CN103154239B - 人胚胎干细胞的分化 - Google Patents
人胚胎干细胞的分化 Download PDFInfo
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Abstract
本发明提供了促进多能干细胞分化成胰岛素产生细胞的方法。具体地讲,本发明提供了产生细胞群的方法,其中所述群体中大于85%的细胞表达定形内胚层谱系特征性标志物。
Description
相关申请的交叉引用
本申请要求2010年8月31日提交的美国临时专利申请序列No.61/378,472的权益,其全文以引用方式并入本文用于所有目的。
技术领域
本发明提供了促进多能干细胞分化成胰岛素产生细胞的方法。具体地讲,本发明提供了产生细胞群的方法,其中所述群体中大于85%的细胞表达定形内胚层谱系特征性标志物。
背景技术
用于I型糖尿病的细胞替代疗法的进展以及可移植胰岛的缺乏已使得注意力集中在开发适于移植物移入的胰岛素产生细胞或β细胞的来源上。一种方法是从多能干细胞,例如胚胎干细胞产生功能性β细胞。
在脊椎动物的胚胎发育中,多能干细胞可在称为原肠胚形成的过程中产生包括三个胚层(外胚层、中胚层和内胚层)的细胞群。诸如甲状腺、胸腺、胰腺、肠和肝脏之类的组织将从内胚层,经由中间阶段发育而来。该过程中的中间阶段是形成定形内胚层。定形内胚层细胞可表达多种标志物,如HNF3β、GATA4、MIXL1、CXCR4和SOX17。
定形内胚层分化成胰腺内胚层导致形成胰腺。胰腺内胚层细胞表达胰-十二指肠同源盒基因PDX1。在不存在PDX1时,胰腺形成腹胰芽和背胰芽后不再发育。因而,PDX1表达标志着胰腺器官发生中的一个关键步骤。除了其它细胞类型,成熟的胰腺还包括外分泌组织和内分泌组织。外分泌和内分泌组织来自胰腺内胚层的分化。
据报道,从小鼠的胚胎细胞衍生了带有胰岛细胞特征的细胞。例如,Lumelsky等人(Science《科学》292:1389,2001)报道了小鼠胚胎干细胞向类似于胰岛的胰岛素分泌结构的分化。Soria等人(Diabetes《糖尿病》 49:157,2000)报道了衍生自小鼠胚胎干细胞的胰岛素分泌细胞使链脲佐菌素诱导的糖尿病小鼠中的血糖变正常。
在一个例子中,Hori等人(PNAS《美国科学院院刊》99:16105,2002)公开了用磷脂酰肌醇3-激酶抑制剂(LY294002)处理小鼠胚胎干细胞产生了类似β细胞的细胞。
在另一个例子中,Blyszczuk等人(PNAS《美国科学院院刊》100:998,2003)报道了从组成型表达Pax4的小鼠胚胎干细胞生成产生胰岛素的细胞。
Micallef等人报道了视黄酸可调节胚胎干细胞定向形成PDX1阳性胰腺内胚层。在对应于胚胎的原肠胚形成末期的期间,加入胚胎干细胞分化第4天的培养物中时视黄酸在诱导Pdx1方面最有效(Diabetes《糖尿病》54:301,2005)。
Miyazaki等人报道了过表达Pdx1的小鼠胚胎干细胞系。他们的结果显示,外源Pdx1表达在所得的分化细胞中明显增强了胰岛素、生长抑素、葡萄糖激酶、神经元素3、p48、Pax6和Hnf6基因的表达(Diabetes《糖尿病》53:1030,2004)。
Skoudy等人报道说,激活素A(TGF-β超家族的成员)能上调小鼠胚胎干细胞中的胰腺外分泌基因(p48和淀粉酶)和内分泌基因(Pdx1、胰岛素和胰高血糖素)的表达。使用1nM激活素A观察到最大效应。他们还观察到胰岛素和Pdx1mRNA的表达水平不受视黄酸的影响;然而,3nM FGF7处理导致了Pdx1的转录水平升高(Biochem.J.《生物化学杂志》379:749,2004)。
Shiraki等人研究了能特征性增强胚胎干细胞分化成PDX1阳性细胞的生长因子的效果。他们观察到,TGF-β2可再现地产生更高比例的PDX1阳性细胞(Genes Cells.2005年6月;10(6):503-16)。
Gordon等人阐明了在没有血清存在有激活素连同Wnt信号传导抑制剂存在的情况下从小鼠胚胎干细胞诱导短尾蛋白(brachyury)[阳性]/HNF3β[阳性]内胚层细胞(US2006/0003446A1)。
Gordon等人(PNAS《美国科学院院刊》,第103卷,第16806页,2006)声称“Wnt和TGF-β/nodal/激活素信号传导同时为生成前原条所必需的”。
然而,胚胎干细胞发育的小鼠模型可能不会完全模拟高等哺乳动物(例如人)中的发育程序。
Thomson等人从人胚泡分离了胚胎干细胞(Science《科学》282:114,1998)。同时,Gearhart和其同事从胎儿生殖腺组织衍生了人胚胎生殖(hEG)细胞系(Shamblott等人,Proc.Natl.Acad.Sci.USA《美国科学院院刊》95:13726,1998)。与可简单通过与白血病抑制因子(LIF)一起培养来防止分化的小鼠胚胎干细胞不一样,人胚胎干细胞必须维持在非常特殊的条件下(美国专利No.6,200,806;WO99/20741;WO01/51616)。
D’Amour等人描述了在高浓度激活蛋白和低血清的存在下产生人胚胎干细胞衍生的定形内胚层的富集培养物(Nature Biotechnology《自然生物技术》2005)。将这些细胞移植到小鼠的肾囊下,导致分化成具有某些内胚层器官的特性的更成熟细胞。在加入FGF-10之后,人胚胎干细胞衍生的定形内胚层细胞可进一步分化成PDX1阳性细胞(US2005/0266554A1)。
D’Amour等人(Nature Biotechnology《自然生物技术》-24,1392-1401(2006))声称:“我们已开发出使人胚胎干(hES)细胞转化成能够合成胰腺激素胰岛素、胰高血糖素、生长抑素、胰多肽和生长激素释放肽的内分泌细胞的分化方法。该方法通过引导细胞经过类似于定形内胚层、肠管内胚层、胰腺内胚层和内分泌前体的阶段转变为表达内分泌激素的细胞来模拟体内胰腺器官发生”。
在另一个例子中,Fisk等人报道了用于从人胚胎干细胞产生胰岛细胞的系统(US2006/0040387A1)。在该情形中,分化途径分成三个阶段。使用丁酸钠和激活素A混合物将人类胚胎干细胞首先分化成内胚层细胞。随后将该细胞用TGF-β拮抗剂例如头蛋白与EGF或乙胞素的混合物培养以生成PDX1阳性细胞。通过烟酰胺诱导终末分化。
因此,仍明显需要开发生成表达胰岛素的功能细胞的体外方法,所述细胞更密切地类似β细胞。本发明采取替代方法,通过生成细胞群来提高使 人胚胎干细胞向胰岛素表达细胞分化的效率,其中所述群体中大于85%的细胞表达定形内胚层谱系特征性标志物。
发明内容
在一个实施例中,本发明提供了细胞群,其中所述群体中大于85%的细胞表达定形内胚层谱系特征性标志物。
在一个实施例中,通过在补充有BSA以及选自胰岛素和IGF-1的因子的培养基中培养多能干细胞,使多能干细胞群分化成表达定形内胚层谱系特征性标志物的细胞群。在一个实施例中,通过用激活素A和Wnt配体处理多能干细胞,达到多能干细胞群向表达定形内胚层谱系特征性标志物的细胞群的分化。
在一个实施例中,通过用GDF-8和至少一种选自下列的其它因子处理多能干细胞,达到多能干细胞群向表达定形内胚层谱系特征性标志物的细胞群的分化:苯胺-吡啶并三嗪、环状苯胺-吡啶并三嗪、N-{[1-(苯基甲基)氮杂 -4-基]甲基}-2-吡啶-3-基乙酰胺、4-{[4-(4-{[2-(吡啶-2-基氨基)乙基]氨基}-1,3,5-三嗪-2-基)吡啶-2-基]氧}丁-1-醇、3-({3-[4-({2-[甲基(吡啶-2-基)氨基]乙基}氨基)-1,3,5-三嗪-2-基]吡啶-2-基}氨基)丙-1-醇、N~4~-[2-(3-氟苯基)乙基]-N~2~-[3-(4-甲基哌嗪-1-基)丙基]吡啶并[2,3-d]嘧啶-2,4-二胺、1-甲基-N-[(4-吡啶-3-基-2-{[3-(三氟甲基)苯基]氨基}-1,3-噻唑-5-基)甲基]哌啶-4-羧酰胺、1,1-二甲基乙基{2-[4-({5-[3-(3-羟丙基)苯基]-4H-1,2,4-三唑-3-基}氨基)苯基]乙基}氨基甲酸酯、1,1-二甲基乙基{[3-({5-[5-(3-羟丙基)-2-(甲氧基)苯基]-1,3-唑-2-基}氨基)苯基]甲基}氨基甲酸酯、1-({5-[6-({4-[(4-甲基哌嗪-1-基)磺酰基]苯基}氨基)吡嗪-2-基]噻吩-2-基}甲基)哌啶-4-醇、1-({4-[6-({4-[(4-甲基哌嗪-1-基)磺酰基]苯基}氨基)吡嗪-2-基]噻吩-2-基}甲基)哌啶-4-羧酰胺、和2-{[4-(1-甲基乙基)苯基]氨基}-N-(2-噻吩-2-基乙基)-7,8-二氢吡啶并[4,3-d]嘧啶-6(5H)-羧酰胺。
附图说明
图1显示了根据实例1中公开的方法分化的,人胚胎干细胞系H1的细胞中指示的基因表达的实时PCR分析。
图2显示了根据实例1中公开的方法分化的,人胚胎干细胞系H1的细胞中指示的蛋白质表达的FACS分析。
图3显示了根据实例2中公开的方法分化的,人胚胎干细胞系H1的细胞中指示的基因表达的实时PCR分析。
图4显示了根据实例2中公开的方法分化的,人胚胎干细胞系H1的细胞中经由免疫荧光的SOX17表达。
图5显示了根据实例2中公开的方法分化的,人胚胎干细胞系H1的细胞中指示的蛋白质表达的FACS分析。
图6显示了根据实例3中公开的方法分化的,人胚胎干细胞系H1的细胞中指示的基因表达的实时PCR分析。
图7显示了根据实例3中公开的方法分化的,人胚胎干细胞系H1的细胞中经由免疫荧光的SOX17表达。
图8显示了根据实例3中公开的方法分化的,人胚胎干细胞系H1的细胞中经由免疫荧光的SOX17表达。
图9显示了根据实例5中公开的方法分化的,人胚胎干细胞系H1的细胞中指示的基因表达的实时PCR分析。
具体实施方式
将本发明的具体实施方式部分分成以下几个分部分,来描述或说明本发明的某些特征、实施例或应用,这是为了使公开内容清楚起见,并非限制本发明。
定义
干细胞是由它们在单细胞水平上既自我更新又分化产生子代细胞的能力来定义的未分化细胞,包括自我更新祖细胞、非更新祖细胞和末端分化细胞。干细胞的特征还在于:其具有在体外由多种胚层(内胚层、中胚层和外胚层)分化成各种细胞谱系的功能细胞以及移植后产生多种胚层的组织和注入胚泡后基本上有助于大部分(如果不是所有的话)组织形成的能力。
干细胞根据其发育潜能分为:(1)全能,指能够产生所有的胚胎和胚胎外细胞类型;(2)多能,指能够产生所有的胚胎细胞类型;(3)专能,指能够产生细胞谱系的亚群,但所有细胞谱系都只分布于特定组织、器官或 生理系统内(例如造血干细胞(HSC)可产生的后代细胞包括:HSC(自我更新)、局限于血细胞的寡能祖细胞以及作为血液正常组分的所有细胞类型和成分(如血小板));(4)寡能,指能够产生比专能干细胞更有限的细胞谱系亚群;以及(5)单能,指能够产生单一细胞谱系(如生精干细胞)。
分化是未特化的(“未定向的”)或特化不足的细胞获得特化细胞(如神经细胞或肌肉细胞)的特征的过程。分化的细胞或诱导分化的细胞是已经在细胞谱系中占据更特化的(“定向的”)位置的细胞。术语“定向的”当应用到分化的过程时,指在分化途径中已经进行到这么一种程度的细胞:在正常环境下,它会继续分化成特定的细胞类型或细胞类型子集,且在正常环境下不能分化成另一细胞类型或回复到分化程度较低的细胞类型。去分化指细胞回复到细胞谱系当中特化(或定向)程度较低的地位的过程。如本文所用,“细胞谱系”限定细胞的遗传关系,即它来自哪些细胞和它能产生什么细胞。细胞谱系将细胞定位于发育和分化的遗传计划内。谱系特异性标志物是指与所关注谱系的细胞表型特异性相关并能够用于评价非定向细胞向所关注谱系的分化的特征。
如本文所用,“表达定形内胚层谱系特征性标志物的细胞”或“第1阶段细胞”或“第1阶段”是指表达至少一种下列标志物的细胞:SOX17、GATA4、HNF3β、GSC、CER1、Nodal、FGF8、短尾蛋白、Mix样同源盒蛋白、FGF4CD48、脱中胚蛋白(eomesodermin,EOMES)、DKK4、FGF17、GATA6、CXCR4、C-Kit、CD99或OTX2。表达定形内胚层谱系特征性标志物的细胞包括原条前体细胞、原条细胞、中内胚层细胞和定形内胚层细胞。
本文所用的“表达胰腺内胚层谱系特征性标志物的细胞”指表达至少一种下列标志物的细胞:PDX1、NKX6.1、HNF1β、PTF1α、HNF6、HNF4α、SOX9、HB9或PROX1。表达胰腺内胚层谱系特征性标志物的细胞包括胰腺内胚层细胞、原肠管细胞和后前肠细胞。
如本文所用,“定形内胚层”指具有在原肠胚形成过程中从上胚层产生的细胞的特性并形成胃肠道及其衍生物的细胞。定形内胚层细胞表达下列 标志物:HNF3β、GATA4、SOX17、Cerberus、OTX2、goosecoid、C-Kit、CD99和MIXL1。
如本文所用,“标志物”是在所关注细胞中差异表达的核酸或多肽分子。关于这一点,差异表达意指阳性标志物的水平增加,而阴性标志物的水平降低。与其它细胞相比,所关注细胞中的核酸或多肽标志物的可检测水平足够高或足够低,使得可以使用本领域已知的多种方法识别所关注细胞,并将所关注细胞与其它细胞区相区分。
本文所用的“胰腺内分泌细胞”或“胰激素表达细胞”或“表达胰腺内分泌谱系特征性标志物的细胞”指能够表达至少一种下列激素的细胞:胰岛素、胰高血糖素、生长抑素和胰多肽。
多能干细胞的分离、扩增和培养
多能干细胞的表征
多能干细胞可表达阶段特征性胚胎抗原(SSEA)3和4以及可用称为Tra-1-60和Tra-1-81的抗体检测的标志物中的一种或多种(Thomson等人,Science《科学》282:1145,1998)。多能干细胞体外分化导致丧失SSEA-4、Tra1-60和Tra1-81的表达(如果存在的话),并增加SSEA-1的表达。未分化的多能干细胞通常具有碱性磷酸酶活性,该酶可通过用4%多聚甲醛固定细胞,然后用Vector Red作为底物显影来检测,如生产商所描述(VectorLaboratories,Burlingame Calif)。未分化的多能干细胞通常也表达OCT4和TERT,这可通过RT-PCR检测。
增殖的多能干细胞的另一理想表型是分化成所有三个胚层即内胚层、中胚层和外胚层组织的细胞的潜能。多能干细胞的多能性可例如通过这样来证实:将细胞注射进重症联合免疫缺陷(SCID)小鼠中,用4%多聚甲醛固定所形成的畸胎瘤,然后对它们进行组织学检验以确定是否存在来自三个胚层的细胞类型。作为另外一种选择,可以通过产生胚状体并评估胚状体中三个胚层相关的标志物的存在来确定多能性。
可以使用标准G带技术并比较已公布的相应灵长类物种的核型来分析增殖的多能干细胞系的核型。希望获得具有“正常核型”的细胞,其意指细胞为整倍体,其中所有人染色体都存在并且没有明显的改变。
多能干细胞的来源
可使用的多能干细胞例如人胚胎干细胞系H1、H7、H9(WiCell)和BG01v(BresaGen,Athens,GA)。
多能干细胞的培养
在一个实施例中,在饲养细胞层上培养多能干细胞,饲养细胞可以多种方式支持多能干细胞。作为另一种选择,在培养系统中培养多能干细胞,所述培养系统基本上不含饲养细胞,但同样支持多能干细胞的增殖而不会进行实质分化。使用通过此前培养另一细胞类型而调理过的培养基来支持多能干细胞在无饲养细胞的培养物中生长而不分化。作为另一种选择,用化学成分确定的培养基来支持多能干细胞在无饲养细胞的培养物中生长而不分化。
在一个实施例中,可以根据如下文献中公开的方法在小鼠胚胎成纤维细胞饲养细胞层上培养多能干细胞:Reubinoff等人(Nature Biotechnology《自然生物技术》18:399-404(2000))。作为另外一种选择,可以根据如下文献中公开的方法在小鼠胚胎成纤维细胞饲养细胞层上培养多能干细胞:Thompson等人(Science《科学》1998年11月6日:第282卷,第5391期,第1145-1147页)。作为另外一种选择,可以在如下文献中公开的任何一种饲养细胞层上培养多能干细胞:Richards等人(Stem Cells《干细胞》21:546-556,2003)。
在一个实施例中,可以根据如下文献中公开的方法在人饲养细胞层上培养多能干细胞:Wang等人(Stem Cells《干细胞》23:1221-1227,2005)。在一个替代实施例中,可以在如下文献中公开的人饲养细胞层上培养多能干细胞:Stojkovic等人(Stem Cells《干细胞》200523:306-314,2005)。作为另外一种选择,可以在如下文献中公开的人饲养细胞层上培养多能干细胞:Miyamoto等人(Stem Cells《干细胞》22:433-440,2004)。作为另外一种选择,可以在如下文献中公开的人饲养细胞层上培养多能干细胞:Amit等人(Biol.Reprod《繁殖生物学》68:2150-2156,2003)。作为另外一种选择,可以在如下文献中公开的人饲养细胞层上培养多能干细胞:Inzunza等人(Stem Cells《干细胞》23:544-549,2005)。
在一个实施例中,可以在根据US20020072117公开的方法所得培养基中培养多能干细胞。作为另外一种选择,可以在根据US6642048公开的方法所得培养基中培养多能干细胞。作为另外一种选择,可以在根据WO2005014799公开的方法所得培养基中培养多能干细胞。作为另外一种选择,可以在根据如下文献公开的方法所得培养基中培养多能干细胞:Xu等人(Stem Cells《干细胞》22:972-980,2004)。作为另外一种选择,可以在根据US20070010011公开的方法所得培养基中培养多能干细胞。作为另外一种选择,可以在根据US20050233446公开的方法所得培养基中培养多能干细胞。作为另外一种选择,可以在根据US6800480公开的方法所得培养基中培养多能干细胞。作为另外一种选择,可以在根据WO2005065354公开的方法所得培养基中培养多能干细胞。
在一个实施例中,可以根据如下文献中公开的方法在人饲养细胞层上培养多能干细胞:Cheon等人(BioReprod《繁殖生物学》DOI:10.1095/biolreprod.105.046870,2005年10月19日)。作为另外一种选择,可以根据如下文献公开的方法培养多能干细胞:Levenstein等人(Stem Cells《干细胞》24:568-574,2006)。作为另外一种选择,可以根据US20050148070公开的方法培养多能干细胞。作为另外一种选择,可以根据US20050244962公开的方法培养多能干细胞。作为另外一种选择,可以根据WO2005086845公开的方法培养多能干细胞。
可将多能干细胞接种至合适的培养基质上。在一个实施例中,合适的培养基质是胞外基质成分,例如衍自基底膜的成分,或者可形成黏着分子受体-配体偶联物的一部分的成分。在一个实施例中,合适的培养基材是 (Becton Dickenson)。是得自Engelbreth-Holm Swarm肿瘤细胞的可溶性制剂,其在室温下胶凝而形成重构的基底膜。
其它的细胞外基质组分和组分混合物适合作为替代物。取决于所扩增的细胞类型,这可包括单独的层粘连蛋白、纤连蛋白、蛋白聚糖、巢蛋白、硫酸乙酰肝素等或者它们的各种组合。
可在存在可促进细胞存活、增殖和保持理想特性的培养基存在的情况下,以合适的分布将多能干细胞接种于所述基质上。所有这些特性可得益于对接种分布的认真考虑并可容易地由本领域技术人员确定。
合适的培养基可用如下组分制备,例如达尔伯克氏改良伊格尔培养基(DMEM),Gibco货号11965-092;敲除达尔伯克氏改良伊格尔培养基(KO DMEM),Gibco货号10829-018;Ham′s F12/50%DMEM基础培养基;200mM L-谷氨酰胺,Gibco货号15039-027;非必需氨基酸溶液,Gibco11140-050;β-巯基乙醇,Sigma货号M7522;人重组碱性成纤维细胞生长因子(bFGF),Gibco货号13256-029。
由多能干细胞形成表达定形内胚层谱系特征性标志物的细胞
本发明提供了由多能干细胞群形成表达定形内胚层谱系特征性标志物的细胞群的方法。在一个实施例中,本发明提供了使表达定形内胚层谱系特征性标志物的细胞进一步分化成表达胰腺内分泌谱系的细胞的方法。在一个实施例中,这通过利用分布分化方案来达到,其中多能干细胞群首先分化成表达定形内胚层谱系特征性标志物的细胞群。接下来,使表达定形内胚层谱系特征性标志物的细胞群随后分化成表达胰腺内胚层谱系特征性标志物的细胞群。接下来,使表达胰腺内胚层谱系特征性标志物的细胞群随后分化成表达胰腺内分泌谱系特征性标志物的细胞群。
本发明提供了细胞群,其中大于85%的细胞表达定形内胚层谱系特征性标志物。细胞群可以进一步处理,以形成表达胰腺内胚层谱系特征性标志物的细胞群。表达胰腺内胚层谱系特征性标志物的细胞群可以进一步处理,以形成表达胰腺内分泌谱系特征性标志物的细胞群。
可通过将处理过的细胞群暴露于可特异性识别由表达所需细胞类型特征性标志物的细胞表达的蛋白质标志物的试剂(例如抗体)来确定分化效率。
用于评估蛋白质标志物和核酸标志物在培养的或分离的细胞中的表达的方法是本领域的标准方法。这些包括定量反转录聚合酶链式反应(RT-PCR)、Northern印迹、原位杂交(参见例如,Current Protocols in Molecular Biology(Ausubel等人编辑,2001增刊))以及免疫测定法,例如分段材料的免疫组织化学分析,Westem印迹、以及用于未受损细胞中容易获得的标志物的流式细胞分析(FACS)(参见例如Harlow和Lane的“Using Antibodies:A Laboratory Manual”,New York:Cold Spring Harbor Laboratory Press(1998))。
多能干细胞的特征是本领域技术人员熟知的,并且其它特征有待继续辨别。多能干细胞标志物包括(例如)一种或多种如下物质的表达:ABCG2、CRIPTO、FOXD3、CONNEXIN43、CONNEXIN45、OCT4、SOX2、NANOG、hTERT、UTF1、ZFP42、SSEA-3、SSEA-4、Tra1-60、Tra1-81。
在用本发明方法处理多能干细胞后,可通过将处理过的细胞群暴露于特异性识别由表达定形内胚层谱系特征性标志物的细胞表达的蛋白质标志物(例如CXCR4)来进行纯化。
适用于本发明的多能干细胞包括例如人胚胎干细胞系H9(NIH编码:WA09)、人胚胎干细胞系H1(NIH编码:WA01)、人胚胎干细胞系H7(NIH编码:WA07)和人胚胎干细胞系SA002(Cellartis,瑞典)。同样适用于本发明的是表达至少一种下列多能细胞特征性标志物的细胞:ABCG2、cripto、CD9、FOXD3、CONNEXIN43、CONNEXIN45、OCT4、SOX2、NANOG、hTERT、UTF1、ZFP42、SSEA-3、SSEA-4、Tra1-60和Tra1-81。
定形内胚层谱系特征性标志物选自SOX17、GATA4、HNF3β、GSC、CER1、Nodal、FGF8、短尾蛋白、Mix样同源盒蛋白、FGF4、CD48、脱中胚蛋白(EOMES)、DKK4、FGF17、GATA6、CXCR4、C-Kit、CD99和OTX2。适用于本发明的是表达至少一种定形内胚层谱系特征性标志物的细胞。在本发明的一个方面,表达定形内胚层谱系特征性标志物的细胞为原条前体细胞。在一个替代方面,表达定形内胚层谱系特征性标志物的细胞为中内胚层细胞。在一个替代方面,表达定形内胚层谱系特征性标志物的细胞为定形内胚层细胞。
胰腺内胚层谱系特征性标志物选自PDX1、NKX6.1、HNF1β、PTF1α、HNF6、HNF4α、SOX9、HB9和PROX1。适用于本发明的是表达至少一种胰腺内胚层谱系特征性标志物的细胞。在本发明的一个方面,表达胰腺内胚层谱系特征性标志物的细胞为胰腺内胚层细胞。
胰腺内分泌谱系特征性标志物选自NGN3、NEUROD、ISL1、PDX1、NKX6.1、PAX4、NGN3和PTF1α。在一个实施例中,胰腺内分泌细胞能够表达以下激素中的至少一种:胰岛素、胰高血糖素、生长抑素和胰多肽。适用于本发明的是表达至少一种胰腺内分泌谱系特征性标志物的细胞。在本发明的一个方面,表达胰腺内分泌系特征性标志物的细胞为胰腺内分泌细胞。胰腺内分泌细胞可为表达胰激素的细胞。作为另外一种选择,胰腺内分泌细胞可为分泌胰激素的细胞。
在本发明的一个方面,胰腺内分泌细胞是表达β细胞谱系特征性标志物的细胞。表达β细胞谱系特征性标志物的细胞可表达PDX1和至少一种下列转录因子:NGN3、NKX2.2、NKX6.1、NEUROD、ISL1、HNF3β、MAFA、PAX4和PAX6。在本发明的一个方面,表达β细胞谱系特征性标志物的细胞是β细胞。
由多能干细胞形成表达定形内胚层谱系特征性标志物的细胞
在本发明的一个方面,通过在缺乏血清且补充有BSA以及选自胰岛素和IGF-1的因子的培养基中培养多能干细胞,可以使多能干细胞群分化成表达定形内胚层谱系特征性标志物的细胞群。在一个实施例中,通过用激活素A和Wnt配体处理多能干细胞,达到多能干细胞群向表达定形内胚层谱系特征性标志物的细胞群的分化。
在替代实施例中,通过用GDF-8和至少一种选自下列的其它因子处理多能干细胞,达到多能干细胞群向表达定形内胚层谱系特征性标志物的细胞群的分化:苯胺-吡啶并三嗪、环状苯胺-吡啶并三嗪、N-{[1-(苯基甲基)氮杂 -4-基]甲基}-2-吡啶-3-基乙酰胺、4-{[4-(4-{[2-(吡啶-2-基氨基)乙基]氨基}- 1,3,5-三嗪-2-基)吡啶-2-基]氧}丁-1-醇、3-({3-[4-({2-[甲基(吡啶-2-基)氨基]乙基}氨基)-1,3,5-三嗪-2-基]吡啶-2-基}氨基)丙-1-醇、N~4~-[2-(3-氟苯基)乙基]-N~2~-[3-(4-甲基哌嗪-1-基)丙基]吡啶并[2,3-d]嘧啶-2,4-二胺、1-甲基-N-[(4-吡啶-3-基-2-{[3-(三氟甲基)苯基]氨基}-1,3-噻唑-5-基)甲基]哌啶-4-羧酰胺、1,1-二甲基乙基{2-[4-({5-[3-(3-羟丙基)苯基]-4H-1,2,4-三唑-3-基}氨基)苯基]乙基}氨基甲酸酯、1,1-二甲基乙基{[3-({5-[5-(3-羟丙基)-2-(甲氧基)苯基]-1,3-唑-2-基}氨基)苯基]甲基}氨基甲酸酯、1-({5-[6-({4-[(4-甲基哌嗪-1-基)磺酰基]苯基}氨基)吡嗪-2-基]噻吩-2-基}甲基)哌啶-4-醇、1-({4-[6-({4-[(4-甲基哌嗪-1-基)磺酰基]苯基}氨基)吡嗪-2-基]噻吩-2-基}甲基)哌啶-4-羧酰胺、和2-{[4-(1-甲基乙基)苯基]氨基}-N-(2-噻吩-2-基乙基)-7,8-二氢吡啶并[4,3-d]嘧啶-6(5H)-羧酰胺。适合于使用的因子的例子可以在美国专利申请序列No.12/494,789中找到。在一个实施例中,至少一种其它因子是14-丙-2-烯-1-基-3,5,7,14,17,23,27-七氮杂四环[19.3.1.1~2,6~.1~8,12~]二十七-1(25),2(27),3,5,8(26),9,11,21,23-壬烯-16-酮。
多能干细胞群可以在缺乏血清且补充有BSA以及选自胰岛素和IGF-1的因子的培养基中培养约一天到约七天。作为另外一种选择,多能干细胞群可以在缺乏血清且补充有BSA以及选自胰岛素和IGF-1的因子的培养基中培养约一天到约六天。作为另外一种选择,多能干细胞群可以在缺乏血清且补充有BSA以及选自胰岛素和IGF-1的因子的培养基中培养约一天到约五天。作为另外一种选择,多能干细胞群可以在缺乏血清且补充有BSA以及选自胰岛素和IGF-1的因子的培养基中培养约一天到约四天。作为另外一种选择,多能干细胞群可以在缺乏血清且补充有BSA以及选自胰岛素和IGF-1的因子的培养基中培养约四天。
在一个实施例中,使用浓度为约5ng/ml至约500ng/ml的GDF-8。在一个替代实施例中,使用浓度为约5ng/ml至约50ng/ml的GDF-8。在一个替代实施例中,使用浓度为约5ng/ml至约25ng/ml的GDF-8。在一个替代实施例中,使用浓度为约25ng/ml的GDF-8。
激活素A可以约1pg/mL至约100μg/mL的浓度使用。在一个替代实施例中,该浓度可以为约1pg/ml至约1μg/ml。在一个替代实施例中,该浓度 可以为约1pg/ml至约100ng/ml。在一个替代实施例中,该浓度可以为约50ng/ml至约100ng/ml。在一个替代实施例中,该浓度可以为约100ng/ml。
Wnt配体可选自Wnt-1、Wnt-3a、Wnt-5a和Wnt-7a。在一个实施例中,Wnt配体是Wnt-1。在一个替代实施例中,Wnt配体是Wnt-3a。
Wnt配体可以约1ng/mL至约1000ng/mL的浓度使用。在一个替代实施例中,Wnt配体可以约10ng/mL至约100ng/mL的浓度使用。在一个实施例中,Wnt配体的浓度是约20ng/mL。
在一个实施例中,胰岛素以约1ng/ml至约100ng/ml的浓度使用。
在一个实施例中,IGF-1以约1ng/ml至约200ng/ml的浓度使用。
表达胰腺内胚层谱系特征性标志物的细胞的形成
在一个实施例中,通过本发明的方法形成的表达定形内胚层谱系特征性标志物的细胞群通过本领域的任何方法进一步分化成表达胰腺内胚层谱系特征性标志物的细胞群。
例如,根据D’Amour等人,Nature Biotechnology《自然生物技术》24,1392-1401(2006)中公开的方法,通过处理表达定形内胚层谱系特征性标志物的细胞群,可以使根据本发明的方法得到的表达定形内胚层谱系特征性标志物的细胞群进一步分化成表达胰腺内胚层谱系特征性标志物的细胞群。
例如,根据美国专利申请序列No.11/736,908中公开的方法,通过处理多能干细胞来使多能干细胞分化成表达定形内胚层谱系特征性标志物的细胞。
表达胰腺内分泌谱系特征性标志物的细胞的形成
在一个实施例中,可采用本领域的任何方法使表达胰腺内胚层谱系特征性标志物的细胞群进一步分化成表达胰腺内分泌谱系特征性标志物的细胞群。
例如,可以根据如下文献中公开的方法对表达胰腺内胚层谱系特征性标志物的细胞群进行处理,使表达胰腺内胚层谱系特征性标志物的细胞群进一步分化成表达胰腺内分泌谱系特征性标志物的细胞群:D’Amour等人,Nature Biotechnology《自然生物技术》,2006)。
例如,可以根据如下文献中公开的方法对表达胰腺内胚层谱系特征性标志物的细胞群进行处理,使表达胰腺内胚层谱系特征性标志物的细胞群进一步分化成表达胰腺内分泌谱系特征性标志物的细胞群:D’Amour等人,Nature Biotechnology《自然生物技术》,2006)。
例如,可以根据美国专利申请No.11/736,908中公开的方法对表达胰腺内胚层谱系特征性标志物的细胞群进行处理,使表达胰腺内胚层谱系特征性标志物的细胞群进一步分化成表达胰腺内分泌谱系特征性标志物的细胞群。
例如,可以根据美国专利申请No.11/779,311中公开的方法对表达胰腺内胚层谱系特征性标志物的细胞群进行处理,使表达胰腺内胚层谱系特征性标志物的细胞群进一步分化成表达胰腺内分泌谱系特征性标志物的细胞群。
例如,可以根据美国专利申请No.60/953,178中公开的方法对表达胰腺内胚层谱系特征性标志物的细胞群进行处理,使表达胰腺内胚层谱系特征性标志物的细胞群进一步分化成表达胰腺内分泌谱系特征性标志物的细胞群。
例如,可以根据美国专利申请No.60/990,529中公开的方法对表达胰腺内胚层谱系特征性标志物的细胞群进行处理,使表达胰腺内胚层谱系特征性标志物的细胞群进一步分化成表达胰腺内分泌谱系特征性标志物的细胞群。
本发明通过(但不限于)以下实例进一步说明。
实例
实例1
胰岛素在人多能干细胞分化成表达定形内胚层谱系特征性标志物的细胞中的作
用:簇种植
先前研究已显示高浓度的FBS对于来自胚胎干细胞的定形内胚层(DE)形成是有害的。参见例如,D’Amour等人,Nature Biotechnology《自然生物技术》,2005年,其中当FBS浓度从10%FBS减少到0.5-2%FBS时,来自人胚胎干细胞的定形内胚层诱导显著增加。报道了类似观察,其中25ng/ml IGF或200ng/ml胰岛素至2%FBS对于在MEF-CM(小鼠胚胎成纤维细胞条件培养基)中培养的ES细胞的添加使在用激活素A的处理后的SOX17表达减少约70%。参见McLean等人,Stem Cells《干细胞》25:29-38,2007。
观察到的抑制效应可能是由于FBS中胰岛素或IGF的存在,触发磷脂酰肌醇3-激酶途径。参见McLean等人,Stem Cells《干细胞》25:29-38,2007。PI-3激酶发信号途径的封闭增加在MEF-CM(小鼠胚胎成纤维细胞条件培养基)中培养的人ES细胞中的Sox17阳性细胞百分比。参见McLean等人,Stem Cells《干细胞》25:29-38,2007。
这些数据暗示预期少至25ng/ml IGF或200ng/ml胰岛素对于含有激活素A和低浓度FBS(0.5-2%FBS)的培养基的添加将阻断定形内胚层的形成。FBS中一般的IGF和胰岛素浓度分别为约70ng/ml(J.Clin.Invest.《临床研究杂志》76:4,1985)和约60ng/ml(InVitro Cell Dev Biol.《体外细胞和发育生物学》32:8-12,1996)。这转变为在2%FBS中约1.4ng/ml IGF和约1.2ng/ml胰岛素。
人胚胎干细胞系H1(p40-p52)的细胞在(1∶30稀释度)(BDBiosciences;目录号356231)包被的皿上补充有16ng/ml FGF2(目录号100-18B,新泽西州的PeproTech公司(PeproTech,NJ))的MEF-CM(小鼠胚胎成纤维细胞条件培养基)中培养,并且如下分化成表达定形内胚层谱系特征性标志物的细胞:
a.补充有2%无脂肪酸BSA(目录号68700,爱荷华州的Proliant公司(Proliant,IA))和100ng/ml激活素A(明尼苏达州的安迪生物科技公司(R&D Systems,MN))加上20ng/ml WNT-3a(目录号1324-WN-002,明尼苏达州的安迪生物科技公司)的RPMI培养基一天,随后
b.补充有2%BSA和100ng/ml激活素A的RPMI培养基另外三天。
在一些培养中,细胞用ITS-X(目录号51500-056,加利福尼亚州的英潍捷基公司)的下列稀释度处理:0、1∶106、1∶5×105、1∶105、1∶104。ITS-X是补充包含1mg/ml胰岛素、0.55mg/ml转铁蛋白、0.00067mg/ml亚硒酸钠和0.2mg/ml乙醇胺的血清替代物。ITS-X的稀释度范围对应于0、1ng/ml、2ng/ml、10ng/ml和100ng/ml胰岛素。作为对照,0.2%FBS(目录号SH30070.03,Hyclone,UT)用于第1天分化,0.5%FBS在第2天,并且2%FBS用于第3-4天。FBS处理的培养物不补充有ITS-X。
在第4天时,收集样品用于FACS和使用实时PCR的基因表达分析。令人惊讶的是,如图1中所示,1-100ng/ml胰岛素对用于分化细胞的培养基的添加不显著影响与定形内胚层相关的标志物(FOXA2、SOX17和CXCR4)、与间充质相关的标志物(T,也称为Brach)、或胚外标志物(SOX7、AFP)的表达。此外,用补充有2%BSA的培养基处理的培养物显示出比用补充有0.5-2%FBS的培养基处理的培养物显著更高的与定形内胚层有关的标志物的表达。
这些观察进一步由如对于多种处理通过FACS测定的CXCR4和CD9的表达支持。参见图2。细胞表面受体CXCR4先前已显示为定形内胚层的标志物。CD9是关于未分化的ES细胞的标志物。因此,在细胞群中CXCR4表达中的增加和CD9表达中的减少指示定形内胚层的形成。如表1中概括的,在测试的任何胰岛素浓度,在用补充有BSA的培养基处理的细胞中未观察到CXCR4或CD9表达中的显著变化。这些数据暗示在这些研究中采用的培养基中测试的浓度下,胰岛素不是抑制性的。
表I
处理 | %CXCR4+CD9- | %CXCR4-CD9+ | %CXCR4-CD9- |
FBS | 56 | 27 | 9 |
BSA | 69 | 13 | 13 |
BSA+1ng/ml胰岛素 | 70 | 13 | 10 |
BSA+5ng/ml胰岛素 | 67 | 15 | 12 |
BSA+10ng/ml胰岛素 | 69 | 13 | 13 |
BSA+100ng/ml胰岛素 | 73 | 12 | 9 |
实例2
胰岛素在人多能干细胞分化成表达定形内胚层谱系特征性标志物的细胞中的作
用:单细胞种植
人胚胎干细胞系H1(p40-p52)的细胞作为单细胞以100000细胞/cm2的密度种植到(1∶30稀释度)(BD Biosciences;目录号356231)包被的皿上补充有16ng/mlFGF2(目录号100-18B,新泽西州的PeproTech公司)和10μM Y-27632(Rho激酶抑制剂,目录号Y0503,密苏 里州的西格玛公司)的MEF-CM(小鼠胚胎成纤维细胞条件培养基)中。种植后72小时,培养物如下分化成定形内胚层(DE):
a.补充有2%无脂肪酸BSA(目录号68700,爱荷华州的Proliant公司)、0.0025g/ml碳酸氢钠(目录号S3187,密苏里州的西格玛公司)、1X GlutaMaxTM(目录号35050-079,加利福尼亚州的英潍捷基公司)和100ng/ml激活素A(明尼苏达州的安迪生物科技公司)加上20ng/ml WNT-3a(目录号1324-WN-002,明尼苏达州的安迪生物科技公司)的MCDB-131(目录号10372-019,加利福尼亚州的英潍捷基公司)培养基一天,随后
b.补充有2%BSA、碳酸氢钠、Glutamax和100ng/ml激活素A的MCDB-131培养基另外三天。
在一些培养中,细胞用下列浓度的胰岛素(目录号I9278,密苏里州的西格玛公司)处理:0、1、10、100、1000或10000ng/ml。在第4天时,收集样品用于FACS和使用实时PCR的基因表达分析。
1-100ng/ml胰岛素对于用于分化细胞的培养基的添加不显著影响与定形内胚层相关的标志物(FOXA2、SOX17、CER1和CXCR4)表达。类似地,胚胎标志物(NANOG)或胚外标志物(SOX7、AFP)的表达不受影响。参见图3。然而,1-10μg/ml胰岛素的添加不增加NANOG的表达。这些数据进一步由对于定形内胚层标志物SOX17(目录号AF1924,明尼苏达州的安迪生物科技公司)的免疫荧光(IF)染色支持(图4)。
图5描述了如通过FACS分析测量的,多种处理的CXCR4和CD9表达概况。如表II中概括的,仅在胰岛素的超生理学浓度(1-10μg/ml),存在CXCR4+CD9-细胞百分比中的减少和CXCR4-CD9+馏分的表达中的增加。这些数据暗示在这个研究中的条件下,仅胰岛素的超生理学浓度抑制定形内胚层的形成。
表II
处理 | %CXCR4+CD9- | %CXCR4-CD9+ | %CXCR4-CD9- |
BSA | 96 | 1.3 | 0.9 |
BSA+1ng/ml胰岛素 | 96 | 1.5 | 0.7 |
BSA+10ng/ml胰岛素 | 95 | 1.4 | 0.7 |
BSA+100ng/ml胰岛素 | 90 | 4.1 | 2 |
BSA+1μg/ml胰岛素 | 90 | 3.6 | 2.2 |
BSA+10μg/ml胰岛素 | 84 | 6.6 | 4.7 |
实例3
IGF在人多能干细胞分化成表达定形内胚层谱系特征性标志物的细胞中的作用:
单细胞种植
人胚胎干细胞系H1(p40-p52)的细胞作为单细胞以100000细胞/cm2的密度种植到(1∶30稀释度)(BD Biosciences;目录号356231)包被的皿上补充有16ng/mlFGF2(目录号100-18B,新泽西州的PeproTech公司)和10μM Y-27632(Rho激酶抑制剂,目录号Y0503,密苏里州的西格玛公司)的MEF-CM(小鼠胚胎成纤维细胞条件培养基)中。种植后72小时,培养物如下分化成定形内胚层(DE):
a.补充有2%无脂肪酸BSA(目录号68700,爱荷华州的Proliant公司)、0.0025g/ml碳酸氢钠(目录号S3187,密苏里州的西格玛公司)、1X GlutaMaxTM(目录号35050-079,加利福尼亚州的英潍捷基公司)和100ng/ml GDF-8(目录号120-00,明尼苏达州的安迪生物科技公司)加上2.5μM GSK3B抑制剂14-丙-2-烯-1-基-3,5,7,14,17,23,27-七氮杂四环[19.3.1.1~2,6~.1~8,12~]二十七-1(25),2(27),3,5,8(26),9,11,21,23-壬烯-16-酮的MCDB-131(目录号10372-019,加利福尼亚州的英潍捷基公司)培养基一天,随后
b.补充有2%BSA、碳酸氢钠、Glutamax和100ng/ml GDF-8的MCDB-131培养基另外三天。
在一些培养中,细胞用下列浓度的IGF(目录号AF100,新泽西州的PeproTech公司)处理:0、1、10、50或200ng/ml。作为对照,代替BSA, 0.2%FBS(目录号SH30070.03,Hyclone,UT)用于第1天分化,并且2%FBS用于第2-4天。一些FBS处理的培养物也用多种浓度的IGF处理。
在第4天时,收集样品用于FACS和使用实时PCR的基因表达分析。
令人惊讶的是,当与不用IGF处理的对照样品相比较时,1-200ng/mlIGF对于BSA处理的培养物的添加不显著影响与定形内胚层相关的标志物(FOXA2、SOX17、CER1和CXCR4)的表达(图6)。对于胚外标志物(SOX7、AFP)观察到相似结果。50-200ng/ml IGF的添加不增加胚胎标志物NANOG的表达。
用补充有FBS的培养基处理的培养物对IGF的抑制效应敏感得多。在这些培养物中,SOX17、HNF3B和CXCR4的表达随着IGF的浓度增加而减少。这些观察进一步由对于DE标志物SOX17(目录号AF1924,明尼苏达州的安迪生物科技公司)的免疫荧光(IF)染色支持(图8-9)。
如表III中概括的,仅在BSA处理的培养物中IGF的超生理学浓度(50-200ng/ml),存在CXCR4+CD9-细胞的表达中的下降和CXCR4-CD9+馏分的表达中的增加。然而,随着IGF的剂量增加,与BSA处理的培养物相比较,FBS处理的培养物显示出CXCr4+CD9-馏分的表达中的更显著下降。上文实例共同显示在不存在FBS的情况下,生理学浓度的IGF或胰岛素对于DE标志物的诱导不是抑制性的。
表III
处理 | %CXCR4+CD9- | %CXCR4-CD9+ | %CXCR4-CD9- |
BSA | 92 | 0.9 | 2.6 |
FBS | 93 | 2.7 | 5.9 |
BSA+1ng/ml IGF | 92 | 0.8 | 3 |
FBS+1ng/ml IGF | 89 | 3 | 3.6 |
BSA+10ng/ml IGF | 90 | 1.3 | 5.3 |
FBS+10ng/ml IGF | 87 | 6.4 | 3.3 |
BSA+50ng/ml IGF | 87 | 6 | 3.5 |
FBS+50ng/ml IGF | 78 | 6.8 | 13.2 |
BSA+200ng/ml IGF | 79 | 10 | 8 |
FBS+200ng/ml IGF | 70 | 13.4 | 12.9 |
实例4
在多个FBS批次中的IGF浓度
IGF-1试剂盒购自Diagnostic Systems Laboratories(DSL)(目录DSL-10-2800)并且由于检测。将二十微升(20μl)血清(一式两份)预处理,并且随后将20μl稀释的样品用于测定。对于培养基样品,20μl样品直接用于测定。测定遵循由试剂盒提供的说明书执行。这个试剂盒可以检测人和牛IGF-1,因为用于该试剂盒的2种单克隆抗体针对同系物肽序列。
测试样品:使用下列测试样品:
4A/5A:Hyclone新生小牛血清;批次AKM12868
4B/5B:NIH-FBS(来自等分试样-20C)
4C/5C:Hyclone FBS,批次:ATK33398
4D/5D:Hyclone FBS,批次:AUK54924
4E/5E:人血清,批次:A70184,来自Valley Biomedical Inc.
4F/5F:敲除血清;英潍捷基公司;批次:557914
4F/5F:F12DMEM,英潍捷基公司;批次:692281
4H/5H:MEF条件培养基,批次:011410(第5天)
对照样品:使用下列对照样品:
本底:“0”IGF-1(阴性对照,来自试剂盒);上文列出的F12样品也充当阴性对照。
2个阳性对照(127ng/ml和241ng/ml;来自试剂盒)。
结果:测定关于血清的灵敏度10ng/ml;并且关于培养基的灵敏度大于0.1ng/ml。
实例5
胰岛素/IGF和FBS在人多能干细胞分化成表达定形内胚层谱系特征性标志物的细
胞中的作用:单细胞种植
人胚胎干细胞系H1(p40-p52)的细胞作为单细胞以100000细胞/cm2的密度种植到(1∶30稀释度)(BD Biosciences;目录号356231)包被的皿上补充有16ng/mlFGF2(目录号100-18B,新泽西州的PeproTech公司)和10μM Y-27632(Rho激酶抑制剂,目录号Y0503,密苏里州的西格玛公司)的MEF-CM(小鼠胚胎成纤维细胞条件培养基)中。种植后72小时,培养物如下分化成定形内胚层(DE):
a.其中补充有0.2%FBS(目录号SH30070.03,Hyclone,UT)、0.0025g/ml碳酸氢钠(目录号S3187,密苏里州的西格玛公司)、1X GlutaMaxTM(目录号35050-079,加利福尼亚州的英潍捷基公司)和100ng/ml GDF-8(目录号120-00,明尼苏达州的安迪生物科技公司)加上2.5μM GSK3B抑制剂14-丙-2-烯-1-基-3,5,7,14,17,23,27-七氮杂四环[19.3.1.1~2,6~.1~8,12~]二十七-1(25),2(27),3,5,8(26),9,11,21,23-壬烯-16-酮的MCDB-131(目录号10372-019,加利福尼亚州的英潍捷基公司)培养基一天,随后
b.补充有FBS、碳酸氢钠、Glutamax和100ng/ml GDF8的MCDB-131培养基另外三天。
0.5%FBS用于第2天,并且2%FBS用于第3-4天。除了常规FBS外,还测试了热处理的FBS(目录号F4135,密苏里州的西格玛公司)和炭 吸附处理的FBS(目录号F6765,密苏里州的西格玛公司)。一些FBS处理的培养物也用多种浓度的IGF(10-100ng/ml)或胰岛素(10-100ng/ml)进行处理。
在第4天时,收集样品用于FACS和实时PCR。与在FBS的存在下的BSA处理的培养物(参见先前实例)形成对比,胰岛素或IGF剂量的添加独立地下调与定形内胚层相关的标志物例如SOX17和CXCR4。参见图9。多能性标志物NANOG的表达也是上调的。
在这个文档自始至终引用的出版物在此全文以引用方式并入。尽管上文已结合实例和优选实施例描述了本发明的各个方面,但应当理解本发明的范围不受上述具体实施方式的限定,而受以下在专利法原则下恰当理解的权利要求书的限定。
Claims (3)
1.一种生成细胞群体的方法,其中所述群体中大于85%的细胞表达定形内胚层谱系特征性标志物,所述方法包括以下步骤:
使所述多能干细胞群在缺乏血清且补充有:(i) BSA和选自具有1-100 ng/ml的浓度的胰岛素和具有1-50 ng/ml的浓度的IGF-1的因子;以及任选(ii)激活素A和Wnt配体或(iii)GDF-8和14-丙-2-烯-1-基-3,5,7,14,17,23,27-七氮杂四环[19.3.1.1~2,6~.1~8,12~]二十七-1(25),2(27),3,5,8(26),9,11,21,23-壬烯-16-酮的培养基中分化成细胞群体,其中所述群体中大于85%的细胞表达定形内胚层谱系特征性标志物。
2.根据权利要求1所述的方法,其中使所述多能干细胞群在缺乏血清且补充有BSA以及选自胰岛素和IGF-1的因子的培养基中分化至少6天的时间。
3.根据权利要求1所述的方法,其中使所述多能干细胞群在缺乏血清且补充有BSA以及选自胰岛素和IGF-1的因子的培养基中分化至少7天的时间。
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