CN1416345A - 灵长类动物胚胎干细胞的无血清培养物 - Google Patents

灵长类动物胚胎干细胞的无血清培养物 Download PDF

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CN1416345A
CN1416345A CN01806235A CN01806235A CN1416345A CN 1416345 A CN1416345 A CN 1416345A CN 01806235 A CN01806235 A CN 01806235A CN 01806235 A CN01806235 A CN 01806235A CN 1416345 A CN1416345 A CN 1416345A
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Abstract

本文公开了培养灵长类动物胚胎干细胞的方法。在长时间和稳定的基础上，在外源提供的成纤维细胞生长因子的存在下和缺乏动物血清的条件下培养这些细胞。较佳的是，培养基中还存在成纤维细胞饲养层。本发明还公开了含有成纤维细胞饲养层和成纤维细胞生长因子的培养基。

Description

灵长类动物胚胎干细胞的无血清培养物
                       相关申请的交叉参考不适用。
                   关于联邦政府资助的研究的申明
                           发明的背景
本发明涉及培养灵长类动物胚胎干细胞培养物的方法和培养中使用到的培养基。
已从植入前胚胎获得灵长类动物(如猴和人)多能胚胎干细胞。参见美国专利第5,843,780号；和J.Thomson等人，282Science，1145-1147(1998)。这些出版物和所有其它本文涉及的出版物的公开内容都被完整地纳入本文作为参考。尽管经过长时间的培养，但这些细胞仍稳定地维持着发育成为所有三种胚胎性胚层的高级衍生物的潜力。
已在人发育生物学、药物开发、药物测试和移植医学中广泛使用灵长类动物(具体是人)的胚胎干细胞系。例如，目前关于植入后人胚胎的知识在很大程度上以有限量的静态组织切片为基础。考虑到伦理方面的因素，人们基本上仍未对控制人胚胎早期的发育选择的基础机制进行研究。
虽然小鼠是实验哺乳动物发育生物学的主要研究对象，并且小鼠和人之间许多控制发育的基本机制是保守的，但是在小鼠和人的早期发育中仍存在明显的差异。因此，灵长类动物/人ES细胞应可提供关于它们的分化和功能的新的重要视点。
可使用灵长类动物ES细胞的分化衍生物来鉴别新药的基因靶标、测试新化合物的毒性或致畸性以及用于移植以替换疾病细胞群。可通过移植ES细胞衍生的细胞来进行治疗的潜在病症包括帕金森综合症、心肌梗塞、青少年发作的糖尿病和白血病。例如可参见J.Rossant等人，17Nature Biotechnology，23-4(1999)和J.Gearhart，282Science，1061-2(1998)。
关于灵长类动物的胚胎干细胞培养物的利用，其关键因素是长期的增殖能力、长时间培养后的发育潜力和核型稳定性。这些细胞的培养物(尤其是培养在成纤维细胞饲养层上的培养物)一般补加动物血清(尤其是胎牛血清)，以便在这样的培养期间产生所需的增殖。
例如，在美国专利第5,453,357、5,670,372和5,690,296中描述了各种培养条件，包括一些同时使用一类碱性成纤维细胞生长因子和动物血清的条件。不幸的是，不同批次的血清倾向于具有不同的特性，从而影响了培养特征。
在WO98/30679中讨论了使用无血清添加剂类替换动物血清，以支持培养中某些胚胎干细胞的生长。该血清替代物包括清蛋白或清蛋白替代品、一种或多种氨基酸、一种或多种维生素、一种或多种运铁蛋白或运铁蛋白的替代品、一种或多种抗氧化剂、一种或多种胰岛素或胰岛素替代品、一种或多种胶原蛋白前体以及一种或多种痕量元素。应注意的是，还可在这种替代物中添加白血病抑制因子、青灰因子或睫状神经营养因子。不幸的是，在关于灵长类动物的胚胎干细胞培养物(尤其是生长在成纤维细胞饲养层上的那些培养物)的内容中，这些培养基并未证明是让人满意的。
在关于营养血清培养基(如胎牛血清)的内容中，WO99/20741讨论了在培养灵长类动物的干细胞中使用各种生长因子(如bFGF)的益处。但是，文中并未描述到不含有营养血清的培养基。
美国专利第5,405,772号描述了用于造血细胞和骨髓基质细胞的生长培养基。为了达到目的，文中建议在无血清的培养基中使用成纤维细胞生长因子。但是，文中并未描述灵长类动物胚胎干细胞的生长条件。
由此可见，还需要不使用动物血清即可稳定地培养灵长类胚胎干细胞的技术。
                            发明概要
一方面，本发明提供一种培养灵长类动物的胚胎干细胞的方法。在基本上没有哺乳动物胎儿血清(较佳还基本上没有任何动物血清)的培养基中、在成纤维细胞生长因子的存在下培养干细胞，其中，所述生长因子的来源不仅仅是成纤维细胞饲养层。在较佳的实施方式中，该培养基还具有成纤维细胞饲养层。
成纤维细胞生长因子是哺乳动物发育的主要分子。目前有9种已知的成纤维细胞生长因子配体和4种针对它们的信号传导的成纤维细胞生长因子受体(以及它们的剪接变体)。综述可参见D.Omitz等人，25J.Biol.Chem.，15292-7(1996)；美国专利第5,453,357号。在不同物种间这些因子会存在细微的差别，因此术语成纤维细胞生长因子不局限于物种。但是，我优选使用人的成纤维细胞生长因子，更优选使用由重组基因产生的人的碱性成纤维细胞生长因子。易于从Gibco BRL-Life Techologies和其它公司获得大量的这种化合物。
应注意的是，为了本专利的目的，所述培养基还需要基本上不含有特定的血清，即使已经从血清中分离出分散的成分(如牛血清清蛋白)并在随后以外来的方式补充。问题在于，当加入血清本身时，差异性问题就会产生。但是，当加入血清的一种或多种已鉴别清楚的纯化成分时，它们就产生差异性的问题。
较佳的是，采用本发明方法培养的灵长类动物胚胎干细胞是人的胚胎干细胞，它们是真正的ES细胞系，因为它们能(i)以未分化的状态在体外无限增殖；(ii)即使在长时间培养后仍可分化成所有3个胚胎性胚层(内胚层、中胚层和外胚层)的衍生物；(iii)在整个长时间的培养过程中维持正常核型。因此，它们是多能的。
上述培养使所述胚胎干细胞可在培养基中稳定地增殖1个月以上(较佳6个月以上，更佳12个月以上)，而同时使所述干细胞维持分化成内胚层、中胚层和外胚层组织的衍生物的潜力，以及维持它们的核型。
另一方面，本发明提供培养灵长类动物胚胎干细胞的另一种方法。在基本上不含有哺乳动物胎儿血清(较佳还基本上不含有任何动物的血清)的培养基中、在能激活成纤维细胞生长因子信号受体的生长因子的存在下培养干细胞，其中，提供所述生长因子的来源不仅仅是成纤维细胞饲养层。虽然所述生长因子较佳是成纤维细胞生长因子，但是它也可以是其它物质，如设计用来激活成纤维细胞生长因子受体的某些合成的小肽(如由重组DNA变体或突变体产生的肽)。综述可参见T.Yamaguchi等人，152Dev.Biol.，75-88(1992)(信号受体)。
又一方面，本发明提供用于培养灵长类动物胚胎干细胞的培养系统。该系统具有成纤维细胞饲养层和人碱性成纤维细胞生长因子，该生长因子的来源不仅仅是该成纤维细胞饲养层。该培养系统基本上不含有动物血清。
又一方面，本发明提供采用上述方法衍生获得的细胞系(较佳是克隆的细胞系)。“衍生”在此使用其广义含义，覆盖了直接或间接衍生的细胞系。
因而，本发明避免了由于动物血清批次差异而产生的差异性。此外，已发现在避免使用动物血清的同时，使用成纤维细胞生长因子可增加克隆的效率。
因此，本发明的一个优点是提供用于灵长类动物胚胎干细胞系的培养条件，其中，所述条件变化少，并使得克隆更有效率。在研究了本说明书和权利要求书后，本发明的其它优点将是显而易见的。
                      优选实施方式的详细描述
在下述实验中，我使用了本发明的方法和培养系统来培养人ES细胞系。两个克隆衍生的人ES细胞系在克隆产生后增殖了8个月以上，并且维持了分化成所有3个胚胎性胚层的高级衍生物的能力。
人ES细胞系H9的初始衍生、培养和表征的技术在J.Thomson等人〔282Science，1145-1147(1998)〕的文章中有所描述。在我的实验中，在完成上述操作后，在经辐射的(35戈瑞的γ辐射过的)小鼠胚胎成纤维细胞上平板培养人ES细胞。此步骤中使用的培养基由80％“KnockOut”Dulbeco改进的Eagle培养基(DMEM)(Gibco BRL，Rockville，MD)、1mM L-谷氨酰胺、0.1mMβ-巯基乙醇和1％非必需氨基酸原液(Gibco BRL Rockville，MD)组成，补充了20％胎牛血清(HyCloneLogan，UT)或20％KnockOut SR(最初优化用于小鼠ES细胞的无血清替代品，Gibco BRL，Rockville，MD)。KnockOut SR的成分是WO98/30679中描述的用于血清替代品的成分。
在另一实验中，培养基中补加血清或者前述血清替代品KnockOut SR，还可含有或不含人重组碱性成纤维细胞生长因子(bFGF，4ng/ml)。培养基中bFGF的浓度范围较佳是0.1ng/ml到500ng/ml之间。
为了测定在不同培养条件下的克隆率，使用0.05％胰蛋白酶/0.25％EDTA，处理7分钟，将H-9培养物解离成单个细胞，然后离心洗涤，之后放到有丝分裂失活的小鼠胚胎成纤维细胞上培养(105个ES细胞/孔，6孔平板)。为了证实单个细胞的生长是由克隆的ES细胞系衍生的，在立体显微镜的直接观察下挑选出各个细胞，然后使用微移液管将其转移到含有小鼠成纤维细胞饲养层以及含有20％血清替代品和4ng/ml bFGF的培养基的96孔平板的各个孔中。
通过每5-7天使用1mg/ml的胶原蛋白酶IV型(Gibco BRL，Rockville，MD)，进行常规的传代培养，使这些克隆得到增殖。衍生6个月后，使用标准的G-显带技术(分析20条染色体的伸展情况)测得H9细胞具有正常的XX核型。但是，衍生后7个月，在单个核型制品中，20条染色体伸展中有16条具有正常的XX核型，而有4条伸展显示出随机的异常性，其中包括1条具有向染色体13短臂的移位，1条具有反向的染色体20，1条具有向染色体4的短臂上的移位，1条具有多个碎片。结果，在衍生8、10和12.75个月后，H9细胞在所检测的所有20条染色体伸展中具有正常的核型。
我们观察到，在先前所述的包括动物血清的培养条件下人ES细胞的克隆率差(不论存在bFGF与否)。我们还观察到，在缺乏动物血清时，克隆率增加，并且存在bFGF时效率增加得更高。
下面的数据是由平板培养的105个单独的ES细胞产生的集落总数+/-平均值的标准误差(集落克隆率％)。含有20％胎儿血清但没有bFGF的培养结果是240±28个。含有20％血清并含有bFGF的培养结果大约相同，为260±12个。缺乏血清(存在20％血清替代品)也没有bFGF所得的结果是633±43个，但含有bFGF的结果是826±61个。因此，血清对克隆率产生不利的影响，存在bFGF而不含有血清的条件对克隆率产生了协同的利益。
在存在血清的条件下长时间培养人ES细胞不需要从外源提供bFGF，并且向含有血清的培养基中加入bFGF也没有明显地提高人ES细胞克隆率(如上所述)。但是，在没有血清的培养基中，bFGF提高了人ES细胞的初始克隆率。
此外，我还发现，对于灵长类动物胚胎干细胞在无动物血清的条件下的连续的未分化繁殖来说，提供外源bFGF是非常重要的。在缺乏bFGF的无血清培养基中，在培养两周后，人ES细胞一律进行分化。(在缺乏bFGF的条件下)其它因子(如LIF)的加入并未能阻止这种分化。
所察觉到的结果特别适用于克隆系。在这方面，在直接的显微镜观察的帮助下，通过将细胞单独放到96孔平板的孔中，可筛选出用于扩增的克隆。在放到96孔平板的孔中培养的192个H-9细胞中，两个克隆成功地扩增(H-9.1和H-9.2)。接着在补充了血清替代品和bFGF的培养基中连续培养这两个克隆。
即使在克隆后连续培养了8个月以上，H9.1和H9.2细胞仍都维持了正常的XX核型。所述H9.1和H9.2克隆即使在无血清培养基中长时间培养后，仍维持形成所有三个胚胎性胚层的衍生物的潜力。在培养6个月后，H9.1和H9.2克隆被证实具有正常的核型，然后用它们给SCID-米色小鼠注射。
H9.1和H9.2细胞都形成了含有所有三个胚胎性胚层的衍生物的畸胎瘤，包括肠上皮(内胚层)，胚肾、横纹肌、平滑肌、骨、软骨(中胚层)和神经组织(外胚层)。H9.1和H9.2细胞的高传代细胞的畸胎瘤中所观察到的分化范围，与低传代的H9亲代细胞形成的畸胎瘤内的分化范围是相当的。
从上面所述内容应可理解，虽然动物血清对生长起支持性作用，但是它是一种复杂的混合物，它可同时含有对人ES细胞培养有利或有害的化合物。而且，不同批次的血清支持人ES细胞有力地以未分化状态增殖的能力极大地不同。使用清楚确定的成分替换血清的做法减少了与这种血清批次的变化有关的结果的差异性，并能进行更精细的分化研究。
此外，含有血清的培养基中的较低的克隆率表明，在常规使用的血清中存在对干细胞生存不利的化合物，尤其是当细胞被分散成单个细胞时。因此非常需要避免使用这些化合物。
上文已结合优选实施方式对本发明进行了描述。这一原理的其它形式也打算包括在权利要求所述的范围内。例如，虽然在上述实验中使用了重组产生的人碱性成纤维细胞生长因子，但是天然分离获得的成纤维细胞生长因子也适合使用。此外，这些技术也应证明可适用于猴和其它灵长类动物的细胞培养物。
因此，为判断本发明的整个范围，应查阅权利要求书。
                           工业应用性
本发明提供用于培养灵长类动物胚胎干细胞的方法，和培养过程中使用到的培养基。

Claims (16)

1.一种培养灵长类动物胚胎干细胞的方法，它包括：
在基本上不含哺乳动物胎儿血清的培养基中、在成纤维细胞生长因子的存在下培养所述干细胞，其中，所述生长因子的来源不仅仅是成纤维细胞饲养层。
2.如权利要求1所述的方法，其特征在于，所述培养基基本上不含有任何动物血清。
3.如权利要求2所述的方法，其特征在于，所述培养基还含有成纤维细胞饲养层。
4.如权利要求2所述的方法，其特征在于，所述成纤维细胞生长因子是碱性成纤维细胞生长因子。
5.如权利要求4所述的方法，其特征在于，所述成纤维细胞生长因子是已由重组基因产生的人碱性成纤维细胞生长因子。
6.如权利要求2所述的方法，其特征在于，所述灵长类动物胚胎干细胞是人的胚胎干细胞。
7.如权利要求2所述的方法，其特征在于，所述培养步骤包括，使所述胚胎干细胞在培养基中增殖1个月以上，同时使其维持分化成内胚层、中胚层和外胚层组织的衍生物的潜力以及维持干细胞的核型。
8.如权利要求2所述的方法，其特征在于，在所述方法的至少一部分中，所述人碱性成纤维细胞生长因子的浓度至少为0.1ng/ml。
9.一种培养灵长类动物胚胎干细胞的方法，它包括：
在基本上不含有哺乳动物胎儿血清的培养基中、在能激活成纤维细胞生长因子信号受体的生长因子的存在下培养干细胞，其中，所述能激活信号受体的生长因子的来源不仅仅是成纤维细胞饲养层。
10.如权利要求9所述的方法，其特征在于，所述培养基基本上不含任何动物血清。
11.如权利要求10所述的方法，其特征在于，所述培养基还含有成纤维细胞饲养层。
12.如权利要求10所述的方法，其特征在于，所述灵长类动物的胚胎干细胞是人的胚胎干细胞。
13.如权利要求10所述的方法，其特征在于，所述培养步骤包括，使所述胚胎干细胞在培养基中增殖1个月以上，同时使其维持分化成内胚层、中胚层和外胚层组织的衍生物的潜力以及维持干细胞的核型。
14.一种用于培养灵长类动物胚胎干细胞的培养系统，它含有：
成纤维细胞饲养层；和
成纤维细胞生长因子，它的来源不仅仅是上述成纤维细胞层；
其中，所述培养系统基本上不含动物血清。
15.采用权利要求1所述的方法衍生获得的细胞系。
16.采用权利要求9所述的方法衍生获得的细胞系。
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