, at least about 50% of the human cells, at least about 55% human cells, at least about 60% human cells, at least about 65% human cells, at least about 70 % human cells, at least about 75% of the human cells, at least about 80% of the human cells, at least about 85% of the human cells, at least about 90% of the human cells, at least about 95% of the human cells or greater than 95% of the human cells, the expression of CXCR4 and SOX17 markers were higher than OCT4, expression of SPARC, AFP, TM and / or SOX7 marker.

应当理解,本发明一些实施方案涉及如细胞培养物或细胞群的组合物,其包括如人定形内胚层细胞的人细胞,在至少约5%至大于至少约95%人细胞中,其中GATA4、MIXL1、HNF3b、GSC、FGF17、VWF、CALCR、FOXQ1、CMKOR1及CRIP1的标志物的表达高于OCT4、SPARC、AFP、TM和/或SOX7标志物的表达。 It should be understood that the present invention Some embodiments relate to cell culture or cell population of the composition, comprising the definitive endoderm cells, such as human human cells, at least about 5% greater than at least about 95% of human cells, wherein GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, and CRIP1 CMKOR1 expression of markers than OCT4, expression of SPARC, AFP, TM and / or SOX7 marker.

本发明其它实施方案涉及诸如细胞培养物或细胞群的组合物,包括诸如人定形内胚层细胞的哺乳动物内胚层细胞,其中在至少在约5%的内胚层细胞中SOX17或CXCR4标志物的表达均大于OCT4、SPARC、α胎蛋白(AFP)、凝血调节蛋白(TM)和/或SOX7标志物的表达。 Other embodiments of the present invention relates to compositions such as cell culture or cell population comprising such as human mammalian endoderm cells definitive endoderm cells, wherein the expression of at least about 5% of the endodermal cells SOX17 or CXCR4 marker greater than OCT4, SPARC, α-fetoprotein (AFP), the expression of protein thrombomodulin (TM) and / or SOX7 marker. 在其它实施方案中,其中在至少在约10%的内胚层细胞中、至少约15%的内胚层细胞中、至少约20%的内胚层细胞中、至少约25%的内胚层细胞中、至少约30%的内胚层细胞中、至少约35%的内胚层细胞中、至少约40%的内胚层细胞中、至少约45%的内胚层细胞中、至少约50%的内胚层细胞中、至少约55%的内胚层细胞中、至少约60%的内胚层细胞中、至少约65%的内胚层细胞中、至少约70%的内胚层细胞中、至少约75%的内胚层细胞中、至少约80%的内胚层细胞中、至少约85%的内胚层细胞中、至少约90%的内胚层细胞中、至少约95%的内胚层细胞中或大于95%的内胚层细胞中,SOX17或CXCR4标志物的表达均大于OCT4、SPARC、AFP、TM和/或SOX7标志物的表达。 In other embodiments, wherein at least about 10% of the endodermal cells, at least about 15% of the endodermal cells, at least about 20% of the endodermal cells, at least about 25% of the endodermal cells, at least about 30% of the endodermal cells, at least about 35% of the endodermal cells, at least about 40% of the endodermal cells, at least about 45% of the endodermal cells, at least about 50% of the endodermal cells, at least about 55% of the endodermal cells, at least about 60% of the endodermal cells, at least about 65% of the endodermal cells, at least about 70% of the endodermal cells, at least about 75% of the endodermal cells, at least endodermal cells to about 80% of the endodermal cells, at least about 85% of the endodermal cells, at least about 90% of the endodermal cells, at least about 95% of the endodermal cells or greater than 95% in SOXl 7 or CXCR4 expression of markers greater than OCT4, expression of SPARC, AFP, TM and / or SOX7 marker.

应当明白,本发明一些实施方案涉及如细胞培养物或细胞群的组合物,其包括哺乳动物内胚层细胞,其中在至少约5%至大于至少约95%所述内胚层细胞中,一种或多种选自GATA4、MIXL1、HNF3b、GSC、FGF17、VWF、CALCR、FOXQ1、CMKOR1及CRIP1的标志物的表达高于OCT4、SPARC、AFP、TM和/或SOX7标志物的表达。 It should be appreciated that some embodiments of the present invention relates to a cell culture or cell population composition comprising mammalian endoderm cells, wherein at least about 5% to about 95%, at least greater than the endodermal cells, one or more selected GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, and CRIP1 CMKOR1 expression of markers than OCT4, expression of SPARC, AFP, TM and / or SOX7 marker.

本发明其它进一步实施方案涉及如细胞培养物或细胞群的组合物,包括诸如人定形内胚层细胞的哺乳动物内胚层细胞,其中在至少在约5%的所述内胚层细胞中SOX17及CXCR4标志物的表达均大于OCT4、SPARC、α胎蛋白(AFP)、凝血调节蛋白(TM)和/或SOX7标志物的表达。 Other further embodiments of the present invention relates to compositions, such as cell culture or cell population comprising a mammalian inner such as a human definitive endoderm cells of mesodermal cells, wherein SOX17 and CXCR4 flag at least endoderm cells in the approximately 5% in the It was greater than expression of OCT4, SPARC, α-fetoprotein (AFP), the expression of protein thrombomodulin (TM) and / or SOX7 marker. 在其它实施方案中,其中在至少约10%的内胚层细胞中、至少约15%的内胚层细胞中、至少约20%的内胚层细胞中、至少约25%的内胚层细胞中、至少约30%的内胚层细胞中、至少约35%的内胚层细胞中、至少约40%的内胚层细胞中、至少约45%的内胚层细胞中、至少约50%的内胚层细胞中、至少约55%的内胚层细胞中、至少约60%的内胚层细胞中、至少约65%的内胚层细胞中、至少约70%的内胚层细胞中、至少约75%的内胚层细胞中、至少约80%的内胚层细胞中、至少约85%的内胚层细胞中、至少约90%的内胚层细胞中、至少约95%的内胚层细胞中或大于95%的内胚层细胞中,SOX17及CXCR4标志物的表达均大于OCT4、SPARC、AFP、TM和/或SOX7标志物的表达。 In other embodiments, wherein at least about 10% of the endodermal cells, at least about 15% of the endodermal cells, at least about 20% of the endodermal cells, at least about 25% of the endodermal cells, at least about 30% of the endodermal cells, at least about 35% of the endodermal cells, at least about 40% of the endodermal cells, at least about 45% of the endodermal cells, at least about 50% of the endodermal cells, at least about 55% of the endodermal cells, at least about 60% of the endodermal cells, at least about 65% of the endodermal cells, at least about 70% of the endodermal cells, at least about 75% of the endodermal cells, at least about endodermal cells 80% of the endodermal cells, at least about 85% of the endodermal cells, at least about 90% of the endodermal cells, at least about 95% of the endodermal cells or greater than 95%, the SOXl 7 and CXCR4 greater than expression of the markers OCT4, expression of SPARC, AFP, TM and / or SOX7 marker.

应当理解,本发明一些实施方案涉及诸如细胞培养物或细胞群的组合物,包括哺乳动物内胚层细胞,其中在至少约5%至大于至少约95%内胚层细胞中,GATA4、MIXL1、HNF3b、GSC、FGF17、VWF、CALCR、FOXQ1、CMKOR1及CRIP1的标志物的表达高于OCT4、SPARC、AFP、TM和/或SOX7标志物的表达。 It should be understood that the present invention Some embodiments relate to compositions such as cell culture or cell population comprising a mammalian endoderm cells, wherein at least about 5% greater than at least about 95% by endodermal cells, GATA4, MIXL1, HNF3b, GSC, FGF17, expression of VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 higher than markers OCT4, expression of SPARC, AFP, TM and / or SOX7 marker.

使用本发明所述的方法,可以制备基本不含其它细胞类型的包括定形内胚层细胞的组合物。 Using the method of the present invention may be prepared substantially free of other cell types of compositions comprising definitive endoderm cells. 关于细胞培养物或细胞群里的细胞,术语“基本不含”指在细胞培养物或细胞群里的特定的细胞不存在、或小于细胞培养物或细胞群里总细胞数的约5%。 On cell culture or cell population in a cell, the term "substantially free" refers to a specific cell in a cell culture or population of cells in the absence of, or less than about 5% of the cell cultures or cell populations in the total number of cells. 在本发明的一些实施方案中,使用本发明所述的方法制备的定形内胚层细胞群或细胞培养物基本不含特异显著表达OCT4、SOX7、AFP、SPARC、TM、ZIC1或BRACH标志物基因的细胞。 In some embodiments of the present invention, prepared using the method of the present invention endoderm cell populations or cell culture substantially free of specific significant expression of OCT4, SOX7, AFP, SPARC, TM, ZIC1 or BRACH marker genes cell.

在本发明一个实施方案中,基于标志物基因的表达,定形内胚层细胞的描述为SOX17高、MIXL1高、AFP低、SPARC低、凝血调节蛋白低、SOX7低、CXCR4高。 In one embodiment of the present invention, based on the expression of marker gene, described definitive endoderm cells is high SOX17, high MIXLl, low AFP, SPARC low, low protein thrombomodulin, SOX7 low, high CXCR4.

定形内胚层的富集、分离和/或纯化关于本发明的其它方面,定形内胚层细胞可使用特异针对这些细胞的亲和标记来富集、分离和/或纯化。 The endoderm enriched, isolated and / or purified with respect to other aspects of the invention, definitive endoderm cells can be labeled using specific affinity for these cells to enrichment, isolation and / or purification. 特异针对定形内胚层细胞的亲和标记的实例为抗体、配体或其它特异针对如多肽的标志物分子的结合试剂,该标志物分子存在于细胞定形内胚层细胞的表面,但基本不存在于根据本发明所述的方法制备的细胞培养基中发现的其它细胞类型中。 Specific for Examples affinity labeled definitive endoderm cells is an antibody, ligand or other specific for such binding agents marker molecule polypeptide, which marker molecules present on the surface endoderm cells within shaped cells, but does not substantially exist in the according to other cell culture medium prepared according to the method of the present invention is found in type. 在一些实施方案中,与CXCR4结合的抗体用于定形内胚层的富集、分离和/或纯化的亲和标记。 In some embodiments, the antibody binds to CXCR4 for definitive endoderm enrichment, isolation and / or purification of an affinity tag. 在其它实施方案中,趋化因子SDF-1或其它基于SDF-1的分子也可用于亲和标记。 In other embodiments, the chemokine SDF-1 or SDF-1 on other molecules are also useful for the affinity tag. 这些分子包括,但不限于SDF-1片断,SDF-1融合物或SDF-1模拟物。 These molecules include, but are not limited to fragments SDF-1, SDF-1 or SDF-1 fusion mimetic.

制备抗体及其用于细胞分离的方法在本技术领域已知,这些方法可利用本发明所述的抗体及细胞来实施。 Preparation of antibodies and methods for separating cells known in the art, these methods may utilize antibodies and cells of the present invention is implemented. 在一个实施方案中,将结合CXCR4的抗体与磁珠结合,然后在细胞培养基中与定形内胚层细胞结合,经酶处理后降低了细胞间及与底物的粘附。 In one embodiment, the antibody binding to CXCR4 binding to magnetic beads, and then combined with the amorphous endoderm cells in cell culture medium, after enzyme treatment and reduced intercellular adhesion with the substrate. 将细胞/抗体/磁珠复合物暴露于移动磁场中以将磁珠结合的定形内胚层细胞与未结合的细胞分开。 The cell / antibody / bead complex is then exposed to a shifting magnetic field to the definitive endoderm cells bound to beads are separated from unbound cells. 一旦定形内胚层细胞在培养基中与其它细胞物理分开,破坏结合的抗体,将细胞重新置于合适的组织培养基中。 Once the definitive endoderm cells physically separated from the other cells in the culture, destroy antibody binding, re-placed in a suitable tissue culture cells.

本发明实施方案考虑了其它定形内胚层细胞培养物或细胞群的富集、分离和/或纯化方法。 Embodiments of the present invention contemplates a cell culture enriched endoderm amorphous or other cell populations, and / or purification process for separating. 例如,在一些实施方案中,CXCR4抗体在包含定形内胚层的细胞培养物中孵育,经处理后降低细胞间及与底物的粘附。 For example, in some embodiments, CXCR4 antibody in a cell culture comprising definitive endoderm incubated, and reduced intercellular adhesion to the substrate after treatment. 然后将细胞洗涤、离心及再悬浮。 Cells were then washed, centrifuged and resuspended. 细胞悬浮物然后再与第二抗体,如能够与第一抗体结合的FITC轭合抗体孵育。 The cell suspension was then reacted with a second antibody, such as a first antibody capable of binding FITC-conjugated antibody incubation. 然后再将细胞洗涤、离心及再悬浮缓冲液中。 Cells were then washed, centrifuged and resuspended in buffer. 随后分析细胞悬浮物,使用荧光活化细胞分选器(FACS)分选。 The cell suspension was then analyzed using a fluorescence activated cell sorter (FACS) sorting. 将CXCR4-阴性细胞分离,收集CXCR4-阳性细胞,由此分离了该类型细胞。 The CXCR4- negative cell separation, CXCR4- positive cells were collected, thereby separating the cell type. 需要时,分离的细胞组合物可进一步使用替代的亲和方法或增加分选数次,使用特异针对定形内胚层的相同或不同标志物。 If desired, the isolated cell compositions may further alternative method of increasing the affinity and sorting several times, using the same or different specific marker for the definitive endoderm.

在本发明其它实施方案中,使用与CXCR4结合的配体或其它分子富集、分离和/或纯化定形内胚层。 In other embodiments of the present invention, a ligand binding to CXCR4 or other molecule enrichment and / or separation of purified definitive endoderm. 在一些实施方案中,所述分子为SDF-1或其片段、融合物或其模拟物。 In some embodiments, the molecule is SDF-1 or a fragment, fusion or mimetic thereof.

在优选实施方案中,当干细胞培养物被诱导向定形内胚层系分化后,自其它非定形内胚层细胞中富集、分离和/或纯化定形内胚层细胞。 In a preferred embodiment, when the cultures are induced stem cells to definitive endoderm after differentiation from other non-definitive endoderm cell enrichment, isolation and / or purification of definitive endoderm cells. 应当明白,上述富集、分离和/或纯化方法可利用任何分化阶段的这种培养物。 It should be understood that the above enrichment, isolation and / or purification methods may utilize this culture was any stage of differentiation.

除上述的方法,定形内胚层细胞也可以其它细胞分离技术分离。 The above-described method, endoderm cells can be isolated, among other cell separation techniques. 此外,定形内胚层细胞也可以在生长条件下通过一系列再培养的方法富集或分离,该生长条件有利于所述的定形内胚层细胞选择性存活或选择性扩增。 Moreover, definitive endoderm cells can be enriched or separated by a series of methods incubated under growth conditions which favor the growth conditions endoderm cell selective survival or selective amplification.

使用本发明所述的方法,可体外自至少经过一些分化的如干细胞培养物或细胞群的多能细胞培养物或细胞群中富集、分离和/或纯化定形内胚层细胞。 Using the method of the present invention, at least in vitro from pluripotent cells after culture or differentiated cell population of some cultures or cell populations such as stem cells enriched and / or purified definitive endoderm cell separation. 在一些实施方案中,所述细胞进行随机分化。 In some embodiments, the cells randomly differentiate. 然而,在一些优选实施方案,所述细胞主要分化为定形内胚层。 However, in some preferred embodiments, the cells mainly differentiated into definitive endoderm. 一些优选的富集、分离和/或纯化方法涉及体外自人胚胎干细胞制备定形内胚层。 Some preferred enrichment, isolation and / or purification methods involving in vitro from human embryonic stem cells to definitive endoderm was prepared. 使用本发明所述的方法,与未处理的细胞群或细胞培养物相比,定形内胚层中富集的细胞群或细胞培养物为至少约2倍至约1000倍。 The method of the present invention is used, compared to untreated cell populations or cell cultures, the definitive endoderm population of cells enriched or cell culture is at least about 2-fold to about 1000-fold. 在一些实施方案中,与未处理的细胞群或细胞培养物相比,富集的定形内胚层至少约5倍至约500倍。 In some embodiments, as compared to untreated cell populations or cell cultures, endoderm enriched at least about 5-fold to about 500-fold. 在其它实施方案中,与未处理的细胞群或细胞培养物相比,富集的定形内胚层为至少约10倍至约200倍。 In other embodiments, as compared to untreated cell populations or cell cultures, endoderm enriched at least about 10-fold to about 200-fold. 在其它实施方案中,与未处理的细胞群或细胞培养物相比,富集的定形内胚层为至少约20倍至约100倍。 In other embodiments, as compared to untreated cell populations or cell cultures, endoderm enriched at least about 20-fold to about 100-fold. 在其它实施方案中,与未处理的细胞群体或细胞培养物相比,富集的定形内胚层为至少约40倍至约80倍。 In other embodiments, as compared to untreated cell population or cell culture, endoderm enriched at least about 40-fold to about 80-fold. 在某些实施方案中,与未处理的细胞群体或细胞培养物相比,富集的定形内胚层为至少约2倍至约20倍。 In certain embodiments, as compared to untreated cell population or cell culture, endoderm enriched at least about 2-fold to about 20-fold.

已经对本发明进行了一般性描述,参考一些具体实施例可进一步理解本发明,本发明的这些实施例仅为示例性说明的目的,而非意在限制本发明。 The present invention has been generally described, with reference to some specific embodiments of the present invention may be further understood, these embodiments of the present invention is merely illustrative purposes, and not intended to limit the present invention.

实施例下述许多实施例描述了多能人细胞的使用。 Example The following example describes the use of a number of pluripotent human cells. 制备多能人细胞的方法为本领域一般技术人员熟知,大量科技出版物,包括美国专利号5,453,357、5,670,372、5,690,926、6,090,622、6,200,806及6,251,671及美国专利申请公开号2004/0229350均有描述,本文将其全部引入,作为参考。 The method of preparation of pluripotent human cells are known to those of ordinary skill are well known, a large number of scientific publications, including U.S. Patent Nos. 5,453,357,5,670,372,5,690,926,6,090,622,6,200,806 and 6,251,671 and U.S. Patent Application Publication No. 2004/0229350 has described herein will be incorporated in its entirety, by reference.

实施例1人ES细胞为研究内胚层发育,我们利用人胚胎干细胞,其为多能干细胞,在培养基中似乎可以无限分裂,同时保持正常的染色体组型。 Example 1 human ES cells embodiment within mesoderm study, we use human embryonic stem cells, which are pluripotent stem cells, it seems to divide indefinitely in culture, while maintaining a normal karyotype. ES细胞衍生于5天大小的胚胎内细胞团,使用免疫学或机械方法分离。 ES cells derived from 5-day-cell mass, isolated immunological or mechanical methods. 特别是,人胚胎干细胞系hESCyt-25来自经患者同意的体外受精周期的额外冰冻胚胎。 In particular, the human embryonic stem cell line hESCyt-25 extra frozen embryos from in vitro fertilization cycles through a patient's consent. 融化后,将孵化的囊胚接种于小鼠胚胎成纤维细胞(MEF)上,使用ES培养基(DMEM、20%FBS、非必需氨基酸、β-巯基乙醇、ITS调节剂)。 After thawing, the hatched blastocyst inoculated mouse embryonic fibroblasts (MEF), the use of ES medium (DMEM, 20% FBS, nonessential amino acids, [beta] -mercaptoethanol, ITS modifier). 大约两周后,胚胎粘附于培养皿中,将未分化hESCs区域转移至MEFs。 After about two weeks, adhered to the dish embryo, undifferentiated hESCs region is transferred to MEFs. 转移以机械切割完成,使用中性蛋白酶简单消化后,以机械移开细胞簇,洗涤、再接种。 Transfer mechanical completion of cutting, the use of a neutral protease digestion easy to mechanically remove cell clusters, washed, then inoculated. 自衍生化后,连续传代hESCyt-25 100次。 Since the derivatization, serial passaging hESCyt-25 100 times. 我们利用hESCyt-25人胚胎干细胞系作为起始材料制备定形内胚层。 We use hESCyt-25 human embryonic stem cell line prepared as an amorphous starting material of the endoderm.

本领域一般技术人员应当明白,干细胞或其它多能细胞也可用作本发明所述的分化方法的起始材料。 This should be understood by those of ordinary skill in the art, stem cells or other pluripotent cells can also be used as starting materials of the differentiation process of the present invention. 例如,胚胎性腺嵴细胞可以根据本领域已知的方法分离,用作多能细胞起始材料。 For example, cells can be isolated embryonic gonadal ridges according to the methods known in the art, as a starting material pluripotent cells.

实施例2hESCyt-25的表征人胚胎干细胞系在培养18个月中一直保持正常的形态学、染色体组型、生长及自我更新特性。 Example Characterization of human embryonic stem 2hESCyt-25 cell lines have been cultured for 18 months to maintain normal morphology, karyotype, growth and self-renewal characteristics. 该细胞系对OCT4、SSEA-4及TRA-1-60抗原显示了强烈的免疫反应性,上述抗原都是未分化hESCs的特征,并显示其它已建立的hESC系相同的碱性磷酸酯活性与形态学。 The cell line OCT4, SSEA-4 and TRA-1-60 antigen showed strong immunoreactivity, the above antigen are characterized in undifferentiated hESCs, and shows the same activity of alkaline phosphatase other established hESC lines and morphology. 此外,当人干细胞系hESCyt-25悬浮培养时,也易于形成类胚胎体(EBs)。 Further, when the human stem cell line hESCyt-25 suspension culture, but also easy to form embryoid bodies (EBs). 由于其多能本质的证明,hESCyT-25分化为代表三种主要胚层的不同细胞类型。 Due to demonstrate the pluripotent nature, hESCyT-25 to differentiate into different cell types represent the three primary germ layers. 以Q-PCR检测ZIC1及免疫细胞化学(ICC)检测巢蛋白及更多成熟的神经标志物来确证外胚层的生成。 In Q-PCR and immunocytochemistry detected ZIC1 (ICC) and more sophisticated detection of nestin marker corroborated neural ectoderm generated. β-III微管的免疫细胞化学染色可在伸长的细胞簇中观察到,具有早期神经元的特征。 Immunocytochemical staining β-III microtubules can be observed in the elongation of the cell clusters, having a characteristic of early neurons. 此前,我们在包含视黄酸的悬浮液中处理EBs可诱导多能干细胞分化为一种胚外系的内脏内胚层(VE)。 Previously, we suspension containing retinoic acid may be treated EBs induced pluripotent stem cells differentiate into extraembryonic visceral endoderm one kind of system (VE). 经过54小时的处理,处理的细胞表达高水平的α胎蛋白(AFP)、SOX7的两个VE标志物。 After 54 hours of treatment, treated cells express high levels of α-fetoprotein (AFP), SOX7 two markers VE. 免疫细胞化学染色显示以单层分化的细胞表达AFP为零星的片状。 Immunocytochemical staining differentiated cells show a monolayer sporadic expression of AFP as a sheet. 如下所述,hESCyT-25细胞系也可形成定形内胚层,通过实时定量聚合酶链反应(Q-PCR)及检测SOX17、无AFP表达的免疫细胞化学确证。 Below, hESCyT-25 cell lines also may be definitive endoderm formation by real-time quantitative polymerase chain reaction (Q-PCR) and detection SOX17, no expression of AFP by immunocytochemistry confirmed. 为了证实分化为中胚层,在几个时间点分析正在分化的EB以检测短尾(Brachyury)基因表达。 To confirm differentiation into mesoderm, differentiation of the EB being analyzed at several time points to detect a short-tailed (Brachyury) gene expression. 在试验过程中,短尾基因表达进行性增加。 During the test, macaque increased gene expression. 如前所述,hESCyT-25系为多能的,能够形成代表三个胚层的细胞。 As described above, hESCyT-25 system is a pluripotent, capable of forming cells represent three germ layers.

实施例3SOX17抗体的制备在hESC培养中识别定形内胚层的主要瓶颈为缺乏适当的工具。 Preparation Example 3SOX17 antibodies recognize a major bottleneck embodiment of definitive endoderm in hESC cultures for lack of proper tools. 我们因而制备抗SOX17蛋白的抗体。 We thus preparing an anti-antibody SOX17 protein.

在原肠胚形成期间形成时,标志物SOX17表达在整个定形内胚层,且其表达保持在肠管(尽管表达水平延AP轴有差异)直至器官开始形成。 When formed during gastrulation, SOX17 expression of markers in the whole embryos amorphous layer, and its expression in the intestinal tract held (although the level of expression of casting axis difference AP) until the organ begins to form. SOX17也表达在一系列胚外内胚层细胞上。 SOX17 is also expressed on a number of extraembryonic endoderm cells. 这种蛋白质在中胚层或外胚层无表达。 This protein was not expressed in the mesoderm or ectoderm. 当与其它标志物结合排除胚外谱系时,现已发现SOX17为定形内胚层谱系的合适的标志物。 When combined with other negative ectodermal lineage marker it has been found that SOX17 definitive endoderm lineage is suitable marker.

如本文详述,为了制备SOX17阳性的定形内胚层细胞,将SOX17抗体用于特异检测各种处理及分化方法的效果。 As detailed herein, for SOX17 positive endoderm cell preparation, SOX17 antibody specific for detecting the effect of various treatments and differentiation method. 其它与AFP、SPARC及凝血调节蛋白反应的抗体也用于排除内脏及体壁内胚层(胚外内胚层)的生成。 Other AFP, SPARC and thrombomodulin antibody reaction is also generated in the internal organs and for excluding parietal endoderm (extraembryonic endoderm) of.

为了制备抗SOX17的抗体,根据抗体制备公司GENOVAC(Freiberg,Germany)开发的方法,与SOX17蛋白(附图2)的羧端氨基酸172-414(SEQNO:2)对应的部分人SOX17 cDNA(SEQIDNO:1)用于大鼠的遗传免疫。 SOX17 antibody for the preparation of an antibody preparation according to the company GENOVAC (Freiberg, Germany) developed a method, and SOX17 protein (Figure 2) carboxyl-terminal amino acids 172-414 (SEQNO: 2) corresponding partially human SOX17 cDNA (SEQIDNO: 1) a genetic immunization of rats. 遗传免疫的方法可见于美国专利号5,830,876、5,817,637、6,165,993及6,261,281及国际专利申请公开号WO 00/29442及WO99/13915,其公开在此引入,以供参考。 Genetic immunization methods can be found in U.S. Patent Nos. 5,830,876,5,817,637,6,165,993 and 6,261,281 and International Patent Application Publication No. WO 00/29442 and WO99 / ​​13915, the disclosure of which is incorporated herein by reference.

遗传免疫的其它方法也可见于非专利文献。 Other methods of genetic immunization is also found in Non-Patent Document. 例如,Barry等所述的根据遗传免疫制备单克隆抗体,Biotechniques 16:616-620,1994,其公开在此全部引入,以供参考。 For example, Barry et al prepared a monoclonal antibody according to genetic immunization, Biotechniques 16: 616-620,1994, the entire disclosure of which is incorporated by reference. 根据遗传免疫制备抗特异蛋白抗体的具体方法,例如,Costaglia等(1998)人促甲状腺素受体的遗传免疫引起甲状腺炎及制备识别自身受体的单克隆抗体,J.Immunol.160:1458-1465;Kilpatrick等人(1998)基因枪传送的基于DNA的免疫介导了Flt-3受体的鼠单克隆抗体的快速制备,Hybridoma 17:569-576;Schmolke等人,(1998),在人血清中通过DNA免疫产生的E2-特异性单克隆抗体识别肝炎G病毒颗粒J,Virol.72:4541-4545;Krasemann等人,(1999)使用非传统的核酸免疫策略产生针对蛋白的单克隆抗体,J.73:119-129;及Ulivieri等人(1996)通过DNA免疫产生幽门螺杆菌空泡毒素确定部分的单克隆抗体,51:191-194,上述公开在此全部引入,以供参考。 According to a particular genetic immunization method of making an anti-antibody specific for the protein, e.g., Costaglia other genetic immunization (1998) caused by human thyrotropin receptor monoclonal antibody preparation and identifying thyroiditis autoreceptors, J.Immunol.160: 1458- 1465; DNA-based immunization Kilpatrick et al (1998) gene transfer mediated gun rapid preparation of the Flt-3 receptor murine monoclonal antibody, Hybridoma 17: 569-576; Schmolke et al., (1998), human produced by DNA immunization sera E2- specific monoclonal antibody recognizing hepatitis G virus particles J, Virol.72: 4541-4545; Krasemann et al., (1999) using a non-traditional nucleic acid immunization strategy to produce monoclonal antibodies against protein , J.73: 119-129; and Ulivieri et al (1996) determined the monoclonal antibody portion of vacuolating toxins produced by DNA immunization, 51: 191-194, all incorporated herein above disclosure, by reference.

如图3的关系树所示,在Sox家族,SOX7及SOX18与SOX17最相近。 Relation tree as shown in FIG. 3, the Sox family, SOX7 and SOX18 SOX17 with the most similar. 我们利用人SOX7多肽作为阴性对照以证实SOX17抗体对SOX17是特异的,不与最相近的家族成员反应。 We use people SOX7 peptide as a negative control to confirm SOX17 SOX17 antibody is specific, and does not react with the closest family members. 特别是,为了证明遗传免疫产生的抗体对SOX17是特异的,将SOX7及其它蛋白表达在人成纤维细胞上,然后通过Western blot及ICC分析与SOX17抗体的总反应活性。 In particular, in order to demonstrate genetic immunization produced antibodies are specific for SOX17, SOX7 and the other protein expression in human fibroblasts, and then analyzed for total SOX17 reactivity with antibodies by Western blot and ICC. 例如,使用下述方法制备SOX17、SOX7及EGFP的表达载体,将其转染入人成纤维细胞并用Western blot来分析。 For example, the following method was prepared SOX17, SOX7 and EGFP expression vectors and transfected into human fibroblast cells were analyzed by Western blot. 用于制备SOX17、SOX7及EGFP的表达载体分别为pCMV6(OriGene Technologies,Inc.,Rockville,MD)、pCMV-SPORT6(Invitrogen,Carlsbad,CA)及pEGFP-N1(Clonetech,Palo Alto,CA)。 For the preparation of SOX17, SOX7 and EGFP expression vector were pCMV6 (OriGene Technologies, Inc., Rockville, MD), pCMV-SPORT6 (Invitrogen, Carlsbad, CA) and pEGFP-N1 (Clonetech, Palo Alto, CA). 为制备蛋白,使用Lipofectamine 2000以超螺旋DNA瞬时转染端粒酶永生化的MDX人成纤维细胞(Invitrogen,Carlsbad,CA)。 For the preparation of protein using Lipofectamine 2000 to supercoiled DNA transient transfection MDX telomerase immortalized human fibroblasts (Invitrogen, Carlsbad, CA). 转染36小时后,收集总细胞裂解物于50mM TRIS-HCl(pH 8)、150mMNaCl、0.1%SDS、0.5%脱氧胆酸及一些蛋白酶抑制剂(Roche DiagnosticsCorporation,Indianapolis,IN)中。 36 hours after transfection, total cell lysates were collected in 50mM TRIS-HCl (pH 8), 150mMNaCl, 0.1% SDS, 0.5% deoxycholate and several protease inhibitors (Roche DiagnosticsCorporation, Indianapolis, IN) in. Western blot分析100μg的细胞蛋白,以SDS-PAGE在NuPAGE(4-12%梯度聚丙烯酰胺,Invitrogen,Carlsbad,CA)分离,通过电印迹转移至PDVF膜上(Hercules,CA),以在10mMTRIS-HCl(pH 8)、150mM NaCl、10%BSA、0.05%Tween-20(Sigma,St.Louis,MO)中稀释至1/1000的鼠SOX17抗血清进行探测,然后以结合碱性磷酸酯酶的抗大鼠IgG处理(Jackson ImmunoResearch Laboratories,West Grove,PA),结果以Vector Black碱性磷酸酯酶染色显示(VectorLaboratories,Burlingame,CA)。 Western blot analysis of cellular proteins 100μg, by SDS-PAGE on NuPAGE (4-12% gradient polyacrylamide, Invitrogen, Carlsbad, CA), transferred to PDVF membrane (Hercules, CA) by electroblotting to the 10mMTRIS- diluted HCl (pH 8), 150mM NaCl, 10% BSA, 0.05% Tween-20 (Sigma, St.Louis, MO) to the mouse antiserum 1/1000 SOX17 probed, and the alkaline phosphatase to bind processing anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), Vector Black results of alkaline phosphatase staining (VectorLaboratories, Burlingame, CA). 使用的蛋白大小标准为宽范围的彩色标志物(Sigma,St.Louis,MO)。 Protein size standards used for a wide range of color markers (Sigma, St.Louis, MO).

在图4中,将来自SOX17、SOX7或EGFP cDNA瞬时转染的人成纤维细胞的蛋白质提取物,以SOX17抗体为探针进行Western blot。 In FIG. 4, from the SOX17, SOX7 EGFP cDNA or transiently transfected into human fibroblast protein extracts, SOX17 antibody to probe Western blot. 仅hSOX17转染的细胞中提取的蛋白质产生了约51Kda的带,该带与预测的人SOX17蛋白质的46Kda分子量最匹配。 HSOX17 only transfected cells produced a protein extracted from the band of about 51Kda, 46Kda and the predicted molecular weight of the person with the best match SOX17 protein. SOX17抗体对来自人SOX7或EGFP转染细胞的提取物不具有反应性。 SOX17 antibody having no reactivity with the extract from human or SOX7 EGFP transfected cells. 此外,SOX17抗体明显地标记了以hSOX17表达构建体转染的人成纤维细胞核,但不标记以EGFP单独转染的细胞。 Further, SOXl 7 clearly labeled human antibody constructs were transfected fibroblasts expressing hSOX17 to the nucleus, but does not separate labeled with EGFP transfected cells. 同样地,SOX17抗体显示通过ICC检测的特异性。 Likewise, SOX17 antibody exhibits specificity by ICC detected.

实施例4SOX17抗体作为定形内胚层标志物的确证根据SOX17抗体对人SOX17蛋白质具有特异性并进一步标志定形内胚层,将部分分化的hESCs与SOX17和AFP抗体共同标记。 Example 4SOX17 antibody further embodiment endoderm markers according SOX17 SOX17 antibody is specific for human proteins, and the partial differentiation of hESCs SOX17 and AFP antibody labeled collectively as confirmation of definitive endoderm markers. 已证明了SOX17、SOX7及AFP各自表达于内脏内胚层中,SOX7为SOX基因家族F亚群密切相关的成员(图3)。 Have demonstrated SOX17, SOX7 and AFP are each expressed in visceral endoderm, SOX7 as SOX gene family subgroup F closely related members (Figure 3). 然而,AFP及SOX7在定形内胚层细胞中的表达不在可被ICC检测的水平,因此,可将其用作bonifide定形内胚层细胞的阴性标志物。 However, AFP expression in SOX7 and definitive endoderm cells can not be ICC levels detected, thus, it can be used as the definitive endoderm cells bonifide negative marker. 据显示,SOX17抗体标志细胞群体以分散的细胞群存在,或与AFP阳性细胞混合。 It has been shown, SOX17 antibody markers cell population dispersed cell populations exist, or mixed with AFP-positive cells. 特别是,图5A显示与AFP共同标志的少量SOX17细胞;然而,也发现了一些区域,其中在SOX17+细胞领域中具有少量或不具有AFP+细胞(图5B)。 In particular, FIG 5A shows the hallmarks of a small amount of AFP and SOX17 cells; however, also found some areas, having the art SOX17 + cells have little or no AFP + cells (FIG. 5B). 类似地,因为还报道了体壁内胚层表达SOX17,可将与SOX17共同标志的抗体与体壁标志物SPARC和/或凝血调节蛋白(TM)一起用于鉴别体壁内胚层的SOX17+细胞。 Similarly, as also reported in parietal endoderm expression SOX17, it may be with SOX17 common marker marker SPARC antibody with a body wall and / or thrombomodulin (TM) used together within the authentication parietal endoderm of SOX17 + cells. 如附图6A-C所示,通过hES细胞的随机分化产生凝血调节蛋白及SOX17共同标志的体壁内胚层细胞。 As shown in FIG. 6A-C, produced by random differentiation of hES cells within the thrombomodulin protein SOX17 common signs and parietal endoderm cells.

根据上述细胞标志试验,可将定形内胚层细胞的特性通过标志物表达谱SOX17hi/AFPlo/[TMlo或SPARClo]建立。 According to the test cell markers, the expression profile can SOX17hi / AFPlo / [TMlo or SPARClo] established characteristic of definitive endoderm cells by markers. 换言之,SOX17标志物的表达高于AFP标志物及TM或SPARC标志物的表达,AFP标志物为内脏内胚层的一种特征,TM或SPARC标志物为体壁内胚层的特征。 In other words, SOX17 expression of a marker above the AFP marker and expression of SPARC (TM) or markers, AFP as a marker characterized visceral endoderm, TM or SPARC markers characteristic of parietal endoderm. 因此,那些对SOX17呈阳性而对AFP呈阴性并对TM或SPARC呈阴性的细胞为定形内胚层。 Accordingly, those of SOX17 and AFP positive and negative SPARC (TM) or negative for definitive endoderm cells.

SOX17hi/AFPlo/TMlo/SPARClo标志物表达谱的特异性可作为定形内胚层预测的进一步证据,将SOX17及AFP基因表达可定量地与抗体标记细胞的相对数量相当。 Specific SOX17hi / AFPlo / TMlo / SPARClo marker expression profile may be used as further evidence that the predicted endoderm, SOX17 and AFP gene will be expressed quantitatively the relative amount of antibody labeled cells considerably. 如图7A所示,以视黄酸(内脏内胚层诱导物)或活化素A(定形内胚层诱导物)处理的hESCs导致了在SOX17 mRNA表达水平的10倍差异。 As shown in FIG. 7A, hESCs to retinoic acid (visceral endoderm inducer) or Activin A (the setting endoderm inducer) treatment resulted in 10-fold difference in SOX17 mRNA expression levels. 该结果反映出SOX17抗体标记的细胞数量的10倍差异(图7B)。 The results reflect SOX17 10-fold difference in the number of cells labeled antibody (FIG. 7B). 此外,如图8A所示,与未处理相比,hESCs的活化素A处理抑制了6.8倍的AFP基因表达。 Further, as shown, compared to the untreated, activin A treatment of hESCs suppressed in FIG. 8A 6.8 fold AFP gene expression. 在这些培养物中AFP标记的细胞数量的急剧减少从视觉上反映了这种变化,如图8B-C所示。 In these cultures drastically reduced the number of cells labeled AFP visually reflect this change, as shown in FIG. 8B-C. 为进一步量化,以流式细胞仪测定,证明了AFP基因表达的约7倍的减少为AFP抗体标记的细胞数量约减少7倍的结果(附图9A-B)。 To further quantify, determined by flow cytometry, we demonstrated approximately 7-fold reduction in gene expression of AFP-AFP antibody labeled number of cells about 7-fold reduction results (Figure 9A-B). 该结果非常重要,表明了Q-PCR中观察到的基因表达的数量变化,反映了通过抗体染色观察的细胞类型特异化典型的变化。 This result is very important, it indicates the amount of changes in gene expression observed in Q-PCR, reflecting cell types through specific antibody staining of typical variations.

hESCs在Nodal家族成员(Nodal、活化素A及活化素B-NAA)存在的条件下培养,导致SOX17抗体标志的细胞随着时间明显增加。 hESCs in the presence of Nodal family members (Nodal, activin A and activin B-NAA) culture, leading to cell marker SOX17 antibody significantly increased with time. 连续以活化素处理5天,有多于50%的细胞被SOX17标记(图10A-F)。 Activin continuously to 5 days, more than 50% of the cells were labeled SOX17 (FIGS. 10A-F). 活化素处理5天后,几乎没有细胞以AFP标志。 Activin 5 days of treatment, cells were almost no AFP marker.

总之,所产生的抗人SOX17蛋白质的碳端242个氨基酸抗体,在Western blots鉴别了人SOX17蛋白质但未识别SOX7,其为最近的Sox家族的近亲。 In short, the carbon produced by the anti-human SOX17 protein of 242 amino acids of the end of antibodies in Western blots but identified human SOX17 protein identification SOX7, which is nearest the Sox family kin. SOX17抗体识别了分化的hESC培养物中的细胞亚群,该亚群主要为SOX17+/AFPlo/-(多于95%的标志细胞)以及少量(<5%)的SOX17、AFP共同标志的细胞(内脏内胚层)。 SOX17 antibody that recognizes a cell subsets differentiated hESC cultures, the subpopulation is mainly SOX17 + / AFPlo / - (more than 95% of the flag cells) and a small amount (<5%) of SOX17, of AFP common flag cell ( visceral endoderm). 以活化素处理hESC培养物,产生了SOX17基因表达和SOX17标记细胞的显著上升,并显著地抑制了AFP mRNA表达及以AFP抗体标记的细胞数量。 Activin treated to hESC cultures, and generate gene expression increased significantly SOX17 SOX17 labeled cells, and significantly suppressed the expression of AFP mRNA in a number-AFP antibody labeled cells.

实施例5Q-PCR基因表达分析在下述试验中,实时定量RT-PCR(Q-PCR)为用于筛查各种处理对hESC分化作用的主要方法。 Example 5Q-PCR Analysis of gene expression in the following tests embodiment, the major method of quantitative real-time processing of the differentiation of hESC for RT-PCR (Q-PCR) screening. 特别是,在多个时间点以Q-PCR实时测定基因表达,分析多个标志基因。 In particular, at a plurality time points measured in real time Q-PCR gene expression analysis of multiple marker genes. 评价期望和不期望的细胞类型的标志基因特征,以获得对细胞群体总体动力学的更好了解。 Expectation and unwanted cell types characteristic marker gene in order to obtain a better understanding of the overall dynamics of cell populations. Q-PCR分析的力量包括其极端灵敏以及因基因组序列易于利用而相对容易能够发展必要的标志物。 Q-PCR analysis, including its extreme strength and sensitivity due to a genomic sequence can be easy to use and relatively easy to develop the necessary markers. 此外,Q-PCR极高的灵敏度允许在较大的群体中检测相对少量细胞的基因表达。 In addition, Q-PCR allow high sensitivity detection of gene expression in a relatively small number of cells in larger groups. 此外,检测极低水平的基因表达的能力提供了群体内的“分化倾向”的指示。 Furthermore, the ability to detect very low levels of gene expression provides a "tendency of differentiation" indication within a population. 在这些细胞表型的明显分化之前,其向具体分化途径的倾向性不能用免疫细胞化学技术识别。 Before significant differentiation phenotype of these cells, the specific differentiation pathway which tendency is not identified by immunocytochemistry. 因而,Q-PCR提供了一种分析方法,该方法至少为免疫细胞活性技术的补充,并潜在地更优于免疫细胞活性技术,用于筛查分化处理成功性。 Thus, Q-PCR analysis is provided a method of at least supplementary immune cell activity technology, and potentially better than the activity of immune cells technique successfully used for screening of the differentiation process. 此外,Q-PCR提供了一种机制,该机制通过半高通量规模上的定量分析评价分化方法是否成功。 In addition, Q-PCR provides a mechanism that analyzes whether evaluation by quantitative differentiation method succeeds half on high-throughput scale.

此处使用的方法相对定量,在Rotor Gene 3000 instrument(CorbettResearch)上使用SYBR Green化学及两步RT-PCR操作。 The method of relative quantification used herein, using SYBR Green two-step RT-PCR and chemical operation on Rotor Gene 3000 instrument (CorbettResearch). 该方法允许储存cDNA样品,以分析未来的其它标志基因,因此,避免样品间反转录效率的变异性。 This method allows storage cDNA sample, analysis of other marker genes in the future, therefore, to avoid inter-sample variability anti transcription efficiency.

若可能,设计的引物定位于外显子-外显子边界或横越至少800bp的内含子,因为这根据经验可消除污染的基因组DNA的扩增。 If possible, the designed primers located in exon - exon boundaries or across at least 800bp intron because it can eliminate amplification of genomic DNA contamination empirically. 当使用不含内含子或不具有假基因的标志基因时,进行RNA样品的DNA酶I处理。 When containing no introns or without pseudogene marker gene, a DNA sample of RNA DNase I treatment.

我们通常使用Q-PCR测定靶标和非靶标细胞类型的多个标志物的基因表达,以提供描述细胞样品基因表达的宽泛表达谱。 We usually use the expression Q-PCR assay target markers and the plurality of non-standard types of target cells, a sample to provide a gene expression broadly described expression profiles. 将hESC分化早期(具体地,外胚层、中胚层、定形内胚层及胚外内胚层)相关标志物及可用的验证引物提供于下述表1中。 The hESC differentiation of early (in particular, the ectoderm, mesoderm, endoderm and endoderm) and associated markers available authentication primers are provided in Table 1 below. 也已证明了这些引物组的人特异性。 It has also been proved that these human-specific primer set. 该事实很重要,因为通常将hESCs生长于小鼠滋养层上。 This fact is important, because usually the hESCs grown on mouse feeder layer. 最常见的是,各条件取3个样品,并独立地分析两次,以评价与各种定量检测相关的生物变异性。 The most common is, taking three samples for each condition and analyzed individually twice to evaluate the biological variability associated with various quantitative detection.

为制备PCR模板,将总RNA用RNeasy(Qiagen)分离,并用RiboGreen(Molecular Probes)定量。 To prepare a template for PCR with total RNA RNeasy (Qiagen) were separated and quantified using RiboGreen (Molecular Probes). 用iScript反转录试剂盒(BioRad)完成350-500ng总RNA的反转录,该试剂盒包含寡聚dT和随机引物的混合物。 350-500ng of total RNA completed with iScript reverse transcription kit (BioRad) reverse transcription, the kit comprises a mixture of oligo dT and random primers. 随后将各20μL反应物稀释至100μL总体积,取3μL用于各10μL Q-PCR反应,该反应包含400nM正向引物和反向引物及5μL 2X SYBR Greenmaster mix(Qiagen)。 20μL of each reaction was then diluted to a total volume of 100μL, 3μL taken for each 10μL Q-PCR reaction which contained 400nM of forward primer and reverse primer and 5μL 2X SYBR Greenmaster mix (Qiagen). 选用两步循环参数,在85-94℃5秒变性(根据各引物组扩增子的熔解温度进行具体选择),然后在60℃退火/延伸45秒。 Selection two step cycle parameters, five seconds denaturation at 85-94 deg.] C (specifically selected according to the melting temperature of each primer set amplicons), then annealed at 60 ℃ / extend for 45 seconds. 在各延伸期的最后15秒期间收集荧光数据。 Fluorescence data was collected during each extension phase of the last 15 seconds. 将10倍稀释系列的三个点用于产生各轮的标准曲线,并基于该标准曲线将循环阈(Ct's)转变为定量数值。 The three-fold dilution series of 10 points for each wheel to generate a standard curve, based on the standard curve and the cycle threshold (Ct's) into quantitative values. 将各样品的数值以看家基因表征校准,然后计算三个样品的平均及标准差。 The value of each sample to characterize the calibration housekeeping gene, and then calculating the average difference between the three samples and standards. 在结束PCR循环时,以熔解曲线分析确定反应的特异性。 At the end of the PCR cycles, melting curve analysis in order to determine the specificity of the reaction. 将单一特异产物显示在对PCR扩增子合适的Tm单一峰处。 The product exhibits suitable monospecific single peak at Tm of PCR amplicons. 此外,将不具有反转录酶的反应作为阴性对照,不扩增。 Further, having no reverse transcriptase reaction was used as a negative control, no amplification.

在建立Q-PCR方法学中的第一步为确定试验体系中合适的看家基因(HGs)。 The first step in establishing Q-PCR methodology in a test system for determining the appropriate housekeeping genes (HgS). 由于HG用于样品间RNA输入、RNA完整性及RT效率校准,为使校准有意义,HG在所有样品类型中随时间恒定表达水平是有价值的。 Since HG for RNA input among the samples, RNA integrity and RT efficiency calibration, the calibration to make sense, in all sample types HG expression level is a constant value over time. 我们测定了在分化hESCs中Cyclophilin G、次黄嘌呤磷酸核糖转移酶1(HPRT)、β-2-微球蛋白(microglobulin)、hydroxymethylbiane合成酶(HMBS)、TATA-结合蛋白(TBP)及glucoronidaseβ(GUS)的表达水平。 We measured in differentiated hESCs in Cyclophilin G, hypoxanthine phosphoribosyl transferase 1 (HPRT), β-2- microglobulin (microglobulin), hydroxymethylbiane synthase (HMBS), TATA- binding protein (TBP) and glucoronidaseβ ( GUS) expression levels. 我们的结果显示了β-2-微球蛋白表达水平在分化过程中提高,因此,我们排除了将该基因用于校准。 Our results show that the expression of β-2- microglobulin levels increase during differentiation, therefore, we exclude this gene for calibration. 其它基因表达水平与随时间和整个处理过程一致。 Consistent levels over time and the entire process to the other genes. 我们通常同时使用Cyclophilin G及GUS来计算所有样品的校准系数。 We usually calibration coefficient is calculated simultaneously for all samples using Cyclophilin G and GUS. 同时使用多个HGs减少校准过程固有的变异,增加相对基因表达值的可靠性。 Using multiple HGs reduce variability inherent calibration process, increasing the reliability of the relative gene expression values.

在获得用于校准的基因后,将Q-PCR用于检测接受不同试验处理后样品,确定许多标志基因的相对基因表达水平。 After obtaining the gene for calibration, the Q-PCR for detecting the received different treatment test sample to determine the relative gene expression levels of many marker genes. 选择使用的标志基因是在早期胚层的代表性特异群体中富集,特别是在定形内胚层和胚外内胚层中有差别表达的多组基因。 Selectable marker gene is used in a representative population of early endoderm-specific enrichment, especially extraembryonic endoderm germ layer and has a plurality of sets of differential gene expression in the setting. 将这些基因及其相关富集特征突出显示于表1中。 These genes and their related Enrichment characterized highlighted in Table 1.

表1 Table 1

因为许多基因在不只一个胚层中表达,在同一试验中定量比较许多基因的表达水平是有用的。 Because many genes are expressed in more than one germ layer, in the same trial quantitative comparison of expression levels of many genes it is useful. SOX17在定形内胚层中表达,在内脏及体壁内胚层中较小程度地表达。 SOX17 expression in the endoderm, the expression to a lesser extent in the visceral endoderm and the body wall. SOX7和AFP在内脏内胚层中在早期的发育时间点表达。 SOX7 and AFP expression in early developmental time points in the visceral endoderm. SPARC和TM在体壁内胚层中表达,以及Brachyury在早期中胚层中表达。 SPARC expression in the TM and the body wall endoderm, mesoderm and Brachyury expression during early.

预测定形内胚层细胞高水平表达SOX17 mRNA,低水平表达AFP和SOX7(内脏内胚层)、SPARC(体壁内胚层)和Brachyury(中胚层)。 Prediction Definitive endoderm cells express high levels of SOX17 mRNA, expressed at low levels and SOX7 AFP (visceral endoderm), SPARC (parietal endoderm) and Brachyury (mesoderm). 此外,本发明将ZIC1用于进一步排除早期外胚层的诱导。 Further, the present invention will be used to further exclude ZIC1 induces an early ectoderm. 最后,GATA4和HNF3b同时在定形及胚外内胚层中表达,因此,与SOX17在定形内胚层中的表达相关(表1)。 Finally, GATA4 and HNF3b expressed simultaneously in endoderm and outer shaping embryos, therefore, SOX17 expression and germ layers (Table 1) within the setting. 代表性试验显示于图11-14中,证实了表1所述的标志物基因如何与各样本彼此相关,因而强调了定形内胚层、胚外内胚层、中胚层以及神经细胞类型的特异分化模式。 Representative experiment are shown in Figures 11-14, confirms the marker genes according to Table 1 and how they relate to each other in each sample, and thus emphasizes the endoderm, extraembryonic endoderm, mesoderm, and differentiation of specific cell types of the neural model .

上述数据清晰表明了活化素剂量增加,导致的SOX17基因表达增加。 The above data clearly show that increasing dose of activin, SOX17 gene expression caused increases. 进一步地,该SOX17表达主要代表定形内胚层,而不是相反的胚外内胚层。 Further, the main representative SOX17 expression of definitive endoderm, mesoderm but not in extraembryonic opposite. 观察的结论为,SOX17基因表达与AFP、SOX7及SPARC反相关。 Observation to the conclusion that, SOX17 gene expression and AFP, SOX7 and SPARC inversely related.

实施例6人ES细胞定向分化为定形内胚层若人ES细胞培养物在不主动地保持其未分化状态下培养,该培养物将随机分化。 Example 6 directed human ES cells differentiate into mesoderm embodiment when the human ES cell culture was incubated not actively maintain their undifferentiated state, the culture differentiation of randomly shaped. 不同的分化导致形成胚外内胚层细胞,其包括体壁及内脏内胚层(AFP、SPARC及SOX7表达)两者,以及以ZIC1、Nestin(外胚层)及Brachyury(中胚层)的表达为标志的外胚层、中胚层衍生物。 Different differentiation results in formation of extraembryonic endoderm cells, which body wall visceral endoderm and comprises (AFP, SPARC and SOX7 expression) of both, and to express ZIC1, Nestin (ectoderm), and Brachyury (mesoderm) is marked ectoderm, mesoderm derivatives. 在ES细胞培养物中,由于缺乏特异抗体标志物,定形内胚层细胞外观未经传统方法检测或确定。 In ES cell cultures, due to the lack of specific antibody markers, the appearance setting endoderm cells without conventional method of detecting or determining. 同样地,由于缺乏手段,从ES细胞的培养物中产生早期定形内胚层未经仔细研究。 Similarly, the lack of means, without careful study of early endoderm generated from ES cells in culture. 由于无良好的、可用的定形内胚层细胞的抗体试剂,绝大多数确证集中在外胚层及胚外内胚层。 Since no good, the antibody reagents available endoderm cells, most concentrated confirmed endoderm and ectoderm germ. 总之,在随机的分化ES细胞培养物中,与SOX17hi定形内胚层细胞相比,有显著大量的胚外及神经外胚层细胞类型产生。 In short, randomly differentiated ES cell cultures, as compared to SOX17hi definitive endoderm cells, there is a significant amount of neuroectodermal and ectodermal cell types produce.

当将未分化的hESC克隆在成纤维细胞滋养物扩充时,克隆的边缘呈现出与克隆内部细胞不同的形态学特征。 When undifferentiated hESC expansion clones were nourished fibroblasts cloned edge inside the cell clone showing the different morphological features. 许多边缘细胞能以其较大的不均一的细胞体型及OCT4的高水平表达来区分。 Many cells can edge with its larger cell size variation and a high level expression of OCT4 be distinguished. 已有描述,当ES细胞开始分化时,它们表达OCT4的水平相对于未分化ES细胞的水平呈上下改变。 Has been described, when the ES cells begin to differentiate, they OCT4 expression levels relative to undifferentiated ES cells at a level goes change. 相对于未分化细胞的OCT4阈值水平,上下改变显示离开多能状态的初始分化状态。 OCT4 threshold level with respect to undifferentiated cells, and down to change the display to leave the initial state of differentiation of a pluripotent state.

当未分化的克隆以SOX17免疫细胞化学检测时,在未分化ESC克隆的四周及连接处偶尔可随机检测到SOX17阳性细胞的10-15个细胞组成的小簇。 When undifferentiated clone detection to SOX17 immunocytochemistry, in and around the joints undifferentiated ESC occasional random clones detected small clusters of 10-15 cells SOX17 positive cells. 如上所述,当克隆体积扩增以致变得拥挤时,克隆外缘分布的袋状似乎是从经典的ESC形态分化的第一批细胞。 As described above, when the amplification of clones that when the volume becomes congested, the outer edge of the bag-like distribution appears clones differentiated from the classic first aspect of the ESC cells. 在克隆的内部及边缘的年龄较小、体积较小的未分化克隆(<1mm;4-5天大小)无SOX17阳性细胞,然而在一些克隆的周边及边缘以内的年龄较大、体积较大克隆(1-2mm直径,>5天大小)含有零星的、游离的片状SOX17阳性、AFP阴性细胞,如上所述,其从经典的hESC形态分化而来。 In the younger age and the interior edge of the clones, clone undifferentiated small volume (<1mm; 4-5 days old) No SOX17 positive cells, but in some large and within the peripheral edge of clone age, larger clone (1-2mm diameter> 5 days of age) containing sporadic, sheet-like positive SOX17 free, of AFP negative cells, as described above, which is differentiated from the classic from hESC morphology. 鉴于这为有效的SOX17抗体的首次开发,在该早期“未分化”ESC培养物中生成的定形内胚层细胞此前尚未证实。 Since this is a valid SOX17 antibody was first developed in the early "undifferentiated" definitive endoderm cells generated ESC culture was not previously been confirmed.

基于Q-PCR测定的SOX17及SPARC基因表达水平的负相关,绝大多数SOX17阳性、AFP阴性细胞将对抗体共标记的体壁标志物呈阴性。 Q-PCR-based assay negative correlation of SPARC and SOX17 expression levels, the vast majority of positive SOX17, of AFP negative cells co-labeled antibody will body wall negative marker. 如图15A-B所示,在表达TM的体壁内胚层细胞得到特异证实。 As shown in FIG. 15A-B, the expression of the TM in parietal endoderm cells obtained specifically confirmed. 与Nodal因子活化素A及B接触导致TM表达强度及TM阳性细胞的数目的剧降。 Nodal factor contacted with activin A and B results in a sharp decline in the number of TM and expression of TM-positive cells. 在活化素处理的培养基上,通过使用SOX17、AFP及TM抗体的三重标记,观察到对AFP及TM也呈阴性的SOX17阳性细胞(图16A-D)。 Activin treatment in a medium by using SOX17, AFP and TM triple-labeled antibody, was observed SOX17 and AFP positive cells are also negative for TM (FIG. 16A-D). 这些都是在分化ESC的培养物上首次细胞证实SOX17阳性定形内胚层细胞(图16A-D及17)。 These are first differentiated cells confirmed the SOX17 positive definitive endoderm cells (FIGS. 16A-D and 17) on the ESC cultures.

使用上述的SOX17抗体及Q-PCR工具,我们已经开发一系列能够有效程序化ESCs变为SOX17hi/AFPlo/SPARC/TMlo定形内胚层细胞的方法。 SOX17 antibody using the above tools and Q-PCR, we have developed a series of effective methods can be programmed ESCs becomes definitive endoderm cells SOX17hi / AFPlo / SPARC / TMlo of. 我们应用了一系列的旨在增加这些细胞数量及增殖能力的分化方法,在群体水平上使用Q-PCR检测SOX17基因表达,在个体细胞上使用SOX17蛋白抗体标记。 We use a range of methods designed to increase differentiation and proliferation of these cell numbers using Q-PCR detection of gene SOX17 expression at the population level, using SOX17 antibody labeled on individual cells.

我们首次分析并描述了TGFβ家族生长因子,如Nodal/活化素/BMP,用于自体外细胞培养的胚胎干细胞中创建定形内胚层细胞的效果。 We first analyze and describe the TGFβ family of growth factors, such as Nodal / activin / BMP, used to create the effect of setting endoderm cells from stem cells in vitro cell culture of embryos. 在典型的试验中,我们将活化素A、活化素B、BMP或其组合加入至未分化的人干细胞系hESCyt-25培养物中开始分化过程。 In a typical experiment, we will activin A, activin B, BMP, or combinations thereof is added to undifferentiated human stem cell line cultures hESCyt-25 differentiation process started.

如图19所示,加入100ng/ml浓度的活化素A分化4天,与未分化的hESCs相比,诱导了19倍SOX17基因表达。 19, the addition of 100ng / ml activin A concentration four days of differentiation, as compared to undifferentiated of hESCs, 19-fold induction of gene SOX17 expression. 与活化素A一起加入活化素家族的第二个成员活化素B通过4天的组合活化素处理,与未分化的hESCs相比,诱导了37倍SOX17基因表达。 Added along with activin A second member of the family of activin activin B by a combination of activin for 4 days, as compared to undifferentiated of hESCs, 37-fold induction of gene SOX17 expression. 与活化素A及活化素B一同加入TGFβ家族Nodal/活化素及BMP亚群的第三个成员BMP4,与未分化的hESCs相比,诱导了57倍SOX17基因表达(图19)。 With activin A and activin B join TGFβ family Nodal / BMP and Activin sub-group a third member BMP4, as compared to undifferentiated hESCs, a 57-fold induction of SOX17 gene expression (FIG. 19). 当以活化素及BMP诱导SOX17时,与不加因子的培养物对照相比,分化4天导致5-,10-及15-倍的诱导。 When to activin and BMP-induced SOX17 when compared to cultures without a control factor, leading to four days of differentiation 5-, 10- and 15-fold induction. 通过5天活化素A、B及BMP的三重处理,SOX17诱导的倍数比hESCs高70倍以上。 Triple prime activation process A, B and BMP by five days, SOX17 induced more than 70 fold higher than that of hESCs times. 这些数据显示,以Nodal/活化素TGFβ家族成员较高剂量、较长的处理时间引起SOX17表达增加。 These data show that, in Nodal / TGFβ family member Activin higher doses, longer processing times causes an increase SOX17 expression.

Nodal及相关活化素A、B及BMP分子促进SOX17的表达、定形内胚层体内体外的形成。 Nodal and related activin A, B and BMP molecules facilitate expression of SOX17, in vitro and in vivo formation of definitive endoderm. 此外,加入BMP引起SOX17诱导升高,可能是通过Nodal共受体Cripto的进一步诱导。 Furthermore, addition of BMP-induced increase caused SOX17, may be further induced by Cripto Nodal co-receptor.

我们已经证实联合使用活化素A、B及BMP4引起SOX17诱导的增加及进而的定形内胚层形成。 We have confirmed that combined use of activin A, B and BMP4 caused SOX17 induced increase further and definitive endoderm formation. 与活化素A及B组合,长期加入BMP4(>4天)可诱导体壁及内脏内胚层及定形内胚层的SOX17增加。 In combination with activin A and B, was added a long BMP4 (> 4 days) and the body wall may induce SOX17 visceral endoderm and endoderm increases. 因此,在本发明的一些实施方案中,在加入处理的4天内除去BMP4是重要的。 Thus, in some embodiments of the present invention, the addition of 4 days, treated to remove BMP4 it is important.

为了在单细胞水平上确定TGFβ因子处理的效果,使用SOX17抗体标记检测加入一个时程的TGFβ因子的效果。 To determine the effect of TGFβ factor treatment at the single cell level, using the mark detecting the effect of added antibody SOX17 a time course of TGFβ factor. 如前图10A-F所示,随着时间进行,SOX17标记的细胞的相对数量出现剧增。 As shown in FIG. 10A-F before, with time, the relative number of SOX17 labeled cells grow dramatically. 相对定量(图20)显示SOX17-标记的细胞增加20倍以上。 Relative quantification (Figure 20) show SOX17- labeled cells increased more than 20 times. 该结果表明,与TGFβ因子暴露的时间增加,细胞数量及基因表达水平均增加。 The results show that the exposure time is increased TGFβ factor, the average increase in cell number and expression of water. 如图21所示,与Nodal、活化素A、活化素B及BMP4接触4天后,SOX17诱导的水平比未分化的hESCs高168倍。 21, and Nodal, activin A, activin B and BMP4 for 4 days in contact, SOXl 7 induced levels 168 times higher than undifferentiated hESCs. 图22表明SOX17阳性细胞的数量也呈剂量依赖性。 SOX17 FIG. 22 shows that the number of positive cells was also dose dependent. 100ng/mL或更高剂量的活化素A能够强有力地诱导SOX17的基因表达及细胞数量增加。 100ng / mL or higher doses of activin A can strongly induce gene expression and increase the number of cells SOX17.

除TGFβ家族成员,Wnt家族分子可能在定形内胚层特异性和/或保持上起作用。 In addition to members of the TGFβ family, Wnt family of molecules may endoderm-specific and / or play a role in holding the setting. 与单用活化素相比,使用活化素+Wnt3a的样本SOX17基因表达增加,表明Wnt分子对hESCs分化为定形内胚层也有益(图23)。 Compared with activin alone, activin + samples using SOX17 expression of Wnt3a increases, indicate that Wnt molecules hESCs differentiate into definitive endoderm also advantageous (FIG. 23).

上述所有试验均在含10%血清及添加因子的组织培养基中进行。 All the above tests were performed in 10% serum-containing tissue culture medium and added factors. 令人吃惊的是,我们发现在添加的活化素存在下,血清浓度对SOX17表达水平有作用,如图24A-C所示。 Surprisingly, we have found that in the presence of added activin, SOX17 expression levels of serum concentration of the role, as shown in FIG. 24A-C. 当血清水平由10%降至2%时,在活化素A及B的存在下,SOX17的表达增加3倍。 When the serum levels from 10% to 2%, in the presence of activin A and B, 3-fold increase in the expression of SOX17.

最后,我们证实活化素诱导SOX17+细胞在培养物中分裂,如图25A-D所示。 Finally, we confirmed that activin induced SOX17 + cells divide in culture, as shown in Figure 25A-D. 箭头显示以SOX17/PCNA/DAPI标记的细胞处于有丝分裂期,证据是PCNA/DAPI-标记的有丝分裂板模式及时相差有丝分裂特征。 The arrow shown in SOX17 / PCNA / DAPI-labeled cells in mitosis, as evidenced PCNA / DAPI- labeled mitotic phase difference plate mode timely mitosis feature.

实施例7趋化因子受体4(CXCR4)的表达与定形内胚层标志物相关、而与中胚层、外胚层或内脏内胚层标志物不相关如上所述,通过使用TGFβ家族及更特异的活化素/nodal亚族的细胞因子,ESCs可被诱导分化至定形内胚层胚层。 Example 7 chemokine receptor 4 (CXCR4) is associated with the expression of definitive endoderm markers, while the mesoderm, ectoderm or visceral endoderm markers unrelated described above, by using the TGFβ family and more specific activation cytokine hormone / nodal subfamily, ESCs can be induced to differentiate to definitive endoderm endoderm. 此外,我们已经表明胎牛血清(FBS)在分化培养基中的比例影响定形内胚层自ESCs分化的效率。 Furthermore, we have shown that fetal bovine serum (FBS) in differentiation medium ratio influence the efficiency of definitive endoderm differentiation from ESCs. 该效果为,在培养基中既定的活化素A浓度的条件下,较高水平的FBS将抑制其最大分化至定形内胚层胚层。 This effect is, at a predetermined medium activin A in the conditions, the higher levels of FBS to inhibit the differentiation of endodermal maximum endoderm. 缺乏外源活化素A时,ESCs分化至定形内胚层谱系的效率极低,且FBS浓度对ESCs的分化过程有较弱的效果。 In the absence of exogenous activin A, ESCs differentiated to an extremely low efficiency of the definitive endoderm lineage, and FBS concentration of a weak effect on the differentiation of ESCs.

在这些试验中,hESCs的分化是在RPMI培养基(Invitrogen,Carlsbad,CA;cat#61870-036)中生长6天,该培养基补充有0.5%、2.0%或10%FBS,包含或不含100ng/mL活化素A。 In these tests, a differentiation of hESCs in RPMI medium (Invitrogen, Carlsbad, CA; cat # 61870-036) 6 days in growth medium supplemented with 0.5%, 2.0% or 10% FBS, with or without 100ng / mL activin A. 此外,在分化的前三天,0.5%-2.0%的梯度FBS也与100ng/mL活化素A联合使用。 Further, in the first three days of differentiation, a gradient of 0.5% -2.0% FBS also be used in combination with 100ng / mL activin A. 6天后,自各培养条件中收集复制物样本,以实时定量PCR分析相对基因表达。 After 6 days, samples were collected from each replicate culture conditions, relative to real-time PCR analysis of gene expression. 将剩余细胞混合,以免疫荧光检测SOX17蛋白。 The remaining cells were mixed, SOX17 protein to detect immunofluorescence.

在使用的7个培养条件下,CXCR4的表达水平差异巨大(图26)。 In seven culture conditions used, a great difference in expression levels of CXCR4 (FIG. 26). 一般地,CXCR4表达在活化素A处理的培养基(A100)中高,而在无外源活化素A(NF)的培养基中低。 Generally, CXCR4 expressed in high activin A treatment medium (A100), low in a medium without exogenous activin A (of NF) of. 此外,在A100处理的培养基中,当FBS浓度最低时,CXCR4表达最高。 Further, in the medium treated A100, when the lowest concentration of FBS, CXCR4 was the highest. 在10%FBS条件下,CXCR4水平显著降低,以致相对表达更吻合无活化素A(NF)的条件。 Under conditions of 10% FBS, CXCR4 levels were significantly reduced, so that no more consistent with the relative expression of activin A (NF) conditions.

如上所述,SOX17、GSC、MIXL1、及HNF3β基因的表达与定形内胚层细胞的特征相一致。 As described above, SOX17, GSC, MIXL1, and gene expression characteristics HNF3β definitive endoderm cells consistent. 这4个基因在7个分化条件下的相对表达影射了CXCR4(图27A-D)的表达。 The relative expression of these four genes in the differentiation condition 7 the mapping of CXCR4 (FIG. 27A-D) expression. 这也证实了CXCR4也为一种定形内胚层标志物。 This is confirmed also CXCR4 as a marker of definitive endoderm.

外胚层及中胚层谱系可通过其表达的各种标志物与定形内胚层区分开。 Ectodermal and mesodermal lineage by their expression of various markers of definitive endoderm separated regions. 早期中胚层表达Brachyury及MOX1基因,然而,初生神经外胚层表达SOX1及ZIC1。 Brachyury expression of mesoderm and MOX1 early genes, however, the primary and neuroectodermal Expression SOX1 ZIC1. 图28A-D证实无外源活化素A的培养物有利于中胚层及外胚层基因表达,在活化素处理的培养物中,10%FBS条件也增加了中胚层及外胚层标志物表达的水平。 FIGS 28A-D cultures was confirmed without exogenous activin A beneficial mesoderm and ectoderm gene expression, in the activin-treated cultures, 10% FBS condition also increases the level of expression of mesoderm and ectoderm markers . 这些表达模式与CXCR4模式相反,显示在该发育时程,CXCR4并不高表达于衍生于ESCs中胚层或外胚层中。 The expression pattern of CXCR4 contrast mode, the display process, CXCR4 is not highly expressed in mesoderm or ectoderm derived ESCs in the development at the time.

在哺乳动物发育早期,也发生了分化至胚外谱系。 In the early mammalian development, differentiation also occurred to the extraembryonic lineage. 内脏内胚层的分化在此具有特异的相关性,其与定形内胚层共同的许多基因,包括SOX17具有相同的表达。 Visceral endoderm differentiation herein with specific correlation with the number of genes within a common setting endoderm, including SOX17 expression of the same. 为了将定形内胚层与胚外内脏内胚层区分,应当检测两者不同的标志物。 To the inner and outer definitive endoderm germ visceral endoderm distinguish two different markers should be detected. SOX7代表表达在内脏内胚层,而不是定形内胚层谱系的标志物。 Representative SOX7 expressed in visceral endoderm, rather than the amorphous endoderm lineage markers. 因而,在无SOX7表达的条件下,显示强SOX17基因表达的培养物条件可能包括定形内胚层,而非内脏内胚层。 Thus, in the absence of expression SOX7 conditions, culture conditions show strong SOX17 gene expression of definitive endoderm may include, instead of visceral endoderm. 如图28E所示,SOX7在无活化素A培养基中高表达,SOX7甚至在活化素A存在的条件下,当FBS包括10%时,也表达增加。 As shown in FIG. 28E, SOX7 expression of activin A in the absence of the medium, SOX7 even in the presence of activin A conditions, including when 10% FBS, also increased expression. 该模式与CXCR4表达模式相反,表明CXCR4在内脏内胚层不高表达。 In contrast with the pattern CXCR4 expression pattern showed no expression of CXCR4 in visceral endoderm.

还检测了上述各分化条件下SOX17免疫活性(SOX17+)细胞的相对数量。 SOX17 immunoreactivity was also tested under the above conditions relative amount each of differentiation (SOX17 +) cells. 当hESCs在高剂量活化素A及低FBS浓度(0.5%-2.0%)下分化时,SOX17+细胞在培养物中普遍分布。 When activin A to differentiate hESCs and a low concentration of FBS (0.5% -2.0%) in high doses, SOX17 + cells were widely distributed in the culture. 当使用高剂量活化素A,而FBS浓度为10%(v/v)时,SOX17+细胞出现的频率降低,经常以孤立的簇出现,而不是均匀发布在培养物中(图29A、C、B及E)。 When a high dose activin A, and FBS concentration of 10% (v / v), SOX17 + frequency cells showed decreases, often in an isolated cluster appears, rather than being evenly released in culture (FIG. 29A, C, B and E). 当无外源活化素A使用时,发现SOX17+细胞进一步降低。 When used without exogenous activin A, SOX17 + cells were found to be further reduced. 在这些条件下,SOX17+细胞也以簇状出现,但这些簇较小且较高活化素A、低FBS处理时少(图29C及F)。 Under these conditions, SOX17 + cells also appeared to clustered, but the smaller the clusters and high activin A, less (FIG. 29C and F) at low FBS treatment. 这些结果表明,CXCR4表达模式不仅在各种条件下符合定形内胚层基因表达,而且符合定形内胚层细胞的数量。 These results indicate that, CXCR4 gene expression patterns not only in the endoderm under various conditions, and in accordance with the number of definitive endoderm cells.

实施例8富集定形内胚层的分化条件增加CXCR4阳性细胞的比例活化素A的剂量也影响了定形内胚层从ESCs衍生的效率。 Increasing the proportion of CXCR4 positive cells enriched conditions in Example 8 definitive endoderm embodiment of activin A dose also affected the definitive endoderm derived from ESCs efficiency. 本实施例增加活化素A的剂量增加了CXCR4+细胞在培养物中的比例。 The present embodiment increases the dose of activin A increased the proportion of CXCR4 + cells in cultures.

将hESCs在添加了0.5%-2%FBS(在分化的前3天,逐渐由0.5%增加至1.0%,再至2.0%)及0、10或100ng/mL活化素A的RPMI培养基中分化。 The differentiation of hESCs supplemented with 0.5% -2% FBS (three days before differentiation, increasing from 0.5% to 1.0% and then to 2.0%) and 0, 10 or 100ng / mL activin A in RPMI medium . 分化7天后,将细胞在不含Ca2+/Mg2+、包含2%FBS及2mM(EDTA)的PBS中室温解离5分钟。 After 7 days of differentiation, the cells in the absence of Ca2 + / Mg2 +, containing 2% FBS and 2mM (EDTA) in PBS at room temperature for 5 minutes dissociation. 以35μm尼龙滤器过滤细胞、计数及沉淀。 Nylon filter to 35μm cell filter, and precipitate counted. 将沉淀再悬浮于50%人血清/50%正常驴血清,在冰上孵育2分钟阻断非特异抗体结合位点。 The precipitate was resuspended in 50% human serum / 50% normal donkey serum, were incubated for two minutes to block non-specific antibody binding site on ice. 向每50μL(包含约105个细胞)悬液加入1μL小鼠抗CXCR4抗体(Abcam,cat#ab10403-100),再在冰上标记45分钟。 1μL of each mouse was added to 50 L (containing approximately 105 cells) suspension of anti-CXCR4 antibody (Abcam, cat # ab10403-100), then labeled on ice for 45 minutes. 加入5mL包含2%人血清(缓冲液)的PBS洗涤细胞、沉淀。 Was added 5mL containing cells were washed PBS 2% human serum (buffer), precipitated. 再以5mL缓冲液洗涤一次后,将细胞再以50μL缓冲液/105细胞浓度悬浮。 After 5mL again washed with buffer, and the cells to 50μL buffer / 105 cells were suspended at a concentration. 加入终浓度为5μg/mL的第二抗体(结合的FITC驴抗小鼠抗体;Jackson Immuno Research,cat#715-096-151),标记30分钟后再以上述缓冲液洗涤2次。 Added to a final concentration of 5μg / mL of secondary antibody (donkey anti-mouse FITC-bound antibody; Jackson Immuno Research, cat # 715-096-151), numeral 30 minutes before washed with the above buffer twice. 将细胞再以5×106细胞/mL悬浮于缓冲液中,由流式细胞仪设备操作人员(The ScrippsResearch Institute)使用FACS Vantage(Beckton Dickenson)分析、分选。 Cells were at 5 × 106 cells / mL suspension in buffer, analysis, sorting by a flow cytometer using the equipment operator (The ScrippsResearch Institute) FACS Vantage (Beckton Dickenson). 将细胞直接收集于RLT裂解缓冲液(Qiagen)供随后的总RNA分离,再以实时定量PCR进行基因表达分析。 The cells are directly collected in RLT lysis buffer (Qiagen) for subsequent Total RNA was isolated, then real-time PCR gene expression analysis.

当活化素A在分化培养基中的剂量(图30A-C)增加时,可观察到流式细胞仪测定的CXCR4+细胞也剧增(图30A-C)。 When the dose of activin A (FIG. 30A-C) increase in the differentiation medium, CXCR4 + cells observed by flow cytometry also rose sharply (FIGS. 30A-C). CXCR4+细胞落入R4门,该门仅使用第二抗体作为对照,R4门中存在0.2%的该对照事件。 R4 CXCR4 + cells falls within a door using only the second antibody as a control, 0.2% of the control gate R4 event exists. 当活化素A的剂量增加时,CXCR4+细胞数量的剧增与定形内胚层基因表达明显增加相关(图31A-D)。 When increasing the dose activin A, CXCR4 + dramatic increase in the number of cells and definitive endoderm gene expression was significantly associated with an increased (FIGS. 31A-D).

实施例9富集分离CXCR4阳性细胞供定形内胚层基因表达、除去表达中胚层、外胚层及内脏内胚层标志物的细胞收集上述实施例8识别的CXCR4+及CXCR4-细胞,分析了其相对基因表达,同时测定了母体细胞群的基因表达。 Example 9 Enrichment isolated CXCR4 positive cells for gene expression of definitive endoderm, mesoderm, ectoderm and cell visceral endoderm markers collected expression in the above Example 8 Identification removed CXCR4 + and CXCR4- cell, analyzes the relative gene expression simultaneous determination of gene expression maternal cell population.

当活化素A的剂量增加时,CXCR4+基因表达的相对水平剧增(图32)。 When increasing the dose activin A, CXCR4 + dramatic increase in the relative levels of gene expression (FIG. 32). 这与活化素A剂量依赖性增加CXCR4+细胞相关良好(图30A-C)。 This activin A dose-dependent increase in cell associated CXCR4 + well (FIG. 30A-C). 也很明显,从各细胞群分离出CXCR4+细胞占据了该细胞群中几乎所有的CXCR4基因表达细胞。 It is also evident from the isolated population of CXCR4 + cells in each cell occupies almost all of the CXCR4 gene expression in a cell population cells. 这证实了FACS方法收集这些细胞方法的效率。 This confirms the efficiency of the method of collecting these cells FACS methods. 基因表达分析表明,CXCR4+细胞不仅包括大部分CXCR4基因表达,而且也包括其它定形内胚层标志物的基因表达。 Gene expression analysis showed that, CXCR4 + cells comprise most of the CXCR4 gene expression not only, but also including other gene expression of definitive endoderm markers. 如图31A-D所示,进一步从SOX17、GSC、HNF3B、及MIXL1的母体A100细胞群富集CXCR4+细胞。 As shown in FIG. 31A-D, a further population of cells from maternal A100 SOX17, GSC, HNF3B, enrichment and MIXL1 CXCR4 + cells. 此外,CXCR4-部分包括极少的这些定形内胚层标志物基因表达。 Further, CXCR4- portion comprises very few of these definitive endoderm marker gene expression. 而且,CXCR4+及CXCR4-细胞群显示了中胚层、外胚层及胚外内胚层标志物基因表达相反模式。 Moreover, CXCR4 + cell population and CXCR4- shows mesoderm, and ectoderm germ marker gene, opposite outer endoderm expression patterns. 图33A-D表明,相对于A100母体细胞群,除去CXCR4+细胞以进行Brachyury、MOX1、ZIC1及SOX7基因表达。 FIGS 33A-D show that, with respect to the parent A100 cells, CXCR4 + cells was removed for Brachyury, MOX1, ZIC1 SOX7 and gene expression. 相对于低或无活化素A的条件,该A100母体细胞群表达这些标志物已经很低。 With respect to the conditions of low or no activin A, the A100 parental cell population expressing these markers are already low. 这些结果表明,高剂量活化素A存在分化条件下自hESCs中分离的CXCR4+细胞获得高度富集的基本纯的定形内胚层细胞。 These results show that isolated from hESCs CXCR4 + cells to obtain substantially pure highly enriched in the definitive endoderm differentiation conditions cells high dose activin A.

实施例10使用CXCR4定量细胞群体中的定形内胚层细胞为了确定细胞培养物或细胞群中定形内胚层细胞的比例定量,根据前述方法或于2003年12月23日提交的美国临时专利申请第60/532,004号,题目为“定形内胚层”所述的方法,其公开在此全部引入以供参考,以FACS分析表达CXCR4及其它定形内胚层标志物的细胞。 Example 10 endoderm cells CXCR4 quantification of cell population to determine cell culture ratio quantification of definitive endoderm cells or population of cells, according to U.S. Provisional Patent Application foregoing method or on December 23, 2003, filed 60 / No. 532,004, entitled method "definitive endoderm" said, the entire disclosure of which is incorporated herein by reference, FACS analysis in cells expressing CXCR4 and the other markers of definitive endoderm.

使用诸如上述实施例所述的方法,将hESCs分化产生定形内胚层。 The method of using the above-described embodiments such as the embodiment, the hESCs differentiate to produce definitive endoderm. 特别是,增加表达在分化细胞培养物的产率及纯度,培养基中血清浓度严格控制如下:第一天0.2%FBS、第二天1.0%FBS及第3-6天2.0%FBS。 In particular, increased expression in differentiated cell cultures yield and purity of the culture medium serum concentration strictly controlled as follows: The first day of 0.2% FBS, 1.0% FBS and the second next day 3-6 days 2.0% FBS. 以FACS使用三种细胞表面抗原决定簇E-钙粘蛋白(Cadherin)、CXCR4及凝血调节蛋白分选分化的培养物。 In FACS using three cell surface epitopes E- cadherin (Cadherin), CXCR4 and thrombomodulin sorting differentiation cultures. 然后以Q-PCR分析分选细胞群以确定定形内胚层、胚外内胚层及其它细胞类型的标志物相对表达水平。 Then Q-PCR analysis of cell populations sorted to determine endoderm, extraembryonic endoderm and other cell types relative expression levels of marker. 从最佳分化培养物获得的CXCR4分选细胞产生了>98%纯度的定形内胚层细胞的分离。 CXCR4 obtained from the optimum differentiation culture were sorted cells isolated within a> 98% purity of the definitive endoderm cells.

表2显示了使用本发明所述的方法自hESCs分化的定形内胚层培养物标志物分析的结果表2定形内胚层培养物的成分 Table 2 shows the ingredients used in the method of the present invention results from the differentiation of hESCs definitive endoderm culture marker analysis in Table 2 endoderm cultured amorphous form of

特别是,表2显示CXCR4及SOX17阳性细胞(内胚层)包括70%-80%的细胞培养物中的细胞。 In particular, Table 2 shows CXCR4 and 70% -80% of the cell culture SOX17 positive cells (endoderm) comprising the cells. 在这些表达SOX17的细胞中,低于2%表达TM(体壁内胚层),低于1%表达AFP(内脏内胚层)。 SOX17 expression in these cells, expression of less than 2% (TM) (parietal endoderm), less than 1% expressed AFP (visceral endoderm). 当从SOX17/CXCR4阳性细胞比例中减去TM阳性及AFP阳性细胞(体壁及内脏内胚层组合;总计3%),可发现约67%-77%细胞培养物为定形内胚层。 When subtracting the positive TM (parietal and visceral endoderm composition; total 3%) and the proportion of AFP positive cells from SOX17 / CXCR4 positive cells, can be found in about 67% -77%, the cell culture is a definitive endoderm. 大约10%的细胞为E-钙粘蛋白(ECAD)阳性,其为hESCs标志物,约10-20%的细胞为其它细胞类型。 Approximately 10% of the cells E- cadherin (the ECAD) positive, which is a marker for hESCs, about 10-20% of the cells into other cell types.

我们也发现,与前述的在整个5-6天的分化操作中将FBS浓度保持在≤0.5%的低血清方法相比,在FACS分离前分化细胞培养物中的定形内胚层的纯度可提高。 We also found that the aforementioned 5-6 days throughout the differentiation operation in the FBS concentration is kept in low serum as compared to the method of ≤0.5%, purity before FACS separation of the differentiated cell cultures of the definitive endoderm can be improved. 然而,在整个5-6天的分化操作中将细胞培养物浓度保持在≤0.5%,也导致了产生的定形内胚层细胞总的数量减少。 However, 5-6 days the whole culture of differentiated cells in the concentration operation is maintained at ≤0.5%, also led to the total number of definitive endoderm cells is reduced.

根据本发明所述的方法制备的定形内胚层细胞在活化素存在下的培养物中保持扩增50天以上而无明显分化。 The definitive endoderm cells produced by the method according to the present invention maintains more than 50 days amplification culture in the presence of activin without significant differentiation. 在这些情况下,培养期间保持SOX17、CXCR4、MIXL1、GATA4、HNF3β的表达。 In these cases, the expression of the culture period to maintain SOX17, CXCR4, MIXL1, GATA4, HNF3β of. 此外,在这些培养物中未检测到TM、SPARC、OCT4、AFP、SOX7、ZIC1及BRACH。 Further, in these cultures was not detected in TM, SPARC, OCT4, AFP, SOX7, ZIC1 and BRACH. 将定形内胚层细胞在活化素存在下的培养物中保持扩增基本上50天以上而无明显分化是可能的。 The definitive endoderm cell cultures in the presence of activin substantially maintained over 50 days amplification without significant differentiation is possible.

实施例11定形内胚层细胞的其它标志物在下述试验中,RNA分离自纯化的定形内胚层及人胚胎干细胞群。 Other markers Example 11 amorphous endoderm cells embodiment in the following experiment, RNA isolated from purified endoderm and human embryonic stem cells. 然后以基因芯片分析来自每一纯化细胞群的RNA。 Microarray analysis of RNA was then purified from each cell population. 采用Q-PCR进一步考察定形内胚层而非胚胎干细胞表达的基因作为定形内胚层标志物的潜力。 Further investigated using Q-PCR of definitive endoderm not embryonic stem cells as potential gene expression of definitive endoderm markers.

将人胚胎干细胞(hESCs)保持在DMEM/F12培养基中,该培养基补充了20%KnockOut血清替代品、4ng/mL重组人基础成纤维生长因子(bFGF)、0.1mM 2-巯基乙醇、L-谷氨酸、非必需氨基酸及青霉素/链霉素。 The human embryonic stem cells (of hESCs) held in DMEM / F12 medium, the medium was supplemented with 20% KnockOut Serum Replacement, 4ng / mL recombinant human basic fibroblast growth factor (bFGF), 0.1mM 2- mercaptoethanol, L - glutamate, non-essential amino acids and penicillin / streptomycin. 将hESCs在RPMI培养基中培养5天分化至定形内胚层,该培养基补充了100ng/mL重组人活化素A、胎牛血清(FBS)及青霉素/链霉素。 The hESCs cultured for 5 days differentiation to definitive endoderm in RPMI medium, the medium was supplemented with 100ng / mL of recombinant human activin A, fetal bovine serum (FBS) and penicillin / streptomycin. FBS各天的浓度变化为:0.1%(第一天)、0.2%(第二天)及2%(第3-5天). Days of the FBS concentration was: 0.1% (the first day), 0.2% (the next day) and 2% (3-5 days).

为获得细胞hESCs及定形内胚层纯的群体进行基因表达分析,以荧光活化细胞分选(FACS)分离。 Gene expression analysis to obtain pure cell hESCs and definitive endoderm population to fluorescence activated cell sorting (FACS) was separated. 使用SSEA4抗原(R&amp;D Systems,cat#FAB1435P)免疫纯化hESCs,使用CXCR4(R&amp;D Systems,cat# FAB170P)纯化定形内胚层。 Use SSEA4 antigen (R & amp; D Systems, cat # FAB1435P) immunopurification hESCs, using CXCR4 (R & amp; D Systems, cat # FAB170P) the purified definitive endoderm. 细胞解离使用胰蛋白酶/EDTA(Invitrogen,cat#25300-054)、以含2%人血清的磷酸盐缓冲液(PBS)洗涤,重新悬浮于100%人血清中并置于冰上10分钟阻断非特异性结合。 Cells were dissociated using trypsin / EDTA (Invitrogen, cat # 25300-054), containing phosphate buffer washed with 2% human serum (PBS), resuspended in 100% human serum and placed on ice for 10 minutes barrier off non-specific binding. 将200μL的结合藻红蛋白的抗体加入800μL的人血清中的5×106细胞中,在冰上染色30分钟。 The antibody binding phycoerythrin was added 800μL 200μL human serum 5 × 106 cells, stained on ice for 30 minutes. 以8mL PBS缓冲液洗涤细胞两次,再悬浮于1mL PBS中。 Cells were washed twice in 8mL PBS buffer, resuspended in 1mL PBS. FACS分离以Scripps研究所的核心设备,使用FACS Vantage(BDBiosciences)进行。 FACS separation of the core equipment Scripps Research Institute, using FACS Vantage (BDBiosciences) carried out. 将细胞直接收集于RLT裂解缓冲液,根据操作说明(Qiagen)以RNeasy分离RNA。 The cells are directly collected in RLT lysis buffer, RNA was isolated according to the instructions RNeasy (Qiagen).

将纯化的RNA送样两次(Durham,NC),使用Affymetrix平台的U133Plus2.0高密度寡核苷酸阵列生成表达特征数据产生表达谱数据。 Purified RNA samples sent twice (Durham, NC), generating an expression using the Affymetrix platform characterized U133Plus2.0 high-density oligonucleotide array data generated expression data. 呈现的数据是一组比较,鉴别hESCs与定形内胚层两个细胞群差异表达的基因。 Data presented is a group, identify gene expression differences between two cell populations hESCs and endoderm. 将表达水平与hESCs发现的基因水平相比强烈升高变化的基因选作新的候选标志物,其具有高度的定形内胚层特征。 The level of gene expression levels is strongly increased compared to hESCs discovered genetic changes selected as a new candidate marker having an inner height characteristic of definitive endoderm. 根据所述的方法使用Q-PCR测定选定的基因,以验证基因芯片上发现的基因表达变化,并考察hESC分化时程间的这些基因的表达模式。 Q-PCR assay using the selected genes according to the method, to verify changes in gene expression found on gene chips, and examine the expression patterns of these genes in the process between the time differentiation of hESC.

图34A-M显示一些标志物基因表达结果。 FIGS 34A-M show some marker gene expression results. 加入100ng/ml活化素A后,于第1、3及5天分析细胞培养物,显示分化操作(CXDE)5天结束后表达CXCR4的定形内胚层细胞及人胚胎干细胞(HESC)的结果。 After addition of 100ng / ml activin A, 1, 3 and 5 days in the analysis of cell cultures, differentiation of the operation display (CXDE) CXCR4 expression amorphous endoderm cells and human embryonic stem cells (HESC) results after 5 days. 图34C及GM比较表明六个标志物基因FGF17、VWF、CALCR、FOXQ1、CMKOR1及CRIP1,显示彼此几乎完全相同的表达模式,其也完全与CXCR4及SOX17/SOX7表达模式相同。 FIGS. 34C and GM comparison shows six marker genes FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIPl, shows the expression pattern almost identical to each other, which is also completely CXCR4 and SOX17 / SOX7 same expression pattern. 如上所述,SOX17在定形内胚层及表达SOX7的胚外内胚层两者中均表达。 As described above, SOX17 in the expression of definitive endoderm and SOX7 both the extraembryonic mesoderm of both expression. 由于SOX7在定形内胚层不表达,SOX17/SOX7的比值可靠的估计了整个群体所表明的定形内胚层中SOX17表达的贡献。 Since SOX7 not expressed in the endoderm amorphous ratio SOX17 / SOX7 reliable estimate of the contribution of the entire population of the definitive endoderm indicated in SOX17 expression. 嵌图GL及M与嵌图C的相似性显示FGF17、VWF、CALCR、FOXQ1、CMKOR1及CRIP1可能为定形内胚层的标志物,且表明了其在胚外内胚层细胞中表达不显著。 Inset GL and M and inset C of similarity is shown FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 may be markers of definitive endoderm, and demonstrates its expression was not significant in the extraembryonic endodermal cells.

应当理解,本文所述Q-PCR结果可进一步以ICC确证。 It should be understood that the Q-PCR results described herein may be further confirmed in ICC.

本发明所述的方法、组合物及设备是优选实施方案的代表,是示例性的,不能视为对本发明范围的限制。 The method according to the present invention, compositions and apparatus are representative of preferred embodiments, are exemplary, not be considered as limiting the scope of the present invention. 本领域技术人员可对其作出变化及作其它应用,这包含在本发明的精神中并由公开的范围来定义。 Those skilled in the art that variations may be made thereto, and for other applications, which comprises in the range of the spirit of the invention be defined by the disclosure. 因此,很明显,本领域技术人员在不偏离本发明范围及精神下,可对本文公开的发明作出替换及变动。 Thus, it is clear that one skilled in the art without departing from the scope and spirit of the invention, substitutions and changes may be made to the invention disclosed herein.

如下述权利要求及全部公开所述,短语“基本上由……组成”指包括短语后所列的任何成分,并限于那些对公开中说明的活性或作用不干扰或无贡献的其它成分。 As disclosed in the following claims and all the phrase "consisting essentially consisting ......" refers to any listed ingredients comprising the phrase, and limited to those active ingredients other disclosed or described effect does not interfere with or without contribution. 因而,短语“基本上由……组成”表明列出的成分是需要的或必需的,但是其它成分任选,视是否影响列出成分的活性或作用选用或不用。 Thus, the phrase "consisting essentially consisting ......" indicates that the listed ingredients are required or necessary, other optional components, depending on whether affect the activity or action listed ingredients selected or not.

参考文献在本发明中引用大量的文献及专利参考,本发明引用的各参考文献在此全部引人,以供参考。 A large number of references cited literature and patent references in the present invention, each of the references cited herein is introduced herein in their entirety, by reference.

一些参考已经在正文中完整引用。 Some have a complete reference cited in the text. 正文中引用的一些其它仅按照作者及年引用的参考文献,其完整引用如下:Alexander,J.,Rothenberg,M.,Henry,GL及Stainier,DY(1999).Casanova plays an early and essential role in endoderm formation in zebrafish(Casanova在斑马鱼内胚层形成中起着早期的基本作用).Dev Biol 215,343-357. Body referenced only by the author and other references cited in its complete reference as follows: Alexander, J., Rothenberg, M., Henry, GL and Stainier, DY (1999) .Casanova plays an early and essential role in endoderm formation in (Casanova plays a fundamental role early in the zebra fish endoderm formation) zebrafish .Dev Biol 215,343-357.

A1exander,J.及Stainier,DY(1999).A molecular pathway leading toendoderm formation in zebrafish(引起斑马鱼内胚层形成的分子通道).Curr Biol 9,1147-1157. A1exander, J. And Stainier, DY (1999) .A molecular pathway leading toendoderm formation in zebrafish (endoderm induced zebrafish passage formed molecule) .Curr Biol 9,1147-1157.

Aoki,TO,Mathieu,J.,Saint-Etienne,L.,Rebagliati,MR,Peyrieras,N.及Rosa,FM(2002).Regulation of nodal signalling and mesendodermformation by TARAM-A,a TGFbeta-related type I receptor(以TARAM-A,一种TGF beta相关的I型受体调节nodal信号系统及中内胚层形成).DevBiol 241,273-288. Aoki, TO, Mathieu, J., Saint-Etienne, L., Rebagliati, MR, Peyrieras, N., And Rosa, FM (2002) .Regulation of nodal signalling and mesendodermformation by TARAM-A, a TGFbeta-related type I receptor (in TARAM-a, one kind of TGF beta receptor type I regulating associated nodal signaling system is formed and the inner mesoderm) .DevBiol 241,273-288.

Beck,S.,Le Good,JA,Guzman,M.,Ben Haim,N.,Roy,K.,Beermann,F.及Constam,DB(2002).Extra-embryonic proteases regulate Nodalsignalling during gastrulation(在原肠胚形成过程中调节Nodal信号的胚外蛋白酶).Nat Cell Biol 4,981-985. Beck, S., Le Good, JA, Guzman, M., Ben Haim, N., Roy, K., Beermann, F. And Constam, DB (2002) .Extra-embryonic proteases regulate Nodalsignalling during gastrulation (gastrulation Nodal signaling in regulating the formation of extraembryonic protease) .Nat Cell Biol 4,981-985.

Beddington,RS,Rashbass,P.及Wilson,V.(1992).Brachyury--a geneaffecting mouse gastrulation and early organogenesis(Brachyury--影响小鼠原肠胚形成及早期器官发生的基因).Dev Suppl,157-165. . Beddington, RS, Rashbass, P and Wilson, V (1992) .Brachyury -. A geneaffecting mouse gastrulation and early organogenesis (Brachyury-- mice Effect of gastrulation and early organogenesis) .Dev Suppl, 157 -165.

Bongso,A.,Fong,CY,Ng,SC及Ratnam,S.(1994).Isolation andculture of inner cell mass cells from human blastocysts(人胚泡内细胞团的分离和培养).Hum Reprod 9,2110-2117). Bongso, A., Fong, CY, Ng, SC and Ratnam, S. (1994) .Isolation andculture of (Isolation and culture of inner cell mass of human blastocysts) inner cell mass cells from human blastocysts .Hum Reprod 9,2110- 2117).

Chang,H.,Brown,CW及Matzuk,MM(2002).Genetic analysis ofthe mammalian transforming growth factor-beta superfamily(哺乳动物转化生长因子-β超家族的遗传分析).Endocr Rev 23,787-823. Chang, H., Brown, CW and Matzuk, MM (2002) .Genetic analysis ofthe mammalian transforming growth factor-beta superfamily (superfamily genetic analysis of mammalian transforming growth factor -β) .Endocr Rev 23,787-823.

Conlon,FL,Lyons,KM,Takaesu,N.,Barth,KS,Kispert,A.,Herrmann,B.及Robertson,EJ(1994).A primary requirement for nodal inthe formation and maintenance of the primitive streak in the mouse(形成及保持小鼠原线对nodal的首要要求).Development 120,1919-1928. Conlon, FL, Lyons, KM, Takaesu, N., Barth, KS, Kispert, A., Herrmann, B., And Robertson, EJ (1994) .A primary requirement for nodal inthe formation and maintenance of the primitive streak in the mouse (holding and forming the primary requirement for nodal lines of the original mouse) .Development 120,1919-1928.

Dougan,ST,Warga,RM,Kane,DA,Schier,AF及Talbot,WS(2003).The role of the zebrafish nodal-related genes squint and cyclops inpatterning of mesendoderm(斑马鱼nodal相关基因squint及cyclops在确定中内胚层模式中的作用).Development 130,1837-1851. Dougan, ST, Warga, RM, Kane, DA, Schier, AF and Talbot, WS (2003) .The role of the zebrafish nodal-related genes squint and cyclops inpatterning of mesendoderm (zebrafish nodal-related genes in the determination squint and cyclops action endoderm mode) .Development 130,1837-1851.

Feldman,B.,Gates,MA,Egan,ES,Dougan,ST,Rennebeck,G.,Sirotkin,HI,Schier,AF及Talbot,WS(1998).Zebrafish organizerdevelopment and germ-layer formation require nodal-related signals(斑马鱼形成体发育及胚层形成需要nodal相关的信号).Nature 395,181-185. Feldman, B., Gates, MA, Egan, ES, Dougan, ST, Rennebeck, G., Sirotkin, HI, Schier, AF and Talbot, WS (1998) .Zebrafish organizerdevelopment and germ-layer formation require nodal-related signals ( zebrafish development and mesoderm formation member needs to form a signal related to nodal) .Nature 395,181-185.

Feng,Y.,Broder,CC,Kennedy,PE及Berger,EA(1996).HIV-1entry cofactor:functional cDNA cloning of a seven-transmembrane,Gprotein-coupled receptor(HIV-1进入共因子:七次跨膜的G蛋白偶联受体的功能性cDNA克隆).Science 272,872-877. Feng, Y., Broder, CC, Kennedy, PE and Berger, EA (1996) .HIV-1entry cofactor: functional cDNA cloning of a seven-transmembrane, Gprotein-coupled receptor (HIV-1 into the co-factors: transmembrane seven functional cDNA clone G protein coupled receptors) .Science 272,872-877.

Futaki,S.,Hayashi,Y.,Yamashita,M.,Yagi,K.,Bono,H.,Hayashizaki,Y.,Okazaki,Y.及Sekiguchi,K.(2003).Molecular basis of constitutiveproduction of basement membrane components:Gene expression profiles ofengelbreth-holm-swarm tumor and F9 embryonal carcinoma cells(基底膜成分的构成产物的分子基础:engelbreth-holm-swarm肿瘤及F9胚胎肿瘤细胞基因表达特征).J Biol Chem. Futaki, S., Hayashi, Y., Yamashita, M., Yagi, K., Bono, H., Hayashizaki, Y., Okazaki, Y. And Sekiguchi, K. (2003) .Molecular basis of constitutiveproduction of basement membrane components: gene expression profiles ofengelbreth-holm-swarm tumor and F9 embryonal carcinoma cells (constituting the product of the molecular basis of basement membrane components: engelbreth-holm-swarm tumor expression profile genes and F9 embryonal carcinoma cells) .J Biol Chem.

Grapin-Botton,A.及Melton,DA(2000).Endoderm development:frompatterning to organogenesis(内胚层发育:从模式化到器官发生).TrendsGenet 16,124-130. . Grapin-Botton, A and Melton, DA (2000) .Endoderm development: frompatterning to organogenesis (endoderm development: from occurring to organs schematically) .TrendsGenet 16,124-130.

Harris,TM及Childs,G.(2002).Global gene expression patterns duringdifferentiation of F9 embryonal carcinoma cells into parietal endoderm(在F9胚胎肿瘤细胞分化为体壁内胚层过程中的全基因表达模式).Funct IntegrGenomics 2,105-119. Harris, TM and Childs, G. (2002) .Global gene expression patterns duringdifferentiation of F9 embryonal carcinoma cells into parietal endoderm (global gene expression patterns in F9 embryonal carcinoma cells to differentiate into parietal endoderm process) .Funct IntegrGenomics 2, 105-119.

Hogan,BL(1996).Bone morphogenetic proteins in development(发育中的骨形态蛋白).Curr Opin Genet Dev 6,432-438. Hogan, BL (1996) .Bone morphogenetic proteins in development (developing bone morphogenetic protein) .Curr Opin Genet Dev 6,432-438.

Hogan,BL(1997).Pluripotent embryonic cells and methods of makingsame(多能胚胎细胞及其复制方法)(USA,Vanderbilt University). Hogan, BL (1997) .Pluripotent embryonic cells and methods of makingsame (pluripotent embryonic cells and replication methods) (USA, Vanderbilt University).

Howe,CC,Overton,GC,Sawicki,J.,Solter,D.,Stein,P.及Strickland,S.(1988).Expression of SPARC/osteonectin transcript in murineembryos and gonads(在鼠胚胎及性腺中SPARC/骨结合素转录的表达).Differentiation 37,20-25. Howe, CC, Overton, GC, Sawicki, J., Solter, D., Stein, P., And Strickland, S. (1988) .Expression of SPARC / osteonectin transcript in murineembryos and gonads (in murine embryonic gonads and SPARC / expression of transcription factors osseointegration) .Differentiation 37,20-25.

Hudson,C.,Clements,D.,Friday,RV,Stott,D.及Woodland,HR(1997).Xsox17alpha and-beta mediate endoderm formation in Xenopus(Xsox17α及β介导非洲蟾蜍内胚层的形成).Cell 91,397-405. Hudson, C., Clements, D., Friday, RV, Stott, D., And Woodland, HR (1997) .Xsox17alpha formation in Xenopus (formed in Xenopus endoderm Xsox17α and β-mediated) and-beta mediate endoderm .Cell 91,397-405.

Imada,M.,Imada,S.,Iwasaki,H.,Kume,A.,Yamaguchi,H.及Moore,EE(1987).Fetomodulin:marker surface protein of fetal development which ismodulatable by cyclic AMP(胎儿调节素:周期AMP模拟的胎发育的标志物表面蛋白).Dev Biol 122,483-491. . Imada, M., Imada, S., Iwasaki, H., Kume, A., Yamaguchi, H and Moore, EE (1987) .Fetomodulin: marker surface protein of fetal development which ismodulatable by cyclic AMP (fetal somatomedin: Periodic analog AMP development of the tread surface marker protein) .Dev Biol 122,483-491.

Kanai-Azuma,M.,Kanai,Y.,Gad,JM,Tajima,Y.,Taya,C.,Kurohmaru,M.,Sanai,Y.,Yonekawa,H.,Yazaki,K.,Tam,PP及Hayashi,Y.(2002).Depletion of definitive gut endodern in Sox17-null mutant mice(缺失Sox17的变异小鼠定形肠内胚层的缺失).Development 129,2367-2379. Kanai-Azuma, M., Kanai, Y., Gad, JM, Tajima, Y., Taya, C., Kurohmaru, M., Sanai, Y., Yonekawa, H., Yazaki, K., Tam, PP and hayashi, Y. (2002) .Depletion of definitive gut endodern in Sox17-null mutant mice (foregut endoderm amorphous deletion mutant mice deletion of Sox17) .Development 129,2367-2379.

Katoh,M.(2002).Expression of human SOX7in normal tissues andtumors(正常组织及肿瘤的人SOX7的表达).Int J Mol Med9,363-368. Katoh, M. (2002) .Expression of human SOX7in normal tissues andtumors (normal and tumor tissues of human expression SOX7) .Int J Mol Med9,363-368.

Kikuchi,Y.,Agathon,A.,Alexander,J.,Thisse,C.,Waldron,S.,Yelon,D.,Thisse,B.及Stainier,DY(2001).casanova encodes a novel Sox-relatedprotein necessary and sufficient for early endoderm formation in zebrafish(casanova编码斑马鱼早期内胚层形成充分必要的一种新的Sox相关蛋白).Genes Dev 15,1493-1505. Kikuchi, Y., Agathon, A., Alexander, J., Thisse, C., Waldron, S., Yelon, D., Thisse, B. And Stainier, DY (2001) .casanova encodes a novel Sox-relatedprotein necessary and sufficient for early endoderm formation in zebrafish Dev 15,1493-1505 (early endoderm formed sufficient zebrafish casanova encoding a novel Sox necessary associated protein) .Genes.

Kim,CH及Broxmeyer,HE(1999).Chemokines:signal lamps fortrafficking of T and B cells for development and effector function(趋化因子:运输具有发育及效应器功能的T及B细胞的信号灯).J Leukoc Biol 65,6-15. Kim, CH and Broxmeyer, HE (1999) .Chemokines: signal lamps fortrafficking of T and B cells for development and effector function (chemokines: Transport development and effector function having the T and B cells for lights) .J Leukoc Biol 65,6-15.

Kimelman,D.及Griffin,KJ(2000).Vertebrate mesendoderm inductionand patterning(脊柱中内胚层诱导及模式化).Curr Opin Genet Dev 10,350-356. Kimelman, D., And Griffin, KJ (2000) .Vertebrate mesendoderm inductionand patterning (mesoderm induction and patterning the spine) .Curr Opin Genet Dev 10,350-356.

Kubo A,Shinozaki K,Shannon JM,Kouskoff V,Kennedy M,Woo S,Fehling HJ,Keller G.(2004)Development of definitive endoderm fromembryonic stem cells in culture(培养物胚胎干细胞定形内胚层的发育).Development.131,1651-62. Kubo A, Shinozaki K, Shannon JM, Kouskoff V, Kennedy M, Woo S, Fehling HJ, Keller G. (2004) Development of definitive endoderm fromembryonic stem cells in culture (culture of embryonic stem development endoderm shaped cells) .Development. 131,1651-62.

Kumar,A.,Novoselov,V.,Celeste,AJ,Wolfman,NM,ten Dijke,P.及Kuehn,MR(2001).Nodal signaling uses activin and transforminggrowth factor-beta receptor-regulated Smads(Nodal信号传导使用活化素及转化生长因子-β受体调节的Smads).J Biol Chem 276,656-661. Kumar, A., Novoselov, V., Celeste, AJ, Wolfman, NM, ten Dijke, P., And Kuehn, MR (2001) .Nodal signaling uses activin and transforminggrowth factor-beta receptor-regulated Smads (Nodal signaling using activated Smads factors and transforming growth factor -β receptor modulation) .J Biol Chem 276,656-661.

Labosky,PA,Barlow,DP及Hogan,BL(1994a).Embryonic germcell lines and their derivation from mouse primordial germ cells(胚胎生殖细胞系及其自小鼠多能生殖细胞衍生).Ciba Found Symp 182,157-168;discussion 168-178. Labosky, PA, Barlow, DP and Hogan, BL (1994a) .Embryonic germcell lines and their derivation from mouse primordial germ cells (embryonic germ cells and germ cells from a mouse-derived pluripotent) .Ciba Found Symp 182,157- 168; discussion 168-178.

Labosky,PA,Barlow,DP及Hogan,BL(1994b).Mouse embryonicgerm(EG)cell lines:transmission through the germline and differences in themethylation imprint of insulin-like growth factor 2 receptor(Igf2r)genecompared with embryonic stem(ES)cell lines(小鼠胚胎生殖(EG)细胞系:种系的传递及与胚胎干(ES)细胞系相比胰岛素样生长因子2受体(Igf2r)基因甲基化印迹的差异).Development 120,3197-3204. Labosky, PA, Barlow, DP and Hogan, BL (1994b) .Mouse embryonicgerm (EG) cell lines: transmission through the germline and differences in themethylation imprint of insulin-like growth factor 2 receptor (Igf2r) genecompared with embryonic stem (ES) cell lines (embryonic germ (EG) cell lines in mice: transmitting germline and embryonic stem (ES) cell lines as compared to insulin-like growth factor 2 receptor (Igf2r) imprinted genes differentially methylated) .Development 120, 3197-3204.

Lickert,H.,Kutsch,S.,Kanzler,B.,Tamai,Y.,Taketo,MM及Kemler,R.(2002).Formation of multiple hearts in mice following deletion ofbeta-catenin in the embryonic endoderm(敲除胚胎内胚层β-连环蛋白小鼠的多心脏的形成).Dev Cell 3,171-181. Lickert, H., Kutsch, S., Kanzler, B., Tamai, Y., Taketo, MM, and Kemler, R. (2002) .Formation of multiple hearts in mice following deletion ofbeta-catenin in the embryonic endoderm (knock endoderm formed multiple heart β- catenin mice) .Dev Cell 3,171-181.

Lu,CC,Brennan,J.及Robertson,EJ(2001).From fertilization togastrulation:axis formation in the mouse embryo(从受精到原肠胚形成:小鼠胚胎轴线形成).Curr Opin Genet Dev 11,384-392. . Lu, CC, Brennan, J and Robertson, EJ (2001) .From fertilization togastrulation: axis formation in the mouse embryo (fertilized formed from gastrula: mouse embryonic axis formation) .Curr Opin Genet Dev 11,384- 392.

Ma,Q.,Jones,D.及Springer,TA(1999).The chemokine receptorCXCR4 is required for the retention of B lineage and granulocytic precursorswithin the bone marrow microenvironment(趋化因子受体CXCR4需要在骨髓微环境中保留B谱系及粒细胞前体).Immunity 10,463-471. Ma, Q., Jones, D., And Springer, TA (1999) .The chemokine receptorCXCR4 is required of B lineage and granulocytic precursorswithin the bone marrow microenvironment (chemokine receptor CXCR4 B need to be retained within the bone marrow microenvironment for the retention and granulocyte lineage precursors) .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 its receptor,CXCR4(趋化因子SDF-1及其受体,CXCR4胚胎表达及功能).Dev Biol.213,442-56. McGrath KE, Koniski AD, Maltby KM, McGann JK, Palis J. (1999) Embryonic expression and function of the chemokine SDF-1 its receptor, CXCR4 (chemokine SDF-1 and its receptor, CXCR4 and embryonic expression and function ) .Dev Biol.213,442-56.

Miyazono,K.,Kusanagi,K.及Inoue,H.(2001).Divergence andconvergence of TGF-beta/BMP signaling(TGF-beta/BMP信号系统的辐射与汇聚).J Cell Physiol 187,265-276. Miyazono, K., Kusanagi, K., And Inoue, H. (2001) .Divergence andconvergence of TGF-beta / BMP signaling (radiation TGF-beta / BMP signaling system and aggregation) .J Cell Physiol 187,265-276.

Nagasawa,T.,Hirota,S.,Tachibana,K.,Takakura,N.,Nishikawa,S.,Kitamura,Y.,Yoshida,N.,Kikutani,H.及Kishimoto,T.(1996).Defects ofB-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking theCXC chemokine PBSF/SDF-1(缺乏CXC趋化因子PBSF/SDF-1小鼠B-细胞淋巴细胞生成及骨髓髓细胞生成缺陷)Nature 382,635-638. Nagasawa, T., Hirota, S., Tachibana, K., Takakura, N., Nishikawa, S., Kitamura, Y., Yoshida, N., Kikutani, H., And Kishimoto, T. (1996) .Defects ofB -cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking theCXC chemokine PBSF / SDF-1 (lacking CXC chemokine PBSF / SDF-1 mouse B- cell lymphopoiesis and bone marrow myelopoiesis defect) Nature 382,635-638 .

Niwa,H.(2001).Molecular mechanism to maintain stem cell renewal ofES cells(保持ES细胞干细胞再生的分子机制).Cell Struct Funct 26,137-148. Niwa, H. (2001) .Molecular mechanism to maintain stem cell renewal ofES cells (ES cells retaining the molecular mechanisms of stem cell regeneration) .Cell Struct Funct 26,137-148.

Ogura,H.,Aruga,J.及Mikoshiba,K.(2001).Behavioral abnormalities ofZic1 and Zic2 mutant mice:implications as models for human neurologicaldisorders(Zic1及Zic2突变小鼠的行为异常:潜在的人神经疾病模型).Behav Genet 31,317-324. . Ogura, H., Aruga, J and Mikoshiba, K (2001) .Behavioral abnormalities ofZic1 and Zic2 mutant mice:. Implications as models for human neurologicaldisorders (Zic1 Zic2 mutant mice and abnormal behavior: potential human neurological disease models) .Behav Genet 31,317-324.

Reubinoff,BE,Pera,MF,Fong,CY,Trounson,A.及Bongso,A.(2000).Embryonic stem cell lines from human blastocysts:somaticdifferentiation in vitro(人胚泡胚胎干细胞系:体外体细胞分化).NatBiotechnol 18,399-404. . Reubinoff, BE, Pera, MF, Fong, CY, Trounson, A and Bongso, A (2000) .Embryonic stem cell lines from human blastocysts:. Somaticdifferentiation in vitro (human embryonic stem cell line blastocysts: somatic differentiation in vitro). NatBiotechnol 18,399-404.

Rodaway,A.及Patient,R.(2001).Mesendoderm.an ancient germ layer(中内胚层.一个古老的胚层)? Rodaway, A., And Patient, R. (2001) .Mesendoderm.an ancient germ layer (endoderm in. An old mesoderm)? Cell 105,169-172. Cell 105,169-172.

Rodaway,A.,Takeda,H.,Koshida,S.,Broadbent,J.,Price,B.,Smith,JC,Patient,R.及Holder,N.(1999).Induction of the mesendoderm in thezebrafish germ ring by yolk cell-derived TGF-beta family signals anddiscrimination of mesoderm and endoderm by FGF(卵细胞衍生的TGF-β家族信号诱导斑马鱼胚环中内胚层及以FGF辨别中胚层及内胚层).Development 126,3067-3078. Rodaway, A., Takeda, H., Koshida, S., Broadbent, J., Price, B., Smith, JC, Patient, R., And Holder, N. (1999) .Induction of the mesendoderm in thezebrafish germ ring by yolk cell-derived TGF-beta family signals anddiscrimination of mesoderm and endoderm by FGF (the TGF-β family signaling in zebrafish embryos induced by egg-derived ring mesoderm and in the mesoderm and endoderm discrimination in FGF) .Development 126,3067- 3078.

Rohr,KB,Schulte-Merker,S.及Tautz,D.(1999).Zebrafish zic1expression in brain and somites is affected by BMP and hedgehog signalling(BMP及hedgehog信号系统影响斑马鱼zic1在脑及体节的表达).MechDev 85,147-159. Rohr, KB, Schulte-Merker, S. And Tautz, D. (1999) .Zebrafish zic1expression in brain and somites is affected (expressed in the brain and the body section of BMP and hedgehog signaling systems affect zebrafish zic1) by BMP and hedgehog signalling .MechDev 85,147-159.

Schier,AF(2003).Nodal signaling in vertebrate development(脊椎动物发育中的Nodal信号系统).Annu Rev细胞Dev Biol 19,589-621. Schier, AF (2003) .Nodal signaling in vertebrate development (Nodal signaling system vertebrate development). Annu Rev Cell Dev Biol 19,589-621.

Schoenwolf,GC及Smith,JL(2000).Gastrulation and earlymesodermal patterning in vertebrates(脊椎动物原肠胚及早期中胚层的图式发育).Methods Mol Biol 135,113-125. Schoenwolf, GC and Smith, JL (2000) .Gastrulation and earlymesodermal patterning in vertebrates (FIG formula vertebrate gastrulation and early mesoderm) .Methods Mol Biol 135,113-125.

Shamblott,MJ,Axelman,J.,Wang,S.,Bugg,EM,Littlefield,JW,Donovan,PJ,Blumenthal,PD,Huggins,GR及Gearhart,JD(1998).Derivation of pluripotent stem cells from cultured human primordial germcells(培养的人原始生殖细胞衍生的多能干细胞).Proc Natl Acad Sci USA 95,13726-13731. Shamblott, MJ, Axelman, J., Wang, S., Bugg, EM, Littlefield, JW, Donovan, PJ, Blumenthal, PD, Huggins, GR and Gearhart, JD (1998) .Derivation of pluripotent stem cells from cultured human primordial germcells (cultured human primordial germ cell-derived pluripotent stem cells) .Proc Natl Acad Sci USA 95,13726-13731.

Shapiro,AM,Lakey,JR,Ryan,EA,Korbutt,GS,Toth,E.,Warnock,GL,Kneteman,NM及Rajotte,RV(2000).Islettransplantation in seven patients with type 1 diabetes mellitus using aglucocorticoid-free immunosuppressive regimen(7例I型糖尿病患者不使用糖皮质激素免疫抑制疗法的胰岛移植).N Engl J Med 343,230-238. Shapiro, AM, Lakey, JR, Ryan, EA, Korbutt, GS, Toth, E., Warnock, GL, Kneteman, NM and Rajotte, RV (2000) .Islettransplantation in seven patients with type 1 diabetes mellitus using aglucocorticoid-free immunosuppressive regimen (7 type I diabetic patients without glucocorticoid islet transplantation immunosuppressive therapy) .N Engl J Med 343,230-238.

Shapiro,AM,Ryan,EA及Lakey,JR(2001a).Pancreatic islettransplantation in the treatment of diabetes mellitus(糖尿病治疗中的胰岛移植).Best Pract Res Clin Endocrinol Metab 15,241-264. Shapiro, AM, Ryan, EA and Lakey, JR (2001a) .Pancreatic islettransplantation in the treatment of diabetes mellitus (diabetes in islet transplantation) .Best Pract Res Clin Endocrinol Metab 15,241-264.

Shapiro,J.,Ryan,E.,Wamock,GL,Kneteman,NM,Lakey,J.,Korbutt,GS及Rajotte,RV(2001b).Could fewer islet cells betransplanted in type 1 diabetes? Shapiro, J., Ryan, E., Wamock, GL, Kneteman, NM, Lakey, J., Korbutt, GS and Rajotte, RV (2001b) .Could fewer islet cells betransplanted in type 1 diabetes? Insulin independence should be dominantforce in islet transplantation(I型糖尿病可移植较少的胰岛细胞?胰岛素自主是胰岛移植中的优先考虑).Bmj 322,861. Insulin independence should be dominantforce in islet transplantation (type I diabetes, islet cells can be transplanted less? Insulin independent is a priority islet transplantation) .Bmj 322,861.

Shiozawa,M.,Hiraoka,Y.,Komatsu,N.,Ogawa,M.,Sakai,Y.及Aiso,S.(1996).Cloning and characterization of Xenopus laevis xSox7 cDNA(非洲爪蛙xSox7 cDNA克隆及确证).Biochim Biophys Acta 1309,73-76. Shiozawa, M., Hiraoka, Y., Komatsu, N., Ogawa, M., Sakai, Y. And Aiso, S. (1996) .Cloning and characterization of Xenopus laevis xSox7 cDNA (Xenopus xSox7 cDNA clone and confirmed ) .Biochim Biophys Acta 1309,73-76.

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

Smith,JC,Armes,NA,Conlon,FL,Tada,M.,Umbhauer,M.及Weston,KM(1997).Upstream and downstream from Brachyury,a generequired for vertebrate mesoderm formation(一个脊椎动物中胚层形成必需基因Brachyury的上游及下游).Cold Spring Harb Symp Quant Biol 62,337-346. Smith, JC, Armes, NA, Conlon, FL, Tada, M., Umbhauer, M., And Weston, KM (1997) .Upstream and downstream from Brachyury, (a vertebrate a generequired for vertebrate mesoderm formation mesoderm formation essential gene Brachyury upstream and downstream) .Cold Spring Harb Symp Quant Biol 62,337-346.

Takash,W.,Canizares,J.,Bonneaud,N.,Poulat,F.,Mattei,MG,Jay,P.及Berta,P.(2001).SOX7 transcription factor:sequence,chromosomallocalisation,expression,transactivation and interference with Wnt signalling(SOX7转录因子:测序、染色体定位、表达、转活及对Wnt信号系统的干扰).Nucleic Acids Res 29,4274-4283. . Takash, W., Canizares, J., Bonneaud, N., Poulat, F., Mattei, MG, Jay, P and Berta, P (2001) .SOX7 transcription factor:. Sequence, chromosomallocalisation, expression, transactivation and interference with Wnt signalling (SOX7 transcription factors: sequencing, chromosome localization, expression, and interference transactivation Wnt signaling system) .Nucleic Acids Res 29,4274-4283.

Taniguchi,K.,Hiraoka,Y.,Ogawa,M.,Sakai,Y.,Kido,S.及Aiso,S.(1999).Isolation and characterization of a mouse SRY-related cDNA,mSox7(小鼠SRY相关cDNA,mSox7的分离及确证).Biochim Biophys Acta 1445,225-231. Taniguchi, K., Hiraoka, Y., Ogawa, M., Sakai, Y., Kido, S. And Aiso, S. (1999) .Isolation and characterization of a SRY-related cDNA mouse, mSox7 (SRY-related mouse cDNA, mSox7 Isolation and confirmation) .Biochim Biophys Acta 1445,225-231.

Technau,U.(2001).Brachyury,the blastopore and the evolution of themesoderm(Brachyury、胚孔及中胚层进化).Bioessays 23,788-794. Technau, U. (2001) .Brachyury, the blastopore and the evolution of themesoderm (Brachyury, blastopore and mesoderm Evolution) .Bioessays 23,788-794.

Thomson,JA,Itskovitz-Eldor,J.,Shapiro,SS,Waknitz,MA,Swiergiel,JJ,Marshall,VS及Jones,JM(1998).Embryonic stem celllines derived from human blastocysts(人胚泡胚胎干细胞系).Science 282,1145-1147. Thomson, JA, Itskovitz-Eldor, J., Shapiro, SS, Waknitz, MA, Swiergiel, JJ, Marshall, VS and Jones, JM (1998) .Embryonic stem celllines derived from human blastocysts (blastocyst human embryonic stem cell line). Science 282,1145-1147.

Tremblay,KD,Hoodless,PA,Bikoff,EK及Robertson,EJ(2000).Formation of the definitive endoderm in mouse is a Smad2-dependent process.(小鼠定形内胚层形成为Smad2依赖过程).Development 127,3079-3090. Tremblay, KD, Hoodless, PA, Bikoff, EK and Robertson, EJ (2000) .Formation of the definitive endoderm in mouse is a Smad2-dependent process. (Endoderm formed into shaped mice Smad2-dependent processes) .Development 127,3079 -3090.

Vandesompele,J.,De Preter,K.,Pattyn,F.,Poppe,B.,Van Roy,N.,DePaepe,A.及Speleman,F.(2002).Accurate normalization of real-timequantitative RT-PCR data by geometric averaging of multiple internal controlgenes(几何平均多个内部控制基因准确校准实时定量RT-PCR的数据).Genome Biol 3,RESEARCH0034. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., DePaepe, A. And Speleman, F. (2002) .Accurate normalization of real-timequantitative RT-PCR data by geometric averaging of multiple internal controlgenes (geometric mean multiple internal control genes accurate real-time quantitative RT-PCR calibration data) .Genome Biol 3, RESEARCH0034.

Varlet,I.,Collignon,J.及Robertson,EJ(1997).nodal expression in theprimitive endoderm is required for specification of the anterior axis duringmouse gastrulation(原始内胚层中的nodal表达为小鼠原肠胚前轴的特异化所必需).Development 124,1033-1044. Varlet, I., Collignon, J., And Robertson, EJ (1997) .nodal expression in specific front axle gastrulation mouse theprimitive endoderm is required for specification of nodal (primitive endoderm in the anterior axis duringmouse gastrulation expressed as of required) .Development 124,1033-1044.

Vincent,SD,Dunn,NR,Hayashi,S.,Norris,DP及Robertson,EJ(2003).Cell fate decisions within the mouse organizer are governed by gradedNodal signals(小鼠形成体中细胞命运决定受分级Nodal信号控制).GenesDev 17,1646-1662. Vincent, SD, Dunn, NR, Hayashi, S., Norris, DP and Robertson, EJ (2003) .Cell fate decisions within the mouse organizer are governed by gradedNodal signals body cell fate (mouse formed by classification decision Nodal signaling control ) .GenesDev 17,1646-1662.

Weiler-Guettler,H.,Aird,WC,Rayburn,H.,Husain,M.及Rosenberg,RD(1996).Developmentally regulated gene expression of thrombomodulinin postimplantation mouse embryos(植入后小鼠胚胎中凝血调节蛋白的发育调控基因的表达).Development 122,2271-2281. Weiler-Guettler, H., Aird, WC, Rayburn, H., Husain, M., And Rosenberg, RD (1996) .Developmentally regulated gene expression of thrombomodulinin postimplantation mouse embryos development (post-implantation mouse embryos of thrombomodulin regulation of gene expression) .Development 122,2271-2281.

Weiler-Guettler,H.,Yu,K.,Soff,G.,Gudas,LJ及Rosenberg,RD(1992).Thrombomodulin gene regulation by cAMP and retinoic acid in F9embryonal carcinoma cells(F9胚胎癌细胞中凝血调节蛋白基因由cAMP及视黄酸调控).Proceedings Of The National Academy Of Sciences Of TheUnited States Of America 89,2155-2159. Weiler-Guettler, H., Yu, K., Soff, G., Gudas, LJ, and Rosenberg, RD (1992) .Thrombomodulin gene regulation by cAMP and retinoic acid in F9embryonal carcinoma cells (F9 embryonic carcinoma cells thrombomodulin gene and a cAMP regulation of retinoic acid) .Proceedings of the National Academy of Sciences of TheUnited States of America 89,2155-2159.

Wells,JM及Melton,DA(1999).Vertebrate endoderm development(脊椎动物内胚层发育).Annu Rev Cell Dev Biol 15,393-410. Wells, JM and Melton, DA (1999) .Vertebrate endoderm development (mesoderm vertebrate) .Annu Rev Cell Dev Biol 15,393-410.

Wells,JM及Melton,DA(2000).Early mouse endoderm is patternedby soluble factors from adjacent germ layers(早期小鼠内胚层为邻近胚层的可溶因子模式化).Development 127,1563-1572. Wells, JM, and Melton, DA (2000) .Early mouse endoderm is patternedby soluble factors from adjacent germ layers (endoderm mice early mesoderm adjacent soluble factors schematically) .Development 127,1563-1572.

Willison,K.(1990).The mouse Brachyury gene and mesoderm formation(小鼠短尾基因及中胚层形成).Trends Genet 6,104-105. Willison, K. (1990) .The mouse Brachyury gene and mesoderm formation (mouse and macaque genes mesoderm formation) .Trends Genet 6,104-105.

Zhao,GQ(2003).Consequences of knocking out BMP signaling in themouse(敲除小鼠BMP信号系统的结果).Genesis 35,43-56. Zhao, GQ (2003) .Consequences of knocking out BMP signaling in themouse (knockout mice results in addition to BMP signaling system) .Genesis 35,43-56.

Zhou,X.,Sasaki,H.,Lowe,L.,Hogan,BL及Kuehn,MR(1993).Nodal is a novel TGF-beta-like gene expressed in the mouse node duringgastrulation(Nodal为一种在原肠胚形成过程中表达在小鼠node中的TGF-β类新基因).Nature 361,543-547. Zhou, X., Sasaki, H., Lowe, L., Hogan, BL and Kuehn, MR (1993) .Nodal is a novel TGF-beta-like gene expressed in the mouse node duringgastrulation (Nodal as a gastrulation formed during the expression of TGF-β genes in the mouse node new class of) .Nature 361,543-547.

序列表&lt;110&gt;诺沃塞尔公司D′Amour,Kevin AllenAgulnick,Alan D. SEQUENCE LISTING & lt; 110 & gt; Novosel company D'Amour, Kevin AllenAgulnick, Alan D.

Baetge,Emmanuel E. Baetge, Emmanuel E.

&lt;120&gt;定形的内胚层&lt;130&gt;CYTHERA.045VPC&lt;150&gt;60/532004&lt;151&gt;2003-12-23&lt;150&gt;60/586566&lt;151&gt;2004-07-09&lt;150&gt;60/587942&lt;151&gt;2004-07-14&lt;160&gt;2&lt;170&gt;FastSEQ for Windows Version 4.0&lt;210&gt;1&lt;211&gt;1245&lt;212&gt;DNA&lt;213&gt;Homo sapiens&lt;400&gt;1atgagcagcc cggatgcggg atacgccagt gacgaccaga gccagaccca gagcgcgctg 60cccgcggtga tggccgggct gggcccctgc ccctgggccg agtcgctgag ccccatcggg 120gacatgaagg tgaagggcga ggcgccggcg aacagcggag caccggccgg ggccgcgggc 180cgagccaagg gcgagtcccg tatccggcgg ccgatgaacg ctttcatggt gtgggctaag 240gacgagcgca agcggctggc gcagcagaat ccagacctgc acaacgccga gttgagcaag 300atgctgggca agtcgtggaa ggcgctgacg ctggcggaga agcggccctt cgtggaggag 360gcagagcggc tgcgcgtgca gcacatgcag gaccacccca actacaagta ccggccgcgg 420cggcgcaagc aggtgaagcg gctgaagcgg gtggagggcg gcttcctgca cggcctggct 480gagccgcagg cggccgcgct gggccccgag ggcggccgcg tggccatgga cggcctgggc 540ctccagttcc ccgagcaggg cttccccgcc ggcccgccgc tgctgcctcc gcacatggg & Lt; 120 & gt; amorphous endoderm & lt; 130 & gt; CYTHERA.045VPC & lt; 150 & gt; 60/532004 & lt; 151 & gt; 2003-12-23 & lt; 150 & gt; 60/586566 & lt; 151 & gt; 2004-07-09 & lt; 150 & gt; 60/587942 & lt; 151 & gt; 2004-07-14 & lt; 160 & gt; 2 & lt; 170 & gt; FastSEQ for Windows Version 4.0 & lt; 210 & gt; 1 & lt; 211 & gt; 1245 & lt; 212 & gt; DNA & lt; 213 & gt; Homo sapiens & lt; 400 & gt; 1atgagcagcc cggatgcggg atacgccagt gacgaccaga gccagaccca gagcgcgctg 60cccgcggtga tggccgggct gggcccctgc ccctgggccg agtcgctgag ccccatcggg 120gacatgaagg tgaagggcga ggcgccggcg aacagcggag caccggccgg ggccgcgggc 180cgagccaagg gcgagtcccg tatccggcgg ccgatgaacg ctttcatggt gtgggctaag 240gacgagcgca agcggctggc gcagcagaat ccagacctgc acaacgccga gttgagcaag 300atgctgggca agtcgtggaa ggcgctgacg ctggcggaga agcggccctt cgtggaggag 360gcagagcggc tgcgcgtgca gcacatgcag gaccacccca actacaagta ccggccgcgg 420cggcgcaagc aggtgaagcg gctgaagcgg gtggagggcg gcttcctgca cggcctggct 480gagccgcagg cggccgcgct gggccccgag ggcggccgcg tggccatgga cggcctgggc 540ctccagttcc ccgagcaggg cttccccgcc ggcccgccgc tgctgcctcc gcacatggg c 600ggccactacc gcgactgcca gagtctgggc gcgcctccgc tcgacggcta cccgttgccc 660acgcccgaca cgtccccgct ggacggcgtg gaccccgacc cggctttctt cgccgccccg 720atgcccgggg actgcccggc ggccggcacc tacagctacg cgcaggtctc ggactacgct 780ggccccccgg agcctcccgc cggtcccatg cacccccgac tcggcccaga gcccgcgggt 840ccctcgattc cgggcctcct ggcgccaccc agcgcccttc acgtgtacta cggcgcgatg 900ggctcgcccg gggcgggcgg cgggcgcggc ttccagatgc agccgcaaca ccagcaccag 960caccagcacc agcaccaccc cccgggcccc ggacagccgt cgccccctcc ggaggcactg 1020ccctgccggg acggcacgga ccccagtcag cccgccgagc tcctcgggga ggtggaccgc 1080acggaatttg aacagtatct gcacttcgtg tgcaagcctg agatgggcct cccctaccag 1140gggcatgact ccggtgtgaa tctccccgac agccacgggg ccatttcctc ggtggtgtcc 1200gacgccagct ccgcggtata ttactgcaac tatcctgacg tgtga 1245&lt;210&gt;2&lt;211&gt;414&lt;212&gt;PRT&lt;213&gt;Homo sapiens&lt;400&gt;2 c 600ggccactacc gcgactgcca gagtctgggc gcgcctccgc tcgacggcta cccgttgccc 660acgcccgaca cgtccccgct ggacggcgtg gaccccgacc cggctttctt cgccgccccg 720atgcccgggg actgcccggc ggccggcacc tacagctacg cgcaggtctc ggactacgct 780ggccccccgg agcctcccgc cggtcccatg cacccccgac tcggcccaga gcccgcgggt 840ccctcgattc cgggcctcct ggcgccaccc agcgcccttc acgtgtacta cggcgcgatg 900ggctcgcccg gggcgggcgg cgggcgcggc ttccagatgc agccgcaaca ccagcaccag 960caccagcacc agcaccaccc cccgggcccc ggacagccgt cgccccctcc ggaggcactg 1020ccctgccggg acggcacgga ccccagtcag cccgccgagc tcctcgggga ggtggaccgc 1080acggaatttg aacagtatct gcacttcgtg tgcaagcctg agatgggcct cccctaccag 1140gggcatgact ccggtgtgaa tctccccgac agccacgggg ccatttcctc ggtggtgtcc 1200gacgccagct ccgcggtata ttactgcaac tatcctgacg tgtga 1245 & lt; 210 & gt; 2 & lt; 211 & gt; 414 & lt; 212 & gt; PRT & lt; 213 & gt; Homo sapiens & lt; 400 & gt; 2

Met Ser Ser Pro Asp Ala Gly Tyr Ala Ser Asp Asp Gln Ser Gln Thr1 5 10 15Gln Ser Ala Leu Pro Ala Val Met Ala Gly Leu Gly Pro Cys Pro Trp20 25 30Ala Glu Ser Leu Ser Pro Ile Gly Asp Met Lys Val Lys Gly Glu Ala35 40 45Pro Ala Asn Ser Gly Ala Pro Ala Gly Ala Ala Gly Arg Ala Lys Gly50 55 60Glu Ser Arg Ile Arg Arg Pro Met Asn Ala Phe Met Val Trp Ala Lys65 70 75 80Asp Glu Arg Lys Arg Leu Ala Gln Gln Asn Pro Asp Leu His Asn Ala85 90 95Glu Leu Ser Lys Met Leu Gly Lys Ser Trp Lys Ala Leu Thr Leu Ala100 105 110Glu Lys Arg Pro Phe Val Glu Glu Ala Glu Arg Leu Arg Val Gln His115 120 125Met Gln Asp His Pro Asn Tyr Lys Tyr Arg Pro Arg Arg Arg Lys Gln130 135 140Val Lys Arg Leu L Met Ser Ser Pro Asp Ala Gly Tyr Ala Ser Asp Asp Gln Ser Gln Thr1 5 10 15Gln Ser Ala Leu Pro Ala Val Met Ala Gly Leu Gly Pro Cys Pro Trp20 25 30Ala Glu Ser Leu Ser Pro Ile Gly Asp Met Lys Val Lys Gly Glu Ala35 40 45Pro Ala Asn Ser Gly Ala Pro Ala Gly Ala Ala Gly Arg Ala Lys Gly50 55 60Glu Ser Arg Ile Arg Arg Pro Met Asn Ala Phe Met Val Trp Ala Lys65 70 75 80Asp Glu Arg Lys Arg Leu Ala Gln Gln Asn Pro Asp Leu His Asn Ala85 90 95Glu Leu Ser Lys Met Leu Gly Lys Ser Trp Lys Ala Leu Thr Leu Ala100 105 110Glu Lys Arg Pro Phe Val Glu Glu Ala Glu Arg Leu Arg Val Gln His115 120 125Met Gln Asp His Pro Asn Tyr Lys Tyr Arg Pro Arg Arg Arg Lys Gln130 135 140Val Lys Arg Leu L ys Arg Val Glu Gly Gly Phe Leu His Gly Leu Ala145 150 155 160Glu Pro Gln Ala Ala Ala Leu Gly Pro Glu Gly Gly Arg Val Ala Met165 170 175Asp Gly Leu Gly Leu Gln Phe Pro Glu Gln Gly Phe Pro Ala Gly Pro180 185 190Pro Leu Leu Pro Pro His Met Gly Gly His Tyr Arg Asp Cys Gln Ser195 200 205Leu Gly Ala Pro Pro Leu Asp Gly Tyr Pro Leu Pro Thr Pro Asp Thr210 215 220Ser Pro Leu Asp Gly Val Asp Pro Asp Pro Ala Phe Phe Ala Ala Pro225 230 235 240Met Pro Gly Asp Cys Pro Ala Ala Gly Thr Tyr Ser Tyr Ala Gln Val245 250 255Ser Asp Tyr Ala Gly Pro Pro Glu Pro Pro Ala Gly Pro Met His Pro260 265 270Arg Leu Gly Pro Glu Pro Ala Gly Pro Ser Ile Pro Gly Leu Leu Ala275 280 285Pro Pro Ser Ala Leu His ys Arg Val Glu Gly Gly Phe Leu His Gly Leu Ala145 150 155 160Glu Pro Gln Ala Ala Ala Leu Gly Pro Glu Gly Gly Arg Val Ala Met165 170 175Asp Gly Leu Gly Leu Gln Phe Pro Glu Gln Gly Phe Pro Ala Gly Pro180 185 190Pro Leu Leu Pro Pro His Met Gly Gly His Tyr Arg Asp Cys Gln Ser195 200 205Leu Gly Ala Pro Pro Leu Asp Gly Tyr Pro Leu Pro Thr Pro Asp Thr210 215 220Ser Pro Leu Asp Gly Val Asp Pro Asp Pro Ala Phe Phe Ala Ala Pro225 230 235 240Met Pro Gly Asp Cys Pro Ala Ala Gly Thr Tyr Ser Tyr Ala Gln Val245 250 255Ser Asp Tyr Ala Gly Pro Pro Glu Pro Pro Ala Gly Pro Met His Pro260 265 270Arg Leu Gly Pro Glu Pro Ala Gly Pro Ser Ile Pro Gly Leu Leu Ala275 280 285Pro Pro Ser Ala Leu His Val Tyr Tyr Gly Ala Met Gly Ser Pro Gly290 295 300Ala Gly Gly Gly Arg Gly Phe Gln Met Gln Pro Gln His Gln His Gln305 310 315 320His Gln His Gln His His Pro Pro Gly Pro Gly Gln Pro Ser Pro Pro325 330 335Pro Glu Ala Leu Pro Cys Arg Asp Gly Thr Asp Pro Ser Gln Pro Ala340 345 350Glu Leu Leu Gly Glu Val Asp Arg Thr Glu Phe Glu Gln Tyr Leu His355 360 365Phe Val Cys Lys Pro Glu Met Gly Leu Pro Tyr Gln Gly His Asp Ser370 375 380Gly Val Asn Leu Pro Asp Ser His Gly Ala Ile Ser Ser Val Val Ser385 390 395 400Asp Ala Ser Ser Ala Val Tyr Tyr Cys Asn Tyr Pro Asp Val405 410 Val Tyr Tyr Gly Ala Met Gly Ser Pro Gly290 295 300Ala Gly Gly Gly Arg Gly Phe Gln Met Gln Pro Gln His Gln His Gln305 310 315 320His Gln His Gln His His Pro Pro Gly Pro Gly Gln Pro Ser Pro Pro325 330 335Pro Glu Ala Leu Pro Cys Arg Asp Gly Thr Asp Pro Ser Gln Pro Ala340 345 350Glu Leu Leu Gly Glu Val Asp Arg Thr Glu Phe Glu Gln Tyr Leu His355 360 365Phe Val Cys Lys Pro Glu Met Gly Leu Pro Tyr Gln Gly His Asp Ser370 375 380Gly Val Asn Leu Pro Asp Ser His Gly Ala Ile Ser Ser Val Val Ser385 390 395 400Asp Ala Ser Ser Ala Val Tyr Tyr Cys Asn Tyr Pro Asp Val405 410

所收到的来自JLH的磁盘形式序列表与提交的CYTHERA.045A中的一致以A:\CYTHERA045V.TXT提交的PCT版本122204 JLH received from the disk in the form of listings of CYTHERA.045A submitted is consistent with A: \ PCT version CYTHERA045V.TXT submitted 122,204

Claims (75)

1.包括人细胞的细胞培养物,其中至少约10%的所述人细胞为定形内胚层细胞,所述的定形内胚层细胞为能分化成肠管细胞或衍生于肠管的器官的多能细胞。 1. The cell comprises a human cell culture, wherein at least about 10% of said human cells are shaped endoderm cells, said definitive endoderm cells capable of differentiating into cells of intestinal bowel or organs derived pluripotent cells.
2.如权利要求1所述的细胞培养物,其中至少约50%的所述人细胞为定形内胚层细胞。 2. The cell culture according to claim 1, wherein at least about 50% of said human cell is a definitive endoderm cell.
3.如权利要求1所述的细胞培养物,其中至少约80%的所述人细胞为定形内胚层细胞。 The cell culture according to claim 1, wherein at least about 80% of said human cell is a definitive endoderm cell.
4.如权利要求1所述的细胞培养物,其中所述的定形内胚层细胞表达选自SOX17和CXCR4的标志物。 4. The cell culture according to claim 1, wherein said shaped inner endoderm cells express a marker selected from the group of CXCR4 and SOX17.
5.如权利要求4所述的细胞培养物,其中在所述定形内胚层细胞中,选自SOX17和CXCR4的所述标志物的表达高于选自OCT4、α-胎蛋白(AFP)、凝血调节蛋白(TM)、SPARC及SOX7的标志物的表达。 5. The cell culture of claim 4, wherein the definitive endoderm cells, and SOX17 expression of the selected markers above the selected CXCR4 OCT4, α- fetoprotein (AFP), blood clotting regulatory protein (TM) expression, SPARC and SOX7 markers.
6.如权利要求4所述的细胞培养物,其中所述定形内胚层细胞不表达选自OCT4、AFP、TM、SPARC和SOX7的标志物。 Cell culture as claimed in claim 4, wherein said definitive endoderm cells do not express selected from OCT4, AFP, TM, SPARC and SOX7 of markers.
7.如权利要求4所述的细胞培养物,其中所述定形内胚层细胞表达选自MIXL1、GATA4和HNF3b的标志物。 7. The cell culture of claim 4, wherein said definitive endoderm cells express selected MIXL1, GATA4 and HNF3b marker.
8.如权利要求4所述的细胞培养物,其中所述定形内胚层细胞表达选自FGF17、VWF、CALCR、FOXQ1、CMKOR1和CRIP1的标志物。 8. The cell culture according to claim 4, wherein said definitive endoderm cells express selected FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 marker.
9.如权利要求1所述的细胞培养物,其中所述定形内胚层细胞表达SOX17和CXCR4。 9. The cell culture according to claim 1, wherein said definitive endoderm cells express SOX17 and CXCR4.
10.如权利要求9所述的细胞培养物,其中在所述的定形内胚层细胞中,所述SOX17和CXCR4的表达高于OCT4、AFP、TM、SPARC和SOX7的表达。 10. The cell culture of claim 9, wherein in said definitive endoderm cells, the expression of CXCR4 and SOX17 than OCT4, expression of AFP, TM, SPARC and the SOX7.
11.如权利要求9所述的细胞培养物,其中所述定形内胚层细胞不表达OCT4、AFP、TM、SPARC和SOX7。 11. A cell culture as claimed in claim 9, wherein said definitive endoderm cells not expressing OCT4, AFP, TM, SPARC and SOX7.
12.如权利要求9所述的细胞培养物,其中所述定形内胚层细胞表达MIXL1、GATA4和HNF3b。 12. The cell culture of claim 9, wherein said definitive endoderm cells express MIXL1, GATA4 and HNF3b.
13.如权利要求9所述的细胞培养物,其中所述定形内胚层细胞表达选自FGF17、VWF、CALCR、FOXQ1、CMKOR1和CRIP1的标志物。 13. The cell culture of claim 9, wherein said definitive endoderm cells express selected FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 marker.
14.如权利要求1所述的细胞培养物,其中在所述的细胞培养物中,对应每一种多能细胞存在至少约2个定形内胚层细胞。 14. The cell culture according to claim 1, wherein said cell culture in the presence of a corresponding plurality endoderm cells can be shaped at least about 2 per one cell.
15.如权利要求14所述的细胞培养物,其中所述多能细胞包括胚胎干细胞。 15. The cell culture according to claim 14, wherein the pluripotent cells include embryonic stem cells.
16.如权利要求15所述的细胞培养物,其中所述的胚胎干细胞衍生于选自自桑椹胚、胚胎的内细胞团(ICM)和胚胎的性腺嵴的组织。 16. A cell culture as claimed in claim 15, wherein the embryonic stem cells derived from tissue selected from the morula embryos inner cell mass (ICM) and gonadal ridge of embryos.
17.如权利要求1所述的细胞培养物进一步包括培养基,所述培养基包含少于约10%的血清。 17. The cell culture of claim 1, further comprising a culture medium comprising less than about 10% serum.
18.如权利要求1所述的细胞培养物进一步包括TGFβ超家族的Nodal/活化素亚群的生长因子。 18. The cell culture of claim 1 further comprising a TGFβ superfamily Nodal / Activin subgroup growth factor.
19.如权利要求1所述的细胞培养物,进一步包括选自Nodal、活化素A、活化素B及其组合的生长因子。 19. The cell culture according to claim 1, further comprising a selected Nodal, Activin A, Activin B growth factors, and combinations thereof.
20.包括细胞的细胞群,其中至少约90%的所述细胞为人定形内胚层细胞,所述的人定形内胚层细胞为多能细胞,所述多能细胞能分化成肠管细胞或衍生于肠管的器官。 20. A cell population comprising cells, wherein at least about 90% of said cells are human definitive endoderm cells, said human definitive endoderm cells are pluripotent cells, the pluripotent cells can differentiate into cells or derived from intestinal bowel organ.
21.如权利要求20所述的细胞群,其中至少约95%的所述细胞为人定形内胚层细胞。 Cell population of claim 20, wherein at least about 95% of said cells are human definitive endoderm cells as claimed in claim 21,.
22.如权利要求20所述的细胞群,其中至少约98%的所述细胞为人定形内胚层细胞。 Cell population of claim 20, wherein at least about 98% of the cells are human definitive endoderm cells as claimed in claim 22,.
23.如权利要求20所述的细胞群,其中所述人定形内胚层细胞表达选自SOX17和CXCR4的标志物。 23. The cell population according to claim 20, wherein said human definitive endoderm cells express a marker selected from the group of CXCR4 and SOX17.
24.如权利要求23所述的细胞群,其中在所述人定形内胚层细胞中,选自SOX17和CXCR4的所述标志物的表达高于选自OCT4、AFP、TM、SPARC和SOX7的标志物的表达。 24. The cell population of claim 23 AFP, TM, SPARC and flag SOX7, wherein in said human definitive endoderm cells, and SOX17 expression of the selected markers above the selected CXCR4 OCT4, expression thereof.
25.如权利要求23所述的细胞群,其中所述人定形内胚层细胞不表达选自OCT4、AFP、TM、SPARC和SOX7的标志物。 25. The cell population according to claim 23, wherein said human definitive endoderm cells do not express selected from OCT4, AFP, TM, SPARC and SOX7 of markers.
26.如权利要求23所述的细胞群,其中所述人定形内胚层细胞表达选自MIXL1、GATA4和HNF3b的标志物。 26. The cell population according to claim 23, wherein said human definitive endoderm cells express selected MIXL1, GATA4 and HNF3b marker.
27.如权利要求23所述的细胞群,其中所述定形内胚层细胞表达选自FGF17、VWF、CALCR、FOXQ1、CMKOR1和CRIP1的标志物。 27. The cell population according to claim 23, wherein said definitive endoderm cells express selected FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 marker.
28.如权利要求20所述的细胞群,其中所述人定形内胚层细胞表达SOX17和CXCR4。 28. The cell population according to claim 20, wherein said human SOX17 expression of definitive endoderm cells, and CXCR4.
29.如权利要求28所述的细胞群,其中在所述的人定形内胚层细胞中,SOX17和CXCR4的表达高于OCT4、AFP、TM、SPARC和SOX7的表达。 29. The cell population according to claim 28, wherein in said human definitive endoderm cells, the expression of CXCR4 and SOX17 than OCT4, expression of AFP, TM, SPARC and the SOX7.
30.如权利要求28所述的细胞群,其中所述人定形内胚层细胞不表达OCT4、AFP、TM、SPARC和SOX7。 30. A cell population according to claim 28, wherein said human definitive endoderm cells not expressing OCT4, AFP, TM, SPARC and SOX7.
31.如权利要求28所述的细胞群,其中所述的人定形内胚层细胞表达MIXL1、GATA4和HNF3b。 31. The cell population according to claim 28, Definitive endoderm cells express the human wherein MIXL1, GATA4 and HNF3b.
32.如权利要求28所述的细胞群,其中所述定形内胚层细胞表达选自FGF17、VWF、CALCR、FOXQ1、CMKOR1和CRIP1的标志物。 32. A cell population according to claim 28, wherein said definitive endoderm cells express selected FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 marker.
33.如权利要求20所述的细胞群,其中在所述的细胞群体中,对应每一种多能细胞存在至少约2个定形内胚层细胞。 33. The cell population according to claim 20, wherein said cell population in the presence of a corresponding plurality endoderm cells can be shaped at least about 2 per one cell.
34.如权利要求33所述的细胞群,其中所述多能细胞包括胚胎干细胞。 34. The cell population according to claim 33, wherein the pluripotent cells include embryonic stem cells.
35.如权利要求34所述的细胞群,其中所述的胚胎干细胞衍生于选自桑椹胚、胚胎的ICM和胚胎的性腺嵴的组织。 35. The cell population according to claim 34, wherein said embryonic stem cells are derived from tissue selected morula, ICM embryos and embryonic gonadal ridge.
36.产生定形内胚层细胞的方法,所述的方法包括下述步骤:获得包括人多能细胞的细胞群;为所述的细胞群提供至少一种TGFβ超家族生长因子,所述生长因子的量足以促进所述多能细胞分化成定形内胚层细胞,所述的定形内胚层细胞为能分化成肠管细胞或衍生于肠管的器官的多能细胞;以及给予足够的时间来形成定形内胚层细胞,其中所述的形成定形内胚层细胞的足够时间以检测所述细胞群中定形内胚层细胞的存在来确定。 36. A method of producing definitive endoderm cells, said method comprising the steps of: obtaining a population of pluripotent cells include human cells; providing the cell population to at least one TGFβ superfamily growth factor, said growth factor an amount sufficient to promote the pluripotent cells are differentiated into endoderm cells shaping said definitive endoderm cells capable of differentiating into intestinal cells or derived from organs of the intestine of pluripotent cells; and given sufficient time to form definitive endoderm cells wherein the time sufficient to form the definitive endoderm cells to detect the presence of said cell population of definitive endoderm cells determined.
37.如权利要求36所述的方法,其中至少约10%的所述多能细胞分化成定形内胚层细胞。 37. The method according to claim 36, wherein at least about 10% of the pluripotent cells are differentiated into definitive endoderm cells.
38.如权利要求36所述的方法,其中至少约50%的所述多能细胞分化成定形内胚层细胞。 38. The method according to claim 36, wherein at least about 50% of the pluripotent cells are differentiated into definitive endoderm cells.
39.如权利要求36所述的方法,其中至少约70%的所述多能细胞分化成定形内胚层细胞。 39. The method according to claim 36, wherein at least about 70% of the pluripotent cells are differentiated into definitive endoderm cells.
40.如权利要求36所述的方法,其中至少约80%的所述多能细胞分化成定形内胚层细胞。 40. The method according to claim 36, wherein at least about 80% of the pluripotent cells are differentiated into definitive endoderm cells.
41.如权利要求36所述的方法,其中检测定形内胚层细胞在所述细胞群中的存在包括在所述细胞群细胞中检测至少一种选自SOX17和CXCR4的标志物的表达,以及至少一种选自OCT4、AFP、TM、SPARC和SOX7的标志物的表达,其中在所述定形内胚层细胞中,选自SOX17和CXCR4的所述标志物的表达高于选自OCT4、AFP、TM、SPARC和SOX7的所述标志物的表达。 41. The method according to claim 36, wherein detecting the presence of definitive endoderm cells in said cell population comprises detecting the expression of at least one selected marker CXCR4 and SOX17 in said cell population of cells, and at least one selected from OCT4, expression of AFP, TM, SPARC and markers SOX7, wherein the definitive endoderm cells, said CXCR4 and SOX17 expression of selected markers above the selected OCT4, AFP, TM expression of the marker and SOX7 of SPARC.
42.如权利要求36所述的方法,其中检测定形内胚层细胞在所述的细胞群中的存在包括在所述细胞群细胞中检测至少一种选自SOX17和CXCR4的标志物的表达,以及至少一种选自AFP、TM和SOX7的标志物的表达,其中在所述定形内胚层细胞中,选自SOX17和CXCR4的所述标志物的表达高于选自AFP、TM和SOX7的所述标志物的表达。 42. The method according to claim 36, wherein detecting the presence of definitive endoderm cells in said cell population comprises detecting the expression of at least one selected marker CXCR4 and SOX17 in said cell population of cells, and express at least one selected from AFP, SOX7 markers (TM) and wherein the definitive endoderm cells, and SOX17 expression of the selected markers above the selected CXCR4 AFP, the TM and the SOX7 expression of the markers.
43.如权利要求42所述的方法,其中至少一种所述的标志物的表达以Q-PCR来测定。 43. The method according to claim 42, wherein at least one of the expression of the marker to be measured Q-PCR.
44.如权利要求42所述的方法,其中至少一种所述的标志物的表达以免疫细胞化学来测定。 44. The method according to claim 42, wherein at least one of the expression of the marker is determined by immunocytochemistry.
45.如权利要求36所述的方法,其中检测在所述的细胞群体中定形内胚层细胞的存在包括在所述细胞群体细胞中检测至少一种选自VWF、CALCR、FOXQ1、CMKOR1和CRIP1的标志物的表达,以及至少一种选自OCT4、AFP、TM、SPARC和SOX7的标志物的表达,其中在所述的定形内胚层细胞中,选自FGF17、VWF、CALCR、FOXQ1和CRIP1的标志物的表达高于选自OCT4、AFP、TM、SPARC和SOX7的标志物的表达。 45. The method according to claim 36, wherein said population of cells detected in the presence of definitive endoderm cells VWF comprises detecting at least one selected cell in said cell population, CALCR, FOXQ1, CMKOR1 and the CRIP1 expression of the marker, and at least one selected from OCT4, expression of AFP, TM, SPARC and markers SOX7, wherein in said definitive endoderm cells, the selected FGF17, VWF, CALCR, FOXQ1 flag and CRIP1 expression was higher than that selected OCT4, expression of AFP, TM, SPARC and SOX7 markers.
46.如权利要求36所述的方法,其中所述的至少一种生长因子为TGFβ超家族的Nodal/活化素亚群。 46. ​​The method according to claim 36, wherein said at least one growth factor is Nodal / Activin subgroup TGFβ superfamily.
47.如权利要求46所述的方法,其中所述的至少一种生长因子选自Nodal、活化素A、活化素B及其组合。 47. A method according to claim 46, wherein said at least one growth factor selected Nodal, Activin A, Activin B, and combinations thereof.
48.如权利要求47所述的方法,其中所述的至少一种生长因子为Nodal。 48. The method of claim 47, wherein at least one growth factor, wherein said Nodal.
49.如权利要求47所述的方法,其中所述的至少一种生长因子为活化素A。 49. The method according to claim 47, wherein said at least one growth factor is activin A.
50.如权利要求47所述的方法,其中所述的至少一种生长因子为活化素B。 50. The method according to claim 47, wherein said at least one growth factor is activin B.
51.如权利要求36所述的方法,其中提供TGFβ超家族的多种生长因子。 51. The method according to claim 36, wherein the TGFβ superfamily provide more growth factors.
52.如权利要求51所述的方法,其中所述的多种生长因子包括Nodal、活化素A及活化素B。 52. A method according to claim 51, wherein the plurality of growth factors include Nodal, Activin A and Activin B.
53.如权利要求36所述的方法,其中所述至少一种生长因子的浓度为至少约10ng/ml。 53. A method according to claim 36, wherein said at least one growth factor in a concentration of at least about 10ng / ml.
54.如权利要求36所述的方法,其中所述至少一种生长因子的浓度为至少约100ng/ml。 54. A method according to claim 36, wherein said at least one growth factor in a concentration of at least about 100ng / ml.
55.如权利要求36所述的方法,其中所述至少一种生长因子的浓度为至少约500ng/ml。 55. The method according to claim 36, wherein said at least one growth factor in a concentration of at least about 500ng / ml.
56.如权利要求36所述的方法,其中所述至少一种生长因子的浓度为至少约1000ng/ml。 56. The method according to claim 36, wherein said at least one growth factor in a concentration of at least about 1000ng / ml.
57.如权利要求36所述的方法,其中所述至少一种生长因子的浓度为至少约5000ng/ml。 57. The method according to claim 36, wherein said at least one growth factor in a concentration of at least about 5000ng / ml.
58.如权利要求36所述的方法,其中所述细胞群生长于包括少于约10%血清的培养基中。 58. The method according to claim 36, wherein the population of cells grown in culture medium comprising less than about 10% serum.
59.如权利要求36所述的方法,其中所述多能细胞包括干细胞。 59. The method according to claim 36, wherein the pluripotent cells comprise stem cells.
60.如权利要求59所述的方法,其中所述的多能细胞包括胚胎干细胞。 60. The method according to claim 59, wherein the pluripotent cells include embryonic stem cells.
61.如权利要求60所述的方法,其中所述的胚胎干细胞衍生于选自桑椹胚、胚胎的ICM和胚胎的性腺嵴的组织。 61. The method according to claim 60, wherein said embryonic stem cells are derived from tissue selected morula, ICM embryos and embryonic gonadal ridge.
62.以权利要求36的方法产生的定形内胚层细胞。 Definitive endoderm cells to 62. The method of claim 36 produced.
63.产生富集的定形内胚层细胞的细胞群方法,包括下述步骤:分化多能人细胞群体中的细胞,以产生定形内胚层细胞,所述的定形内胚层细胞为多能细胞,该多能细胞能分化成肠管细胞或衍生于肠管的器官;向所述的细胞群提供试剂,所述试剂与标志物结合,所述标志物表达于所述的定形内胚层细胞中而基本上不表达于所述细胞群中的其它细胞类型中;及将与所述试剂结合的所述定形内胚层细胞与位于所述细胞群中的所述其它细胞类型分离,从而产生富集的定形内胚层细胞的细胞群。 63. A method of generating a population of cells of the endoderm cell enrichment, comprising the steps of: pluripotent cells capable cell populations to produce amorphous endoderm cells, said definitive endoderm cells are pluripotent cells that pluripotent cells capable of differentiating into cells or organs derived from intestinal bowel; providing the cell population of the agent, the agent binds with the marker, the marker in the expression of the definitive endoderm cells without substantially expression in other cell types in the population of cells; and the binding of said definitive endoderm cells with the agent located in the cell population isolated from other cell types, thereby generating definitive endoderm enriched cell populations of cells.
64.如权利要求63所述的方法,其中分化步骤进一步包括,获得包括多能人细胞的细胞群,向所述细胞群体提供所述至少一种TGFβ超家族生长因子,所述生长因子的量足以促进将所述多能细胞分化成定形内胚层细胞,所述的定形内胚层细胞为多能细胞,所述多能细胞能分化成肠管细胞或衍生于肠管的器官,及给予足够的时间形成定形内胚层细胞,其中所述的形成定形内胚层细胞的足够时间以检测所述细胞群体中定形内胚层细胞的存在来确定。 64. The method according to claim 63, wherein the differentiating step further comprises obtaining an amount of pluripotent human cell population comprising providing said at least one TGFβ superfamily of growth factors to the cell population, said growth factor sufficient to promote the pluripotent cells are differentiated into endoderm cells shaping said definitive endoderm cells are pluripotent cells, the pluripotent cells capable of differentiating into intestinal cells or organs derived from the intestinal tract, and given enough time to form endoderm cells, sufficient time wherein the forming of the amorphous endoderm cells to detect the presence within the population of cells to definitive endoderm cells is determined.
65.如权利要求63所述的方法,其中检测包括,在所述细胞群体的细胞中检测至少一种选自SOX17和CXCR4的标志物的表达,以及至少一种选自OCT4、AFP、TM、SPARC和SOX7的标志物的表达,其中在所述的定形内胚层细胞中,选自SOX17和CXCR4的标志物的表达高于选自OCT4、AFP、TM、SPARC和SOX7的标志物的表达。 65. The method according to claim 63, wherein the detecting comprises detecting in a cell of the population of cells expressing at least one selected marker CXCR4 and SOX17, and at least one member selected from OCT4, AFP, TM, SPARC expression markers and SOX7, wherein in said definitive endoderm cells, the expression of selected markers CXCR4 and SOX17 higher than the selected OCT4, expression of AFP, TM, SPARC and SOX7 of markers.
66.如权利要求63所述的方法,其中检测包括,在所述细胞群体的细胞中检测至少一种选自SOX17和CXCR4的标志物的表达,以及至少一种选自AFP、TM和SOX7的标志物的表达,其中在所述的定形内胚层细胞中,选自SOX17和CXCR4的标志物的表达高于选自AFP、TM和SOX7的标志物的表达。 66. The method according to claim 63, wherein the detecting comprises detecting the expression of at least one selected marker CXCR4 and SOX17, and at least one selected from AFP, TM, and SOX7 in cells of said cell population in expression of a marker, wherein in said definitive endoderm cells, selected markers CXCR4 and SOX17 expression than the selected AFP, the expression of markers (TM) and the SOX7.
67.如权利要求63所述的方法,其中检测包括,在所述细胞群体的细胞中检测至少一种选自FGF17、VWF、CALCR、FOXQ1、CMKOR1和CRIP1的标志物的表达,以及至少一种选自OCT4、AFP、TM、SPARC和SOX7的标志物的表达,其中在所述的定形内胚层细胞中,选自FGF17、VWF、CALCR、FOXQ1、CMKOR1和CRIP1的标志物的表达高于选自OCT4、AFP、TM、SPARC和SOX7的标志物的表达。 67. The method of claim 63 and expressing CMKOR1 CRIP1 markers, and at least one claim, wherein the detecting comprises detecting at least one selected FGF17, VWF, CALCR, FOXQ1 in the cells of said cell population, is selected from OCT4, expression of AFP, TM, SPARC and markers SOX7, wherein in said definitive endoderm cells, the selected FGF17, expression of VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 marker selected above expressing OCT4, AFP, TM, SPARC and SOX7 of markers.
68.如权利要求63所述的方法,其中至少约95%的所述细胞为定形内胚层细胞。 68. The method according to claim 63, wherein at least about 95% of the cell is definitive endoderm cells.
69.如权利要求63所述的方法,其中至少约98%的所述细胞为定形内胚层细胞。 69. A method according to claim 63, wherein at least about 98% of the cell is definitive endoderm cells.
70.如权利要求63所述的方法,其中所述的标志物为CXCR4。 70. A method according to claim 63, wherein said marker is CXCR4.
71.如权利要求63所述的方法,其中所述的试剂为抗体。 71. A method according to claim 63, wherein said agent is an antibody.
72.如权利要求71所述方法,其中所述的抗体对CXCR4具有亲和性。 72. The method as claimed in claim 71, wherein said antibody has an affinity for CXCR4.
73.权利要求63所述的方法产生的富集定形内胚层细胞的细胞群。 73. The method of setting the enrichment of claim 63 produced by a cell population endoderm cells.
74.如权利要求4或9的任一权利要求所述的细胞培养物,其中所述的定形内胚层细胞不显著表达选自OCT4、AFP、TM、SPARC和SOX7的标志物。 74. 4 or any one of the 9 cell culture according to one of the preceding claims AFP, TM, SPARC and SOX7 marker as claimed in claim wherein said definitive endoderm cells do not substantially express selected from OCT4,.
75.如权利要求23或28的任一权利要求的细胞群,其中所述的定形内胚层细胞不显著表达选自OCT4、AFP、TM、SPARC和SOX7的标志物。 75. 23 or 28 of any one cell population claims AFP, TM, SPARC and SOX7 marker as claimed in claim wherein said definitive endoderm cells do not substantially express selected from OCT4,.
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