CN110709092B - 用于产生人少突胶质细胞和研究体外髓鞘形成的个性化3d神经培养*** - Google Patents
用于产生人少突胶质细胞和研究体外髓鞘形成的个性化3d神经培养*** Download PDFInfo
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Abstract
使人多能干细胞在体外分化为包含少突胶质细胞的少突球状体,以用于分析、筛选程序等。
Description
联邦政府资助的研发
本发明是根据由美国国立卫生研究院授予的授权号MH107800在美国政府支持下完成的。政府享有本发明的某些权利。
背景技术
理解人中枢神经***的复杂发育以及阐明患者的神经和精神障碍的机制的进展极大地受限于对功能性人脑组织的限制接触。尽管对啮齿动物和其他哺乳动物的研究为神经发育的基本原理提供了重要的见解,但是我们对负责灵长类动物前脑大规模扩张的细胞和分子过程知之甚少,对其许多人类特定的特征也知之甚少。近年来,随着细胞重编程的引入,本领域实现了范式转变,所述细胞重编程是在此期间终末分化的体细胞可以转化为多能干细胞(称为人诱导多能干细胞(hiPSC))的过程。这些hiPSC可以从任何个体产生,并且重要的是,可以定向用于在体外分化为所有胚层衍生物,包括神经细胞。
尽管产生hiPSC的方法和效率跨实验室已经得到了显著改善和标准化,但是用于衍生特定神经和神经胶质细胞类型的方法仍具有挑战性。在过去的十年里,单层多能干细胞的神经特化和分化方案的改善导致了多种细胞类型的产生。然而,二维(2D)方法不太可能重演正在发育的三维(3D)神经***的细胞结构或体内神经网络和回路的复杂性和功能性。此外,这些方法费力且昂贵,效率有限并且产生相对不成熟的神经元。
少突胶质细胞是中枢神经***中的在脑功能中起关键作用的神经胶质细胞。少突胶质细胞扩展了用绝缘层包裹神经元轴突的过程,从而允许神经元之间的信号传输更加迅速。少突胶质细胞还通过向神经元提供营养因子和通过缓冲细胞外环境来发挥支持作用。髓鞘形成的丧失可能导致神经元死亡和神经功能受损。因此,许多人类疾病与髓鞘形成的丧失或减少有关,如多发性硬化症或消失的脑白质病。
对健康和疾病时髓鞘形成的研究受限于适当模型的可得性。啮齿动物通常用作研究人类生物学各个方面的替代物;然而,人脑的髓鞘形成比啮齿动物的脑广泛得多。另外,来自人类患者的健康脑样品的有限可得性排除了广泛研究人脑中髓鞘形成的可能性。近年来,已经尝试从人胚胎和诱导多能干细胞(hESC、hiPSC)产生少突胶质祖细胞(OPC)和少突胶质细胞,用于研究体外髓鞘形成。
hESC和hiPSC衍生的髓鞘形成模型不仅对于研究正常和疾病条件下少突胶质细胞发育和髓鞘形成的过程至关重要,而且还可以用于药物筛选。这些模型既可以用于筛选校正与某些疾病状态相关的特定异常的化合物,又可以在人体暴露之前测试新的治疗性化合物和化学物质的毒性。特别地,在神经毒性领域,对于人类细胞仍然缺乏能够评估神经元或神经胶质功能受损的测定。
因此,研发重演高度功能性人体组织(包括神经元的髓鞘形成)的体外筛选平台具有极为重要的意义。
出版物。将灵长类动物分化的体细胞重编程为多能状态的方法包括分化的体细胞核转移、分化的体细胞与多能干细胞融合以及直接重编程以产生诱导多能干细胞(iPS细胞)(Takahashi K等人(2007)Cell 131:861-872;Park IH等人(2008)Nature 451:141-146;Yu J等人(2007)Science 318:1917-1920;Kim D等人(2009)Cell Stem Cell 4:472-476;Soldner F等人(2009)Cell.136:964-977;Huangfu D等人(2008)NatureBiotechnology 26:1269-1275;Li W等人(2009)Cell Stem Cell 4:16-19)。
发明内容
提供了用于体外产生人OPC和髓鞘性少突胶质细胞的组合物和方法,所述人OPC和髓鞘性少突胶质细胞可以与中枢神经***神经元一起在三维***中培养。本发明的一个特征是能够从患者样品产生OPC和少突胶质细胞,从而允许为所述细胞疾病相关地产生和筛选治疗性药物和治疗方案。所述方法利用诱导人多能干细胞(hiPSC),其可以获自患者或载体细胞样品,例如脂肪细胞、角质形成细胞、成纤维细胞等。诱导hiPSC在体外发展外胚层命运,随后分化为含有少突胶质细胞的球状体-人少突球状体(hOS)以及神经祖细胞、星形胶质细胞和神经元。所述细胞群体可以分离自hOS,或者完整的hOS可以用作使细胞群体相互作用的模型。hOS和从其衍生的细胞可以用于移植、用于实验评估、用作谱系和细胞特异性产物的来源等。在一些实施方案中,所述细胞培养物不含饲养层且不含异种。
在本发明的一些实施方案中,提供了纯化的人OPC或少突胶质细胞(包括但不限于疾病相关少突胶质细胞)的群体,其中所述细胞分化自诱导人多能干细胞(hiPSC)。在一些实施方案中,提供了一组这样的体外衍生的细胞,其中所述组包括两种或更多种遗传上不同的细胞。在一些实施方案中,提供了一组这样的细胞,其中所述细胞可以经受多种候选试剂或多个剂量的候选试剂。候选试剂包括小分子(即药物)、增加或减少目的RNA表达的遗传构建体、电性变化等。在一些实施方案中,组(panel)是指利用来自两个或更多个不同条件,并且可以是三个或更多个、四个或更多个、五个或更多个、六个或更多个、七个或更多个遗传上不同的条件的患者特异性细胞的***或方法。
在本发明的一些实施方案中,提供了用于确定候选试剂对来自hOS的少突胶质细胞的活性的方法,所述方法包括使所述候选试剂与一个或一组从诱导人多能干细胞(hiPSC)分化的纯化的神经元、星形胶质细胞、OPS或少突胶质细胞群体接触。所述细胞群体任选地包含至少一个等位基因,其编码与髓鞘性或脱髓鞘性疾病或少突胶质细胞发育障碍相关或者引起髓鞘性或脱髓鞘性疾病或少突胶质细胞发育障碍的突变;并且确定所述试剂对形态、遗传或功能参数(包括但不限于基因表达谱分析)的影响。所述筛选方法可以与免疫效应细胞组合以确定少突胶质细胞的这样的免疫细胞的活性和髓鞘形成,或者hOS中的少突胶质细胞、星形胶质细胞与来自患者的自体免疫细胞之间的相互作用。单细胞水平的分析方法特别令人感兴趣,例如髓鞘形成测定、单细胞基因表达、少突胶质细胞对神经元或星形胶质细胞的作用以及神经元细胞信号传递等。候选试剂包括免疫效应细胞(例如T细胞、小神经胶质细胞、巨噬细胞、NK细胞等)以及免疫效应蛋白(例如IFN-γ、TGF-β、细胞因子、干扰素等),特别是怀疑参与炎性脱髓鞘性疾病的这样的细胞和蛋白质。
本发明的方法利用在少突胶质细胞的分化和髓鞘的形成中神经细胞与祖细胞之间的天然相互作用。在一些实施方案中,从hiPSC向少突胶质细胞的分化是在基本上无血清的培养基中进行的。
在hOS中分化之后,可以出于各种目的分离单独的目的细胞类型,包括但不限于少突胶质细胞。在适当的发育阶段收获所述细胞,这可以基于所需细胞类型的标志物表达和表型特征来确定。可以通过针对目的标志物的存在进行免疫染色或基因表达、通过形态学确定等凭经验对培养物进行测试。任选地在阳性选择步骤之前或之后通过药物选择、淘选、密度梯度离心等富集所述细胞。在另一个实施方案中,进行阴性选择,其中所述选择是基于在人ES细胞、成纤维细胞、神经细胞、上皮细胞等上发现的一种或多种标志物的表达。选择可以利用淘选方法、磁性粒子选择、粒子分选器选择等。
各种体细胞可用作hiPSC的来源;特别令人感兴趣的是脂肪衍生的干细胞、成纤维细胞、角质形成细胞、外周血细胞等。特别令人感兴趣的是使用来自不同基因型(特别是潜在地与神经和精神障碍相关的基因型)的个体的hiPSC。使hiPSC解离为单细胞,聚集成特定细胞数的球状体,然后悬浮生长;然后通过抑制BMP和TGFβ途径向神经命运诱导。然后将球状体在存在FGF2和EGF的情况下移至培养基中,并且用Wnt途径抑制剂或视黄酸以及音猬因子(sonic hedgehog)途径的激活剂进行图案化。为了促进分化,将球状体更换到包含PDGF-AA、IGF-1、HGF、胰岛素、BDNF、NT3、cAMP、T3和生物素的培养基中。这样培养之后,可以在不存在生长因子的情况下将球状体在含有胰岛素、抗坏血酸、cAMP、T3和生物素的神经培养基中维持延长的时间段,例如维持1、2、3、4、5、6、7、8、9、10、11、12个月或更长时间。
在阅读下面更全面描述的主题方法和组合物的细节后,本发明的这些和其他目的、优点和特征对于本领域技术人员将变得清楚。
附图说明
当结合附图阅读时,根据以下具体实施方式可以最好地理解本发明。要强调的是,根据惯例,附图的各个特征不是按比例的。相反,为了清楚起见,各个特征的尺寸被任意扩大或缩小。附图中包括以下附图。
图1a至图1f.图1a在3D人iPSC衍生的寡球状体(hOS)中产生的少突胶质祖细胞和成熟少突胶质细胞。NKX2.2/OLIG2+少突胶质祖细胞存在于51日龄的hOS的固定切片中。图1b在100日龄的hOS的固定切片中的O4+和O1+少突胶质细胞的实例。图1c展示了MBP+成熟少突胶质细胞在115日龄的hOS的固定切片中的分布的免疫荧光标记。图1d在115日龄的hOS的固定切片中物理地相互作用的MBP+少突胶质细胞过程和GFAP+星形胶质细胞过程的实例。图1e在115日龄的hOS的固定切片中包裹神经丝+轴突的MBP+少突胶质细胞过程的实例。图1f在人iPSC衍生的hOS中髓鞘形成的实例。在第100天体外拍摄有髓轴突的透射电子显微术图像。
具体实施方式
在描述本发明的组合物和方法之前,应理解本发明不限于所描述的特定组合物和方法,因为它们当然可以变化。还应理解本文使用的术语仅是为了描述具体实施方案的目的,而并不旨在是限制性的,因为本发明的范围将仅由所附权利要求限定。
在提供值的范围的情况下,应理解除非上下文另外清晰地规定,还具体公开了该范围的上限与下限之间的每个中间值(直到下限单位的十分之一)。在规定范围内的任何规定值或中间值与该规定范围内的任何其他规定值或中间值之间的每个较小范围都包括在本发明内。这些较小范围的上限和下限可以独立地被包括在所述范围内或被排除在所述范围外,并且其中任一个、没有或两个限值被包括在较小范围内的每个范围也都被包括在本发明内,但以规定范围内的任何明确排除的限值为准。在规定范围包括一个或两个限值的情况下,排除那些被包括的限值的任一个或两个的范围也被包括在本发明内。
除非另外定义,本文所用的所有技术术语和科学术语具有与本发明所属领域的普通技术人员通常理解的含义相同的含义。虽然类似于或等同于本文描述的那些的任何方法和材料也可以用于本发明的实践或测试中,现在将描述一些潜在且优选的方法和材料。本文提及的所有出版物都通过引用并入本文,以公开和描述与引用的出版物有关的方法和/或材料。应理解在存在矛盾的范围内,本公开取代了并入的出版物的任何公开。
必须注意的是除非上下文另外清晰地规定,如本文和所附权利要求所用,单数形式“一个/一种(a、an)”和“所述(the)”包括复数指代物。因此,例如,提及“一种重编程因子多肽”包括多种这样的多肽,并且提及“诱导多能干细胞”包括提及一种或多种诱导多能干细胞和本领域技术人员已知的其等同物,等等。
仅在本申请的提交日期之前提供本文讨论的出版物的公开。本文中的任何内容均不得解释为承认本发明无权凭借在先发明而早于此类出版物。此外,所提供的公开日期可能与实际公开日期不同,实际公开日期可能需要独立地确认。
定义
“多能性”和多能干细胞意味着这样的细胞具有分化为生物体中所有类型细胞的能力。术语“诱导多能干细胞”包括如下多能细胞,它们像胚胎干细胞(ESC)一样可以在长时间培养的同时保持分化为生物体中所有类型细胞的能力,但是与ESC不同,它们衍生自分化的体细胞(即具有更窄、更确定的潜能的细胞),并且在不存在实验操作的情况下它们不能产生生物体中所有类型的细胞。hiPSC具有类似于人ESC的形态,生长为具有大核质比、限定边界和突出核的扁平集落。另外,hiPSC表达本领域普通技术人员已知的几种多能性标志物,包括但不限于碱性磷酸酶、SSEA3、SSEA4、SOX2、OCT3/4、NANOG、TRA-160、TRA-181、TDGF1、DNMT3B、TERT和ZFP42。另外,hiPSC能够形成畸胎瘤。它们能够在活生物体中形成或促进外胚层、中胚层或内胚层组织。
如本文所用,“重编程因子”是指作用于细胞以改变转录从而将细胞重编程为多潜能性或多能性的生物活性因子中的一种或多种(即混合物)。可以将重编程因子提供给细胞,例如来自具有心脏病家族史或心脏病的目的遗传组成的个体的细胞,如成纤维细胞、脂肪细胞等;单独地或作为重编程因子的单一组合物(即作为预混合组合物)。可以按相同的摩尔比或按不同的摩尔比提供因子。在培养本发明的细胞的过程中可以一次或多次提供因子。在一些实施方案中,重编程因子是转录因子,包括但不限于Oct3/4;SOX2;KLF3;c-MYC;NANOG;和LIN-28。
使体细胞与如上定义的重编程因子以足以将细胞重编程为多能性的组合和量接触。可以将重编程因子单独地或作为重编程因子的单一组合物(即作为预混合组合物)提供给体细胞。在一些实施方案中,重编程因子作为载体上的多个编码序列来提供。如本领域已知的,体细胞可以是成纤维细胞、脂肪细胞、基质细胞等。体细胞或hiPSC可以获自细胞库、正常供体、患有目的神经或精神疾病的个体等。
诱导多能性后,根据任何便利的方法培养hiPSC,例如在辐照的饲养细胞和市售培养基上培养。可以通过用蛋白酶(例如accutase)消化使hiPSC与饲养层解离,优选地浓度和时间足以使单细胞从板上分离。
出于各种目的,例如用于替换具有功能丧失突变的基因、提供标志物基因等,可以将基因引入体细胞或从其衍生的hiPSC中。可选地,引入表达反义mRNA或核酶的载体,从而阻断不需要的基因的表达。基因治疗的其他方法是引入耐药基因(例如多耐药基因(MDR))以使正常祖细胞具有优势并承受选择压力或抗凋亡基因(如BCL-2)。可以使用本领域已知的各种技术(例如,电穿孔、钙沉淀的DNA、融合、转染、脂质转染、感染等)将核酸引入靶细胞中,如上所讨论。引入DNA的特定方式对于本发明的实践并不是关键的。
术语“少突胶质细胞”、“少突胶质祖细胞”等可以包括少突胶质细胞谱系的细胞,即最终产生少突胶质细胞、少突胶质前体细胞以及成熟的和髓鞘性少突胶质细胞的神经祖细胞,出于本发明的目的,它们通过实验操作源自非少突胶质细胞。少突胶质细胞可以通过对少突胶质细胞谱系的细胞具有特异性的标志物来鉴定,如下所讨论。少突胶质细胞可能具有功能特征,也就是说,它们可能具有使神经元髓鞘化的能力等等。“少突胶质细胞前体”或“少突胶质祖细胞”定义为能够产生包括少突胶质细胞的后代的细胞。
少突胶质细胞是中枢神经***的髓鞘形成细胞。少突胶质细胞扩展了许多过程,这些过程接触并反复包封轴突段。少突胶质细胞膜的这些包裹层的后续凝聚形成了髓鞘。一个轴突可能含有来自许多不同少突胶质细胞的髓鞘区段。
髓鞘形成在少突胶质细胞谱系的成熟中需要许多连续步骤。这些步骤伴随着细胞表面抗原表达的协调变化。少突胶质祖细胞的标志物包括例如血小板衍生的生长因子α-受体(PDGFR-α)。少突胶质细胞的其他标志物包括巢蛋白(Nestin)、蛋白脂蛋白、多唾液酸化形式的神经细胞粘附分子(NCAM)、神经节苷脂GD3和碳酸酐酶II(CA-II)。一些标记物(如CA-II)涵盖了谱系的所有阶段,并且也是成体少突胶质细胞的标志物。半乳糖基神经酰胺和磺基半乳糖基神经酰胺同样是仍然存在于成熟少突胶质细胞表面的早期标志物。编码特定髓鞘蛋白的其他基因在少突胶质细胞分化和成熟的不同阶段表达。例如,2′,3′-环核苷酸-3′-磷酸水解酶(CNP)、髓鞘碱性蛋白(MBP)、PLP/DM-20、髓鞘相关糖蛋白(MAG)和髓鞘/少突胶质细胞糖蛋白(MOG)基因以及其他次要的髓鞘蛋白都是成熟少突胶质细胞的标志物。
据推测,少突胶质细胞的成熟和存活需要许多因子。这些因子可以用本发明的人少突胶质细胞培养物来测试。目的因子可能包括PDGF、碱性FGF、***I(IGF-I)、神经营养蛋白3(NT-3)、神经胶质生长因子(GGF)、睫状神经营养因子(CNTF)、IL-6、转化生长因子(TGF)-β和IL-2。
髓鞘构成脊椎动物神经***中最丰富的膜结构。髓鞘中丰富的脂质和低水含量允许轴突的电绝缘,并且有髓区域的独特分段结构负责神经冲动的跳跃式传导。这允许髓鞘支持脊椎动物中枢神经***相对较薄的轴突中的快速神经传导。高速传导、长距离传输信号的保真度和空间经济性是髓鞘对脊椎动物神经***赋予的主要优势。
可以用本发明的细胞和培养物研究髓鞘形成的机制和调节这个复杂过程的信号。有后续步骤,包括少突胶质细胞向将在3D球形体内被髓鞘化的轴突的迁移;少突胶质细胞过程对轴突的粘附;以及所述过程在具有预定数量的髓鞘的轴突周围的旋生和不被髓鞘化的空间的识别。这些步骤中的每一个都可以在所提出的发明中的活细胞中进行研究和操作。此外,发育期间少突胶质细胞与星形胶质细胞之间的相互作用可以在此处介绍的3D球状体中进行研究。球状体的长培养寿命还允许研髓鞘可塑性和这些相互作用随时间的变化。
少突胶质细胞在脑区之间的迁移也可以在提出的发明中进行研究。在脑发育期间,少突胶质细胞的早期波在皮下层产生,并且迁移到皮质或皮层。可以将含有少突胶质细胞的球状体与皮质球状体融合,以模拟控制少突胶质细胞迁移到皮质的迁移动力学、方向提示和相反信号。这个***还可以用于研究异常少突胶质细胞迁移或分布可能起作用的疾病。少突胶质细胞迁移在提出的3D模型中可能会得到更好的保留,因为少突胶质细胞在体内通过组织迁移,而不是像2D少突胶质细胞模型那样沿平坦表面迁移。
除了作为体外培养细胞的各种用途外,还可以在合适的动物模型中测试少突胶质细胞。在一个层面上,评估细胞在体内存活和维持其表型的能力。将细胞组合物给予免疫缺陷动物(如裸鼠或者化学地或通过辐照使免疫缺陷的动物)。在再生长一段时间后收获组织,并且评估所给予的细胞或其后代是否仍然存在,并且可以针对对目的治疗的响应进行表型分析。还可以在动物模型中通过评估损伤后接着发生的或在疾病背景下由于用本发明的分化细胞进行治疗而引起的恢复程度来确定适合性。
疾病相关性。许多病理与少突胶质细胞功能障碍有关。人类遗传性髓鞘疾病脑白质营养不良可能是髓鞘形成异常、髓鞘形成不足或脱髓鞘的结果。如在不同形式的佩梅病(Pelizaeus-Merzbacher disease)中观察到的,髓鞘形成异常和髓鞘形成不足是胎儿生命或幼儿早期期间无法发生髓鞘形成。脱髓鞘(髓鞘分解)是代谢性脑白质营养不良的特征,所述代谢性脑白质营养不良是例如克拉伯病(Krabbe's disease)、异染性脑白质营养不良、ALD、卡纳万病(Canavan disease)、亚历山大病(Alexander disease)、正染性脑白质营养不良或线粒体障碍。髓鞘形成异常和脱髓鞘在一些形式的脑白质营养不良中可能组合。
一些遗传性疾病可能会引起脱髓鞘继发于血管、线粒体或神经元改变的脑白质病,或者可能与可具有普遍征兆的代谢疾病有关。伴皮质下梗死和脑白质病的大脑常染色体显性动脉病(CADASIL)是一种常染色体显性大脑动脉病。MRI证实了多发皮质下梗死,其中白质的脱髓鞘或多或少可能是广泛的。MELAS(线粒体脑肌病伴乳酸酸中毒和卒中样发作)表现为乳酸酸中毒并且血清和CSF中乳酸与丙酮酸的比例增加。MRI显示存在白质改变和皮质萎缩。
苯丙酮尿症可能与脱髓鞘有关。中间代谢异常也可能引起脱髓鞘。一些神经元遗传性疾病可能影响髓鞘(GM2神经节苷脂沉积症、威尔逊病(Wilson's disease)和CNS退行性疾病)。
血脑屏障的破坏是CNS脱髓鞘性疾病的病理表现中的主要事件,所述CNS脱髓鞘性疾病是例如多发性硬化症(MS)、脱髓鞘性形式的EAE和病毒引起的脱髓鞘。T细胞在这个过程中起关键作用。激活的T细胞进入CNS可能是炎性细胞、巨噬细胞和小神经胶质细胞以及促炎细胞因子(如TNF-α和干扰素-γ)释放的原因。
术语“治疗(treatment、treating、treat)”等在本文中用于一般性地指代获得所需的药理和/或生理作用。就完全或部分预防疾病或其症状而言,作用可以是预防性的;和/或就部分或完全稳定或治愈疾病和/或归因于疾病的副作用而言,作用可以是治疗性的。如本文所用的“治疗”涵盖哺乳动物(特别是人)的疾病的任何治疗,并且包括:(a)防止疾病或症状发生在可能易患疾病或症状但尚未诊断为患有疾病或症状的受试者中;(b)抑制疾病症状,即阻止其发展;或(c)减轻疾病症状,即引起疾病或症状的消退。
术语“个体”、“受试者”、“宿主”和“患者”在本文可互换使用,并且是指希望对其进行诊断、处理或治疗的任何哺乳动物受试者,特别是人。
本发明的方法
提供了用于获得和使用人少突球状体(hOS)和其中所含的细胞(特别是包括少突胶质细胞和神经元)的体外细胞培养物的方法,其中所述细胞分化自诱导人多能干细胞(hiPSC)。在一些实施方案中,hiPSC衍生自从神经学上正常的个体获得的体细胞。在其他实施方案中,hiPSC衍生自得从如下个体获得的体细胞,所述个体包含至少一个编码与神经疾病(包括但不限于上述髓鞘形成相关疾病)相关的突变的等位基因。在一些实施方案中,提供了一组这样的少突胶质细胞,其中所述组包括两种或更多种遗传上不同的少突胶质细胞。在一些实施方案中,提供了一组这样的少突胶质细胞,其中所述少突胶质细胞经受多种候选试剂或其他治疗干预或者多个剂量的候选试剂或其他治疗干预。候选试剂包括但不限于小分子(即药物)、增加或减少目的RNA表达的遗传构建体、电性变化等。
还提供了用于确定候选试剂对疾病相关细胞的活性的方法,所述方法包括使所述候选试剂与分化自人多能干细胞(例如分化自hESC或hiPSC)的一种或一组细胞接触,其中所述多能干细胞任选地包含至少一个编码与神经疾病相关的突变的等位基因;以及确定所述试剂对形态、遗传或功能参数(包括但不限于基因表达谱分析)的影响。除了遗传性疾病模型外,这些方法还可以用于影响少突胶质细胞成熟和髓鞘形成的环境操作,包括但不限于氧张力、温度和施加力的变化。
从体细胞产生人少突球状体(hOS)和其中所含的细胞(包括例如神经祖细胞、少突胶质祖细胞(OPC)、星形胶质细胞、髓鞘性少突胶质细胞和神经元)采用多步过程。最初,hiPSC可以获自任何便利的来源,或者可以使用本领域公认的方法从体细胞产生。使hiPSC与饲养层解离,并且在不存在FGF2的情况下,优选地在作为单细胞解离时,使其悬浮培养生长。在某些实施方案中,培养物不含饲养层,例如当在玻连蛋白包被的容器上生长时,并且hiPSC作为单细胞悬浮液解离并且聚集成特定大小的球状体。培养物还可以不含非人组分,即不含异种。悬浮生长任选地在培养的初始阶段在培养基中包括有效剂量的选择性Rho相关激酶(ROCK)抑制剂,例如长达约6小时、约12小时、约18小时、约24小时、约36小时约48小时(参见例如,Watanabe等人(2007)Nature Biotechnology 25:681 686)。可用于这样的目的的抑制剂包括但不限于Y-27632;Thiazovivin(Cell Res,2013,23(10):1187-200);法舒地尔(HA-1077)HCl(J Clin Invest,2014,124(9):3757-66);GSK429286A(Proc Natl AcadSci USA,2014,111(12):E1140-8);RKI-1447;AT13148;等。
然后将hiPSC的悬浮培养物向神经命运诱导。这种培养物可以不含饲养层。用于神经诱导,将有效剂量的BMP和TGFβ途径的抑制剂添加到培养基中,持续至少约2天、至少约3天、至少约4天、至少约5天且至多约10天、至多约9天、至多约8天、至多约7天、至多约6天、至多约5天的时间。例如,可以按至少约0.1μM、至少约1μM、至少约5μM、至少约10μM、至少约50μM、至多约100μM浓度的有效剂量添加多索***(dorsomorphin,DM),其抑制骨形态发生蛋白(BMP)I型受体(ALK2、ALK3和ALK6)。其他有用的BMP抑制剂包括但不限于A 83-01;DMH-1;K02288;ML 347;SB 505124;等。可以按至少约0.1μM、至少约1μM、至少约5μM、至少约10μM、至少约50μM、至多约100μM浓度的有效剂量添加SB-431542,其抑制TGFβ信号传导,但是对BMP信号传导没有影响。TGFβ的其他有用抑制剂包括但不限于LDN-193189(J Clin Invest,2015,125(2):796-808);加鲁尼替尼(Galunisertib)(LY2157299)(Cancer Res,2014,74(21):5963-77);LY2109761(Toxicology,2014,326C:9-17);SB525334(Cell Signal,2014,26(12):3027-35);SD-208;EW-7197;Kartogenin;DMH1;LDN-212854;ML347;LDN-193189HCl(Proc Natl Acad Sci USA,2013,110(52):E5039-48);SB505124;吡非尼酮(HistochemCell Biol,2014,10.1007/s00418-014-1223-0);RepSox;K02288;橙皮素;GW788388;LY364947等。
可以在第2、3、4、5、6或7天开始在培养基中包括有效剂量的Wnt抑制剂,例如浓度为约0.1μM至约100μM,并且可以是约1μM至约25μM,这取决于所选择的抑制剂的活性。示例性抑制剂包括但不限于:XAV-939在无细胞测定中通过以11nM/4nM的IC50抑制端锚聚合酶1/2来选择性地抑制Wnt/β-连环蛋白介导的转录;ICG-001拮抗Wnt/β-连环蛋白/TCF介导的转录,并且以3μM的IC50特异性地结合至元件结合蛋白(CBP);IWR-1-endo是Wnt途径抑制剂(在表达Wnt3A的L细胞中IC50为180nM),通过稳定轴蛋白脚手架破坏复合体来诱导轴蛋白2蛋白水平并促进β-连环蛋白磷酸化;Wnt-C59(C59)是Wnt3A介导的多聚TCF结合位点驱动荧光素酶的激活的PORCN抑制剂(在HEK293细胞中IC50为74pM);LGK-974是有效且具特异性的PORCN抑制剂,并且在TM3细胞中以0.4nM的IC50抑制Wnt信号传导;KY02111通过抑制Wnt信号传导促进hPSC向心肌细胞的分化,可能在APC和GSK3β的下游起作用;IWP-2是Wnt加工和分泌的抑制剂(在无细胞测定中IC50为27nM),选择性地阻断Porcn介导的Wnt棕榈酰化,一般不影响Wnt/β-连环蛋白,并且对Wnt刺激的细胞反应无影响;IWP-L6是高效的Porcn抑制剂(EC50为0.5nM);WIKI4是新型端锚聚合酶抑制剂(对TNKS2的IC50为15nM),并且抑制Wnt/β-连环蛋白信号传导;FH535是Wnt/β-连环蛋白信号传导抑制剂,也是双PPARγ和PPARδ拮抗剂。代替Wnt抑制剂,视黄酸可以按10nM至1μM的浓度被包括在培养基中。
也可以在第10、11、12、13、14或15天开始添加音猬因子途径的激动剂。潜在的激动剂包括SAG和2,6,9-三元取代嘌呤(purmorphamine),以范围从100nm至10μM的浓度使用。
在悬浮培养约5天、约6天、约7天、约8天、约9天、约10天之后,将漂浮的球状体移至神经培养基中以使神经祖细胞分化。培养基补充有有效剂量的FGF2和EGF。可以按各自至少约0.5ng/ml、至少约1ng/ml、至少约5ng/ml、至少约10ng/ml、至少约20ng/ml、至多约500ng/ml、至多约250ng/ml、至多约100ng/ml的浓度提供生长因子。
为了促进早期祖细胞向少突胶质细胞的分化,在FGF2/EGF暴露后约1周、约2周、约3周、约4周之后,将神经培养基改变为用有效剂量的PDGF-AA、IGF-1、HGF、BDNF和NT3替换FGF2、EGF、IWP-2和SAG。可以按各自至少约0.5ng/ml、至少约1ng/ml、至少约5ng/ml、至少约10ng/ml、至少约20ng/ml、至多约500ng/ml、至多约250ng/ml、至多约100ng/ml的浓度提供生长因子。培养基还可以包含例如胰岛素、T3、cAMP类似物、生物素等,对于胰岛素,浓度为至多约50μg/ml、至多约25μg/ml。
在暴露于分化因子后约4周、约5周、约6周、约7周之后,可以在不存在生长因子的情况下将球状体在神经培养基中维持延长的时间段,例如维持1、2、3、4、5、6、7、8、9、10、11、12个月或更长时间。神经培养基可以包含抗坏血酸、25μg/mL胰岛素、1μM cAMP类似物、60ng/mL T3和100ng/mL生物素,其中每4-5天更换一次培养基。
可以通过任何便利的方法(包括流式细胞术、磁性免疫选择、免疫淘选等)从球状体中分离细胞群体。便利地,PDGFR和/或MBP分别用作少突胶质祖细胞和少突胶质细胞的阳性选择标志物。如此分离的细胞可以重悬于可接受的培养基中,并且维持培养,冷冻,分析目的参数;移植到人类或动物模型中;等等。少突胶质祖细胞或少突胶质细胞的群体是令人感兴趣的,例如在CNS神经元的髓鞘再生的方法中,例如在创伤性损伤后神经元的再生中,在诸如多发性硬化症等脱髓鞘性疾病的治疗性治疗中,其中向有需要的患者提供有效剂量的细胞。
筛选测定
在小分子的筛选测定中,用一组细胞和细胞环境测试向培养的细胞中添加候选试剂的作用,其中细胞环境包括以下中的一种或多种:电刺激(包括离子性的改变)、用目的候选试剂刺激、与其他细胞(包括但不限于神经元和神经祖细胞、免疫效应细胞(如T细胞)、小神经胶质细胞、巨噬细胞等)接触等,并且其中在暴露于目的环境之前、在所提供试剂的剂量中等少突胶质细胞组的基因型可能有所不同。通常包括至少一个对照,例如阴性对照和阳性对照。细胞的培养通常在无菌环境中进行,例如在含有湿润的92%-95%空气/5%-8%CO2气氛的培养箱中在37℃下进行。细胞培养可以在含有不确定的生物液(如胎牛血清)的营养混合物或完全确定且无血清的培养基中进行。通过监测多个输出参数(包括形态、功能和遗传变化)来评估环境变化的影响。
在遗传因子的筛选测定中,可以将多核苷酸添加到一组细胞中的一个或多个中,以改变细胞的遗传组成。监测输出参数以确定表型是否存在变化。以这种方式,鉴定编码或影响目的途径中蛋白质表达的遗传序列。可以将结果输入数据处理器中以提供筛选结果数据集。将算法用于比较和分析在不同条件下获得的筛选结果。
单细胞水平的分析方法特别令人感兴趣,例如如上所述:原子力显微术、单细胞基因表达、单细胞RNA测序、钙成像、流式细胞术、髓鞘形成、电子显微术、实时成像等。可以测量各种参数以确定药物或治疗对少突胶质细胞的作用。
参数是细胞的可量化组分,特别是可以合意地在高通量***中准确测量的组分。参数也可以是任何细胞组分或细胞产物,包括细胞表面决定簇、受体、蛋白质或其构象或翻译后修饰、脂质、碳水化合物、有机或无机分子、核酸(例如mRNA、DNA等)或衍生自这种细胞组分的部分或其组合。尽管大多数参数将提供定量读数,但是在一些情况下,半定量或定性结果是可接受的。读数可以包括单个确定的值,或者可以包括均值、中值或方差等。预期会有变化,并且将使用标准统计方法和用于提供单个值的通用统计方法来获得测试参数集的每个测试参数的值的范围。
目的参数包括检测细胞质、细胞表面或分泌的生物分子,经常是生物聚合物,例如多肽、多糖、多核苷酸、脂质等,包括如本文公开的各种髓鞘组分。细胞表面、髓鞘和分泌的分子是优选的参数类型,因为它们介导细胞通讯和细胞效应子反应,并且可以更容易地测定。在一个实施方案中,参数包括特异性表位。通常使用特异性单克隆抗体或受体探针来鉴定表位。在一些情况下,包含表位的分子实体来自两种或更多种物质,并且包含确定的结构。参数可以是特异性修饰的蛋白质或寡糖的检测。参数可以由特异性单克隆抗体或配体或受体结合决定簇决定。
目的候选试剂是包括许多化学类别(主要是有机分子)的生物活性剂,其可以包括有机金属分子、无机分子、遗传序列等。本发明的一个重要方面是评估候选药物、选择治疗性抗体和基于蛋白质的治疗剂,所述候选药物、治疗性抗体和基于蛋白质的治疗剂具有优选的生物学反应功能。候选试剂包含与蛋白质进行结构相互作用(特别是氢键合)所必需的官能团,并且通常包括至少一个胺、羰基、羟基或羧基,通常至少两个官能化学基团。候选试剂通常包含被一个或多个上述官能团取代的环状碳或杂环结构和/或芳族或多芳族结构。在生物分子中也发现了候选试剂,所述生物分子包括肽、多核苷酸、糖、脂肪酸、类固醇、嘌呤、嘧啶、其衍生物、结构类似物或组合。
包括药理活性药物、遗传活性分子等。目的化合物包括化学治疗剂、抗炎剂、激素或激素拮抗剂、离子通道修饰剂和神经活性剂。适用于本发明的药剂的实例是在“ThePharmacological Basis of Therapeutics,”Goodman和Gilman,McGraw-Hill,New York,New York,(1996),第九版中在以下章节下描述的那些:在突触和神经效应器交界处作用的药物(Drugs Acting at Synaptic and Neuroeffector Junctional Sites);心血管药物(Cardiovascular Drugs);维生素(Vitamins);皮肤病学(Dermatology);和毒理学(Toxicology),全部通过引用并入本文。
测试化合物包括上述所有类别的分子,并且还可以包含未知含量的样品。令人感兴趣的是衍生自天然来源(如植物)的天然存在的化合物的复杂混合物。尽管许多样品将包含溶解状态的化合物,但是也可以测定可以溶解在合适的溶剂中的固体样品。目的样品包括环境样品,例如地下水、海水、开矿废料等;生物样品,例如从农作物制备的裂解物、组织样品等;制造样品,例如药品制备期间的时间进程;以及准备分析的化合物文库;等等。目的样品包括待评估潜在治疗价值的化合物,即候选药物。
术语样品还包括已添加了另外的组分(例如影响离子强度、pH、总蛋白质浓度等的组分)的上述流体。另外,可以处理样品以实现至少部分分级或浓缩。如果注意减少化合物的降解,则可以例如在氮气、冷冻或其组合下存储生物样品。所使用的样品体积足以允许可测量的检测,通常约0.1∶l至1ml的生物样品是足够的。
化合物(包括候选试剂)获自各种各样的来源,包括合成或天然化合物的文库。例如,许多手段可用于各种各样的有机化合物(包括生物分子)的随机和定向合成,包括表达随机化的寡核苷酸和寡肽。可选地,可获得或容易地产生呈细菌、真菌、植物和动物提取物形式的天然化合物的文库。另外,天然或合成产生的文库和化合物通过常规化学、物理和生化手段容易地修饰,并且可以用于产生组合文库。已知的药理学试剂可以经受直接或随机化学修饰,如酰化、烷基化、酯化、酰胺化等,以产生结构类似物。
如本文所用,术语“遗传因子”是指多核苷酸及其类似物,所述因子通过将遗传因子添加到细胞中在本发明的筛选测定中来测试。遗传因子的引入导致细胞总遗传组成的改变。遗传因子(如DNA)通常可以通过将序列整合到染色体中而在细胞基因组中导致实验引入的变化。遗传变化也可以是短暂的,在这种情况下外源序列没有整合,而是作为附加体来维持。遗传因子(如反义寡核苷酸)也可以通过干扰mRNA的转录或翻译来影响蛋白质的表达,而不会改变细胞的基因型。遗传因子的作用是增加或减少细胞中一种或多种基因产物的表达。
编码多肽的表达载体的引入可以用于在缺乏所述序列的细胞中表达编码的产物,或者使产物过表达。可以使用组成型的或经受外部调控的各种启动子,其中在后者的情况下,可以启动或关闭基因的转录。这些编码序列可以包括全长cDNA或基因组克隆、从其衍生的片段或将天然存在的序列与其他编码序列的功能或结构域组合的嵌合体。可选地,引入的序列可以编码反义序列;是反义寡核苷酸;RNAi,编码天然序列的显性负突变或显性或组成性活性突变;改变调控序列等。
反义和RNAi寡核苷酸可以通过本领域已知的方法化学地合成。优选的寡核苷酸是从天然磷酸二酯结构进行化学修饰的,以增加其细胞内稳定性和结合亲和力。文献中已经描述了许多这样的修饰,它们改变了骨架、糖或杂环碱基的化学。在骨架化学中有用的变化包括硫代磷酸酯;二硫代磷酸酯,其中两个非桥接氧都被硫取代;亚磷酰胺;烷基磷酸三酯和硼酸磷酸酯。非手性磷酸酯衍生物包括3’-O’-5’-S-硫代磷酸酯、3’-S-5’-O-硫代磷酸酯、3’-CH2-5’-O-膦酸酯和3’-NH-5’-O-氨基磷酸酯。肽核酸用肽键替换了整个核糖磷酸二酯骨架。糖修饰(例如吗啉代寡核苷酸类似物)也用于增强稳定性和亲和力。可以使用脱氧核糖的α-异头物,其中碱基相对于天然β-异头物是倒置的。核糖的2’-OH可以改变形成2’-O-甲基或2’-O-烯丙基糖,从而提供抗降解性而不包含亲和力。
通过在一种或多种环境条件下(例如在用β-肾上腺素能激动剂刺激后、电或机械刺激后等),向至少一种细胞中并且通常是多种细胞中添加试剂来筛选所述试剂的生物活性。测量响应于试剂的参数读数的变化,理想地将其归一化,然后可以通过与参考筛选结果(例如与具有其他目的突变的细胞、正常少突胶质细胞、衍生自其他家族成员的少突胶质细胞等)进行比较来评估所得的筛选结果。参考筛选结果可以包括在存在和不存在不同环境变化的情况下的读数、用其他试剂(其可以包括或可以不包括已知的药物)获得的筛选结果等。
便利地将试剂以溶解状态或易溶形式添加到培养的细胞培养基中。可以在流过***中作为间歇或连续的流添加试剂,或者可选地,将大剂量的化合物单独地或增量地添加到原本静态的溶液中。在流过***中,使用两种流体,其中一种是生理中性溶液,并且另一种是添加了测试化合物的相同溶液。第一流体流经细胞,随后第二流体流经细胞。在单溶液法中,将大剂量的测试化合物添加到细胞周围的大量培养基中。培养基的各组分的总浓度不应随大剂量的添加或在流过法中的两种溶液之间显著变化。
优选的试剂配方不包括可能对整个配方有显著影响的另外的组分,如防腐剂。因此,优选的配方基本上由生物活性化合物和生理学上可接受的载体(如水、乙醇、DMSO等)组成。然而,如果化合物是液体而没有溶剂,则配方可以基本上由化合物本身组成。
可以用不同试剂浓度并行进行多种测定,以获得对各种浓度的差异反应。如本领域已知的,确定试剂的有效浓度通常使用由1:10或其他对数分度稀释产生的浓度范围。如果需要,可以用第二系列稀释进一步精确浓度。通常,这些浓度之一用作阴性对照,即在零浓度下或低于试剂的检测水平或在不会导致表型的可检测变化的试剂浓度下或低于所述浓度。
除了上述功能参数外,各种方法还可以用于量化所选参数的存在。为了测量存在的分子的量,便利的方法是用可检测部分标记分子,所述可检测部分可以是荧光的、发光的、放射性的、酶活性的等,特别是对以高亲和力结合至参数具有特异性的分子,荧光部分可容易地用于标记几乎任何生物分子、结构或细胞类型。免疫荧光部分可以被引导至不仅与特定蛋白质结合,还可以与特定构象、切割产物或位点修饰(像磷酸化)结合。可以例如通过将单独的肽和蛋白质表达为细胞内的绿色荧光蛋白嵌合体来将它们工程化为自发荧光(关于综述,参见Jones等人(1999)Trends Biotechnol.17(12):477-81)。因此,可以对抗体进行基因修饰以提供荧光染料作为其结构的一部分。
根据所选择的标记,可以使用诸如放射免疫测定(RIA)或酶联免疫吸附测定(ELISA)、均相酶免疫测定等免疫测定技术和相关的非酶技术使用荧光标记以外的其他标记来测量参数。这些技术利用特异性抗体作为报告分子,由于它们对单个分子靶标的高度特异性,因此特别有用。美国专利号4,568,649描述了采用闪烁计数的配体检测***。这些技术对于蛋白质或修饰的蛋白质参数或表位或碳水化合物决定簇特别有用。蛋白质和其他细胞决定簇的细胞读数可以使用荧光或以其他方式标记的报告分子获得。基于细胞的ELISA或相关的非酶或基于荧光的方法使得能够测量细胞表面参数和分泌参数。捕获ELISA和相关的非酶方法通常采用两种特异性抗体或报告分子,并且可用于测量溶解状态的参数。流式细胞术方法可用于测量细胞表面和细胞内参数以及形状变化和粒度,并且可用于分析用作抗体或探针连接试剂的珠粒。来自这样的测定的读数可以是与单独的荧光抗体检测到的细胞表面分子或细胞因子相关的均值荧光,或者是平均荧光强度、中值荧光强度、荧光强度的方差或它们之间的某种关系。
在本领域使用通过定量成像以及荧光和共聚焦显微术鉴定输入细胞类型并读取参数的单细胞多参数和多细胞多参数多重测定,参见Confocal Microscopy Methods andProtocols(Methods in Molecular Biology第122卷)。Paddock编辑,Humana Press,1998。这些方法描述在于1999年11月23日发布的美国专利号5,989,833中。
核酸(尤其是信使RNA)的定量作为参数也是令人感兴趣的。这些可以通过依赖于核酸核苷酸序列的杂交技术来测量。技术包括聚合酶链式反应方法以及基因阵列技术。关于实例,参见Current Protocols in Molecular Biology,Ausubel等人编辑,John Wiley&Sons,New York,NY,2000;Freeman等人(1999)Biotechniques 26(1):112-225;Kawamoto等人(1999)Genome Res 9(12):1305-12;和Chen等人(1998)Genomics 51(3):313-24。
从测试化合物获得的筛选结果与一种或多种参考筛选结果的比较是通过使用合适的推导方案、AI***、统计比较等完成的。优选地,将筛选结果与参考筛选结果的数据库进行比较。可以编译参考筛选结果的数据库。这些数据库可以包括来自包括已知试剂或试剂组合的组的参考结果以及来自在去除或专门改变了单个或多个环境条件或参数的环境条件下处理的细胞的分析的参考结果。参考结果也可以从含有具有选择性地靶向或调节特定细胞途径的遗传构建体的细胞的组中产生。
读数可以是均值、平均值、中值或方差,或者是与测量相关的其他统计或数学得出的值。通过直接与相应的参考读数进行比较,可以进一步精确参数读数信息。在相同条件下为每个参数获得的绝对值将显示出活生物***固有的可变性,并且还反映了个体细胞的可变性以及个体之间固有的可变性。
为了方便起见,本发明的***能以试剂盒来提供。试剂盒可以包括可冷冻、冷藏或以一些其他方式处理以维持生存力的待使用的细胞、用于测量参数的试剂以及用于准备筛选结果的软件。软件将接收结果并进行分析,并且可以包括参考数据。软件还可以用来自控制培养物的结果对结果进行归一化。可以将组合物任选地包装在合适的容器中,所述容器带有用于所需目的(如筛选方法等)的书面说明。
为了进一步阐述可用于本发明的实践的通用技术,从业人员可以参考细胞生物学、组织培养、胚胎学和神经生物学方面的标准教科书和综述。关于组织培养和胚胎干细胞,读者可能希望参考Teratocarcinomas and embryonic stem cells:A practicalapproach(E.J.Robertson编辑,IRL Press Ltd.1987);Guide to Techniques in MouseDevelopment(P.M.Wasserman等人编辑,Academic Press 1993);Embryonic Stem CellDifferentiation in Vitro(M.V.Wiles,Meth.Enzymol.225:900,1993);Properties anduses of Embryonic Stem Cells:Prospects for Application to Human Biology andGene Therapy(P.D.Rathjen等人,Reprod.Fertil.Dev.10:31,1998)。
分子和细胞生物化学方面的通用方法可以在诸如以下等标准教科书中找到:Molecular Cloning:A Laboratory Manual,第3版(Sambrook等人,Harbor LaboratoryPress 2001);Short Protocols in Molecular Biology,第4版(Ausubel等人编辑,JohnWiley&Sons 1999);Protein Methods(Bollag等人,John Wiley&Sons 1996);NonviralVectors for Gene Therapy(Wagner等人编辑,Academic Press 1999);Viral Vectors(Kaplift和Loewy编辑,Academic Press 1995);Immunology Methods Manual(I.Lefkovits编辑,Academic Press 1997);以及Cell and Tissue Culture:LaboratoryProcedures in Biotechnology(Doyle和Griffiths,John Wiley&Sons 1998)。本公开中提及的用于基因操作的试剂、克隆载体和试剂盒可从商业供应商(如BioRad、Stratagene、Invitrogen、Sigma-Aldrich和ClonTech)获得。
在本说明书中引用的每个出版物出于所有目的通过引用以其整体特此并入。
应理解本发明不限于所描述的特定方法、方案、细胞系、动物物种或属以及试剂,因为它们可以变化。还应理解本文所用的术语仅是为了描述具体实施方案的目的,而并不旨在限制本发明的范围,本发明的范围将仅由所附权利要求限定。
如本文所用,单数形式“一个/一种(a、an)”和“所述(the)”包括复数指代物。因此,例如,提及“一种细胞”包括多种这样的细胞,并且提及“培养物”包括提及一种或多种培养物和本领域技术人员已知的其等同物,等等。除非另外明确说明,本文所用的所有技术术语和科学术语具有与本发明所属领域的普通技术人员通常理解的含义相同的含义。
提出以下实施例以向本领域普通技术人员提供有关如何制备和使用本发明的完整公开和描述,并且不旨在限制本发明人认为是其发明的内容的范围,也不旨在它们代表以下实验是全部或仅进行的实验。虽然已经努力确保关于所使用的数字(例如量、温度等)的准确性,但是仍应考虑一些实验误差和偏差。除非另外说明,份是重量份,分子量是重均分子量,温度是以摄氏度计,并且压力是大气压或接近大气压。
实验
实施例1
在二维(2D)培养物中已经进行了产生人类少突胶质细胞和模拟体内髓鞘形成过程的先前方法。在这些方案中,髓鞘形成是在少突胶质细胞的包裹在合成纳米柱周围进行时研究的。这些方法不允许就像在人脑中以三维过程发生那样来研究髓鞘形成。可选地,人类干细胞来源的少突胶质细胞已被移植到啮齿动物中,但是尚不清楚小鼠环境在多大程度上影响人类少突胶质细胞的生物学和髓鞘形成,并且这些方法费力且不允许进行高通量筛选。下述方法首次在仅源自人hiPSC或hESC的三维(3D)培养物中体外产生髓鞘性少突胶质细胞的方法,从而可以在健康和疾病条件下在体外研究人的髓鞘形成。
hiPSC维持和聚集。在iPSC培养基(含100U/mL青霉素和100μg/mL链霉素的完全E8培养基)中,在用玻连蛋白(0.5μg/cm2)包被的组织培养处理的板上培养人诱导多能干细胞(hiPSC)系。为了形成漂浮的聚集体,首先通过将500μL的hiPSC培养基加ROCK抑制剂Y-27632(10nM)于2,000g离心5分钟来制备Aggrewell板。将hiPSC用Y-27632预处理1小时,然后用预热的accutase处理5-7分钟使其解离。一旦解离,收集hiPSC,并在hiPSC培养基中稀释,然后计数。将300万个hiPSC的等分试样转移到15mL falcon管中,以2,000g离心5分钟,然后重悬于1.5mL具有Y-27632的hiPSC培养基中。然后将300万个细胞悬浮液添加到制备的Aggrewell板的各个孔中,并将板以100g离心3分钟。然后将Aggrewell板在具有5%CO2的37℃培养箱中保存过夜。
第二天,通过用P-1000移液管用切割吸头将hiPSC聚集体从Aggrewell中冲洗掉。将去除的聚集体使用40μm细胞过滤器过滤,并转移到100mm超低附着组织培养皿中,所述超低附着组织培养皿包含具有100U/mL青霉素和100μg/mL链霉素的E6培养基。
少突胶质细胞生成的神经诱导和促进。为了进行神经诱导,在最初6天内每天添加多索***(5μM)和SB-431542(10μM)。产生两种球状体变体,其中一些从第三天开始每天暴露于全反式视黄酸(RA,100nM),而其他的则从第四天开始暴露于Wnt途径抑制剂IWP-2(5μM)。在悬浮的第六天,将漂浮的球状体移至含有DMEM/F12、N-2补充剂、无维生素A的B-27血清替代品、GlutaMax(1:100)、MEM非必需氨基酸溶液(1:100)、0.1nMβ-巯基乙醇、100U/mL青霉素和100μg/mL链霉素的神经培养基(NM)。NM还补充有20ng/ml FGF2和20ng/ml EGF,持续19天,前10天每天更换培养基,随后9天每隔一天更换一次。在第11天,还每天将平滑化的激动剂SAG(1μM)添加到培养基中。为了促进神经祖细胞分化为少突胶质细胞,从第25天开始去除了FGF2、EGF、IWP-2、RA和SAG并将10ng/mL PDGF-AA、10ng/mL IGF-1、5ng/mL HGF、25μg/mL胰岛素、1μM cAMP类似物、60ng/mL T3、100ng/mL生物素、20ng/ml BDNF和20ng/ml NT3添加到NM培养基中。从第25天到第43天每隔一天更换一次培养基。从第43天往后,在具有20μg/mL抗坏血酸、25μg/mL胰岛素、1μM cAMP类似物、60ng/mL T3和100ng/mL生物素的NM中培养球状体,其中每4-5天更换一次培养基。
结果:
图1a至图1b.3D培养物中少突胶质细胞的衍生。培养51天后,将球状体在4%PFA中固定过夜。将球状体包埋在OCT中,进行切片,并使用针对NKX2.2和OLIG2(二者为共同鉴定少突胶质祖细胞的标志物)的抗体进行免疫荧光标记。相同的程序用于固定和制备来自100日龄hOS的切片,然后用O4(幼稚和成熟少突胶质细胞的表面标志物)和O1(成熟少突胶质细胞的表面标志物)进行免疫荧光标记。NKX2.2/OLIG2+细胞以及多极O4+和O1+细胞的存在表明,少突胶质祖细胞和成熟少突胶质细胞二者均存在于通过上述方法衍生的人球状体中。
图1c至图1e.3D培养中细胞类型之间的相互作用。从115日龄hOS制备切片,并用针对MBP(参与髓磷脂形成的蛋白质和成熟少突胶质细胞的标志物)、GFAP(星形胶质细胞的标志物)和神经丝(轴突标志物)的抗体标记。MBP+和GFAP+过程之间的重叠指示少突胶质细胞与星形胶质细胞相互作用。MBP+过程围绕神经丝+过程的包裹表明,少突胶质细胞将轴突包裹在115日龄的hOS中。
图1f.人hiPSC衍生的3D培养物(hOS)中的髓鞘形成。培养100天后,将通过所述方法衍生的球状体固定并处理以用于透射电子显微术。围绕轴突的环状层的存在与髓鞘形成是一致的。
意义和应用。髓鞘形成对于正常的脑功能是必不可少的,并且髓鞘形成中的畸变是许多神经障碍的基础。在此方法中描述的人神经3D球状体首次包含了髓鞘性少突胶质细胞,并且可用于***地研究健康和疾病中髓鞘形成的过程。此方法尤其适合疾病建模,因为可以直接从患有脱髓鞘性障碍的患者的皮肤细胞中重新编程此方案中使用的hiPSC。另外,由于方法的可扩展性,这些培养物还可用于药物筛选,以鉴定在健康和特定疾病情况下都影响髓鞘形成的化合物。此外,这也是首个允许研究人发育中神经元与少突胶质细胞、少突胶质细胞与星形胶质细胞等之间的关键细胞间相互作用的***。最后,源自这些培养物的少突胶质细胞可以在未来脱髓鞘性障碍的治疗中用作移植的细胞来源。
前面仅说明本发明的原理。应理解本领域的技术人员将能够设计不同的安排,这些不同的安排虽然没有在本文明确地描述或显示,但是体现本发明的原理并且被包括在其精神和范围内。此外,本文叙述的所有实施例和条件性语言主要旨在帮助读者理解本发明人所贡献的本发明的原理和概念以推动本领域发展,并且将被解释为而不限于这些特别叙述的实施例和条件。此外,引用本发明的原理、方面和实施方案的所有本文的陈述连同其具体实施例旨在包括其结构和功能等同物两者。另外,预期这样的等同物包括当前已知的等同物以及未来开发的等同物两者,即不论结构而执行相同功能的开发的任何要素。因此,本发明的范围不旨在受限于本文显示和描述的示例性实施方案。相反地,本发明的范围和精神由所附权利要求体现。
Claims (15)
1.一种用于产生人相互作用的髓鞘性少突胶质细胞、神经元和星形胶质细胞的3D培养物的方法,所述方法包括:
(i)通过在包含有效剂量的BMP抑制剂和TGFβ抑制剂的培养基中培养多能干细胞的完整集落或单细胞聚集体达2到10天的时段在多能干细胞悬浮培养物中诱导神经命运,以生成漂浮球状体的群体;
(ii)通过在包含成纤维细胞生长因子2(FGF2)和表皮生长因子(EGF)的培养基中以悬浮培养物形式进行培养1到4周而使漂浮球状体的群体分化为神经祖细胞的球状体;
(iii)通过在缺乏FGF2和EGF并且包含有效剂量的PDGF-AA、IGF-1、HGF、BDNF、NT3、胰岛素、cAMP、T3和生物素的神经培养基中培养而使神经祖细胞的球状体分化为包括少突胶质细胞的人少突球状体(hOS);
(iv)将所述hOS球状体在神经培养基中维持延长的时间段,以产生包括相互作用的髓鞘性少突胶质细胞、神经元和星形胶质细胞的3D培养物,其中,所述神经培养基缺乏生长因子但包括抗坏血酸、胰岛素、cAMP、T3和生物素。
2.如权利要求1所述的方法,其中所述细胞包含至少一个与髓鞘障碍相关的等位基因。
3.如权利要求1-2中任一项所述的方法,其中所述多能干细胞是诱导多能干细胞。
4.如权利要求1-3中任一项所述的方法,其中所述BMP抑制剂为多索***,以及所述TGFβ抑制剂为SB-431542。
5.如权利要求4所述的方法,其中(i)中的所述悬浮培养物不含饲养层。
6.如权利要求1所述的方法,其中,从培养的第二天开始,(i)中的所述培养基还包含有效剂量的wnt抑制剂。
7.如权利要求1所述的方法,其还包括用音猬因子途径激动剂和视黄酸或用音猬因子途径激动剂和Wnt抑制剂对步骤(i)中的球状体进行培养。
8.如权利要求1所述的方法,其还包括通过流式细胞术、磁性免疫选择或免疫淘选从少突球状体中分离少突胶质细胞。
9.如权利要求8所述的方法,其中将分离的少突胶质细胞暴露于目的候选试剂或治疗。
10.如权利要求9所述的方法,其中将所述少突胶质细胞与神经元或神经祖细胞组合。
11.一种用于确定候选试剂对人少突胶质细胞的影响的方法,所述方法包括:使所述候选试剂与根据权利要求7所述的方法分化自人诱导多能干细胞(hiPSC)的一种或一组所述3D培养物接触,其中所述少突胶质细胞任选地包含至少一个编码与神经疾病相关的突变的等位基因;以及确定所述试剂对形态、遗传或功能参数的影响。
12.如权利要求11所述的方法,其中所述候选试剂是免疫效应细胞。
13.如权利要求11所述的方法,其中所述候选试剂是免疫调节剂。
14.如权利要求11所述的方法,其中一组少突胶质细胞包括至少两种具有不同基因型的少突胶质细胞。
15.如权利要求14所述的方法,其中所述一组少突胶质细胞包含在不同环境条件下的少突胶质细胞。
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WO2019246436A1 (en) * | 2018-06-22 | 2019-12-26 | The Board Of Trustees Of The Leland Stanford Junior University | Functional cortico-spinal-muscle assembled spheroids |
US20230021826A1 (en) * | 2019-12-17 | 2023-01-26 | Corestem Co.,Ltd. | Differentiation method for procuring large amount of oligodendrocytes by disassembling 3d organoids generated from human pluripotent stem cells |
KR102657342B1 (ko) * | 2020-05-20 | 2024-04-15 | 고려대학교 산학협력단 | 희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 희소돌기아교세포를 포함하는 오가노이드의 제조방법, 상기 방법으로 제조된 오가노이드 및 이의 용도 |
WO2022059793A1 (ja) * | 2020-09-18 | 2022-03-24 | 国立大学法人 筑波大学 | 神経束および神経束の製造方法 |
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CN113444787A (zh) * | 2021-07-05 | 2021-09-28 | 中山大学附属第七医院(深圳) | 一种自闭症的脂质生物标志物及其应用 |
US20240043798A1 (en) * | 2022-08-08 | 2024-02-08 | Trailhead Biosystems Inc. | Methods and compositions for generating oligodendrocyte progenitor cells |
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