CN108893444A - 诱导多能干细胞向皮层神经细胞分化的无血清培养体系 - Google Patents

诱导多能干细胞向皮层神经细胞分化的无血清培养体系 Download PDF

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CN108893444A
CN108893444A CN201810818078.2A CN201810818078A CN108893444A CN 108893444 A CN108893444 A CN 108893444A CN 201810818078 A CN201810818078 A CN 201810818078A CN 108893444 A CN108893444 A CN 108893444A
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甄威
刘子铖
康健
周海荣
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93 Pole Constant Biological Medicine Science And Technology Jiangsu Co Ltd
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Abstract

本发明属于干细胞生物学和细胞培养技术领域，提出了促进诱导多能干细胞向皮层神经细胞分化的无血清培养体系，具体包括无血清分化基本培养基及促进皮层神经细胞分化的添加物。其中，无血清基本培养基必要成分包括：DMEM/F12培养基，人血清白蛋白，L‑谷氨酰胺，D‑葡萄糖，维生素C，N2/B27细胞培养添加剂，非必需氨基酸。促进皮层神经细胞分化的添加物，具体包括：SB431542，皮啡肽，成纤维生长因子‑2，维甲酸，BDNF，GDNF，CTNF，DAPT，阿糖胞苷。用此培养基可以帮助提高细胞混合体系中的皮层神经细胞分化，减少向其他类型细胞的分化，从而最终得到高纯度的皮层神经细胞。

Description

诱导多能干细胞向皮层神经细胞分化的无血清培养体系
技术领域
本发明属于干细胞生物学和细胞培养技术领域，具体来说，涉及用于一种用于促进诱导多能干细胞向皮层神经细胞分化的无血清培养体系。
背景技术
神经变性疾病为一类缓慢起病、病程呈进行性发展、预后不良的疾病，迄今尚缺乏有效的根治方法。临床常见的神经变性疾病有帕金森病、肌萎缩侧索硬化、脊髓性肌萎缩症和阿尔茨海默病等。这些疾病虽病因各异但在病理上均有中枢神经不同部位及不同程度的神经元脱失和功能异常。替代丢失的神经元，使之功能恢复，是治疗这类疾病的新思路。近年来的研究发现体外培养的干细胞不仅可以增殖、分化,而且还可以发生“转分化”，也即一种组织来源的细胞可以分化为其他不相关的组织细胞类型。例如，造血干细胞、间充质干细胞等在一定的体外条件下可以向成熟神经细胞分化。体内实验也证实神经干细胞或非神经组织来源的干细胞移植入受损的脑组织内可以分化为成熟的神经细胞，新生的神经细胞能替代缺失的细胞发挥功能，为这类神经***难治性疾病提供了新的治疗方法。
在组织学工程和临床治疗中，诱导性多能干细胞理论上可以诱导分化成各类神经细胞，这些细胞可广泛应用于组织神经***修复与再造萎缩，坏死，缺损的神经元以及神经胶质。而由诱导性干细胞诱导出具有特定神经递质表型的功能性神经元亚型为细胞移植和基因治疗的载体提供了很好的应用手段，对于中枢神经疾病的治疗有着广泛的应用前景。
多能干细胞培养以及向神经细胞分化的培养基中大多含有胎牛血清成分，血清的成分未知而且复杂，由于不同厂家的血清产品批次性差异很大，导致实验的重复性极低。此外，血清还可能携带病原体和其他致病因子，极大降低了分化的神经细胞在药物筛选检测或细胞治疗等方面的应用价值及安全性。
发明内容
本发明的目的是在无血清培养基的基础上，添加一系列物质来促进诱导多能干细胞向皮层神经细胞定向分化，可以得到大量的皮层神经细胞应用于药物筛选，疾病模型的建立以及干细胞再生医学领域。
本发明的培养基，可将诱导多能干细胞向皮层神经细胞分化，抑制未分化细胞以及向其他类型细胞的生长，在分化阶段的后期得到高纯度的皮层神经细胞，用于科研领域的研究以及商业应用。
本发明提供了一种促进诱导多能干细胞向皮层神经细胞分化的无血清培养体系，具体包括无血清分化基本培养基及促进皮层神经细胞分化的添加物。其中，无血清基本培养基，必要成分包括：基础培养基，血清白蛋白，L-谷氨酰胺，D-葡萄糖，维生素C，N2细胞培养添加剂，B27细胞培养添加剂和非必需氨基酸。促进皮层神经细胞分化的添加物，具体成分包括：SB431542，皮啡肽，成纤维生长因子-2，维甲酸，脑源性神经营养因子，胶质细胞源性神经营养因子，睫状节神经细胞营养因子，DAPT，阿糖胞苷。
在一个或多个实施方案中，所述基本培养基中的基础培养基选自DMEM/F12，Neurobasal，RPMI 1640，DMEM和IMDM培养基中的一种或几种。
在一个或多个实施方案中，所述无血清基本培养基成分中血清白蛋白，L-谷氨酰胺，D-葡萄糖，维生素C，N2细胞培养添加剂，B27细胞培养添加剂和非必需氨基酸的浓度分别为：血清白蛋白为0.25 g/L-25 g/L，L-谷氨酰胺为1-10nM， D-葡萄糖为1-10g/L，维生素C为100-1000μg/L，0.5-5%的N2细胞培养添加剂，0.5-5%的B27细胞培养添加剂和非必需氨基酸为10-100μM。
在一个或多个实施方案中，所述促进皮层神经细胞分化的添加物中SB431542，皮啡肽，成纤维生长因子-2，维甲酸，脑源性神经营养因子，胶质细胞源性神经营养因子，睫状节神经细胞营养因子，DAPT，阿糖胞苷的浓度分别为：SB431542为5-30μM，皮啡肽为1-10μM，成纤维生长因子-2为5-30μg/L，维甲酸为1-10μM，脑源性神经营养因子为1-10μg/L，胶质细胞源性神经营养因子为1-10μg/L，睫状节神经细胞营养因子为1-10μg/L，DAPT为1-5μM，阿糖胞苷为1-5μM。
采用本发明提出用于促进诱导多能干细胞向皮层神经细胞分化的无血清培养体系，其优点是：皮层神经细胞分化培养基去除了动物源血清成分，减小了分化的批次性差异，提高了干细胞来源的皮层神经细胞的安全性和应用价值；皮层神经细胞可以从诱导性干细胞诱导分化而成，而诱导分化体系为不同细胞类型的混合体系，用此培养基可以帮助提高细胞混合体系中的皮层神经细胞分化，减少向其他类型细胞的分化，从而最终得到高纯度的皮层神经细胞；该体系制备出来的皮层神经细胞可以应用于药物筛选，疾病模型的建立以及干细胞再生医学领域。
附图说明
图1 用来检测皮层神经细胞分化成熟的标志物基因（Islet, Map2, Tuj1, Oct4）的表达情况。
具体实施方式
下面结合附图和实施例，更具体地对本发明进行阐述，但不应该被认为仅限于在此阐述的实施例。
以DMEM/F12为基础培养基，向其中添加以下成分，配制得到无血清基本培养基：2.5g/L的血清白蛋白，5nM的L-谷氨酰胺，2g/L的D-葡萄糖，200μg/L的维生素C，1%的N2细胞培养添加剂，2%的B27细胞培养添加剂，100μM的非必需氨基酸。
促进皮层神经细胞分化的添加物，具体成分浓度为：10μM的SB431542，1μM的皮啡肽，10μg/L的成纤维生长因子-2，1μM的维甲酸，1μg/L的脑源性神经营养因子，1μg/L的胶质细胞源性神经营养因子，1μg/L的睫状节神经细胞营养因子，2μM的DAPT，2μM的阿糖胞苷。
人诱导多能干细胞在无血清基本培养基体系中进行培养，形成拟胚体形态。在基本培养基体系中加入10μM的SB431542，1μM的皮啡肽，10μg/L的成纤维生长因子-2，持续培养1周之后，在基本培养基体系中加入1μM的维甲酸，1μg/L的脑源性神经营养因子，2μM的DAPT，2μM的阿糖胞苷，继续培养1周，每2-3天根据培养基消耗情况更换培养基。之后将拟胚体细胞进行酶解分离，在基本培养基体系中加入1μg/L的脑源性神经营养因子，1μg/L的胶质细胞源性神经营养因子，1μg/L的睫状节神经细胞营养因子，进行贴壁培养。
采用实时定量PCR方法检测与皮层神经细胞分化成熟相关的标志基因的表达情况。
结果如图1所示，在本发明的无血清培养体系中，诱导多功能干细胞分化成为皮层神经细胞。
上述实施例为本发明的一种实施方式，但本发明的实施方式并不受本实施方式的限制，其它任何未背离本发明的实质与原理情况下所做的改变、替代、组合、简化以及修饰，均应为等效替换方式，都包含在本发明的保护范围之内。

Claims (6)

1.诱导多能干细胞向皮层神经细胞分化的无血清培养体系，其特征在于，其中所述的无血清培养体系包括无血清分化基本培养基及促进皮层神经细胞分化的添加物。
2.根据权利要求1所述的诱导多能干细胞向皮层神经细胞分化的无血清培养体系，其特征在于，所述的无血清分化基本培养基包括基础培养基和培养基添加剂：基础培养基包括DMEM/F12，Neurobasal，RPMI 1640，DMEM和IMDM培养基中的一种或几种；培养基添加剂包括0.25 g/L-25 g/L的血清白蛋白，1-10nM的L-谷氨酰胺，1-10g/L的D-葡萄糖，100-1000μg/L的维生素C，0.5-5%的N2细胞培养添加剂，0.5-5%的B27细胞培养添加剂，10-100μM的非必需氨基酸。
3.根据权利要求1所述的诱导多能干细胞向皮层神经细胞分化的无血清培养体系，其特征在于，所述的促进皮层神经细胞分化的添加物，具体包括：5-30μM的SB431542，1-10μM的皮啡肽，5-30μg/L的成纤维生长因子-2，1-10μM的维甲酸，1-10μg/L的脑源性神经营养因子，1-10μg/L的胶质细胞源性神经营养因子，1-10μg/L的睫状节神经细胞营养因子，1-5μM的DAPT，1-5μM的阿糖胞苷。
4.根据权利要求2所述的无血清分化基本培养基，其特征在于，所述的非必需氨基酸选自L-丙氨酸、L-精氨酸、L-天冬氨酸、L-天冬酰胺、L-半胱氨酸、L-谷氨酰胺、L-谷氨酸、甘氨酸、L-组氨酸、L-脯氨酸、L-丝氨酸、L-络氨酸中的一种或几种。
5.根据权利要求1所述的诱导多能干细胞向皮层神经细胞分化的无血清培养体系，其特征在于，诱导多能干细胞在不同的培养时间点，在培养体系中加入无血清基本培养基以及一种或几种促进皮层神经细胞分化的添加物，持续培养2周，进行皮层神经细胞成熟度分析。
6.根据权利要求5所述的皮层神经细胞成熟度分析，其特征在于，检测技术包括实时定量PCR和免疫染色。
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