CN116574672A - 一种诱导化学诱导多能干细胞向生血内皮细胞分化的培养基及方法 - Google Patents
一种诱导化学诱导多能干细胞向生血内皮细胞分化的培养基及方法 Download PDFInfo
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Abstract
本发明提出了一种诱导化学诱导多能干细胞向生血内皮细胞分化的培养基及方法，属于干细胞与再生医学领域。包括第一阶段培养基和第二阶段培养基，第一阶段培养基包括：基础培养基DMEM/F12、非必须氨基酸、谷氨酰胺、硫代甘油、胰岛素、转铁蛋白、***钠、乙醇胺、维生素C、脂质混合物、人白蛋白、WNT通路抑制剂、ROCK通路抑制剂，第二阶段培养基包括：基础培养基DMEM/F12、非必须氨基酸、谷氨酰胺、硫代甘油、胰岛素、转铁蛋白、***钠、乙醇胺、维生素C、脂质混合物、人白蛋白、重组人骨形态发生蛋白‑4、碱性纤维生长因子、血管内皮生长因子。本发明诱导培养体系方便进行调控和标准化，促进了其分化下游细胞的应用。
Description
技术领域
本发明涉及基因工程技术领域，具体涉及一种诱导化学诱导多能干细胞向生血内皮细胞分化的培养基及方法。
背景技术
化学诱导重编程是指利用化学小分子将人体细胞转变为多能干细胞，称为化学诱导多能干细胞（hCiPSCs）。与胚胎干细胞和诱导多能干细胞相同，hCiPSCs具备自我更新、无限增殖和分化成所有功能细胞类型的能力。利用多能干细胞的分化特性可以用来研究体内功能细胞和疾病的发育。
造血干细胞移植和免疫细胞疗法等目前已经在临床上显示出广泛的治疗前景和疗效，用于治疗多种血液疾病和肿瘤。然而其来源受限，批量之间差异大，成本高，难以满足治疗的需要量；多能干细胞可以作为无限来源用于制备造血干细胞和各种类型的免疫细胞。等经过多年研究，利用多能干细胞分化能力已经能够制备各种血液谱系，在这个过程中对于血液和内皮谱系的发育关系也有了更深入的理解。在胚胎发育过程中，血液细胞与内皮细胞发育相关联。在卵黄囊血岛（yolk sac）、胚内腹侧中胚层-主动脉性腺-中肾（AGM区）和背侧主动脉的腹侧壁均发现血细胞团与内皮细胞一起形成分化。进而产生了一种假设，即血细胞与内皮细胞有共同的祖细胞-成血管血液干细胞（hemangioblast）。尽管成血管血液干细胞的性质仍有争议，但越来越多的证据表明生血内皮细胞（hemogenicendothelial）是一种短暂的中间产物，但对于胚胎发生过程中多能造血干细胞的从头产生是非常重要的。如何在体外高效制备生血内皮细胞对于进一步获得功能更好的造血干祖细胞以及下游免疫细胞至关重要。
已有的制备技术包内含2D和3D的分化方法，两种方法均有采用，但这些方法均有一些缺点。
已有的制备技术包括3D体系如EB（embryoid body类胚体）、2D体系如与饲养层基质细胞共培养分化或直接定向分化，通过添加诱导因子到特定的分化培养基，但是因为分化方式不同，分化效率存在明显差异。一方面分化体系中含有血清、异源成分、基质细胞，会导致微生物污染的风险；另一方面，成分复杂且不明确、周期长、不易调控和标准化，严重地限制了其分化下游细胞的临床研究和应用。
发明内容
本发明的目的在于提供一种诱导化学诱导多能干细胞向生血内皮细胞分化的培养基及方法。该培养基成分明确、简单、无动物源、成本低，采用定向2D分化方法，操作简单、效率高，有利于未来规模化和产业化。
本发明的技术方案是这样实现的：
本发明提供一种诱导化学诱导多能干细胞向生血内皮细胞分化的培养基，包括第一阶段培养基和第二阶段培养基，所述第一阶段培养基由以下成分组成：基础培养基DMEM/F12、非必须氨基酸、谷氨酰胺、硫代甘油、胰岛素、转铁蛋白、***钠、乙醇胺、维生素C、脂质混合物、人白蛋白、WNT通路抑制剂、ROCK通路抑制剂，所述第二阶段培养基由以下成分组成：基础培养基DMEM/F12、非必须氨基酸、谷氨酰胺、硫代甘油、胰岛素、转铁蛋白、***钠、乙醇胺、维生素C、脂质混合物、人白蛋白、重组人骨形态发生蛋白-4、碱性纤维生长因子、血管内皮生长因子。
作为本发明的进一步改进，所述WNT通路抑制剂为CHIR99021；所述ROCK通路抑制剂为Y27632。
作为本发明的进一步改进，所述第一阶段培养基的配方如下：
作为本发明的进一步改进，所述第二阶段培养基的配方如下：
本发明进一步保护一种诱导化学诱导多能干细胞向生血内皮细胞分化的方法，包括以下步骤：
S1.准备分化用单层化学诱导多能干细胞，培养；
S2.细胞完全换液，换成上述第一阶段培养基，培养，得到中胚层样细胞；
S3.细胞完全换液，将所有培养基替换成上述第二阶段培养基，培养，得到CD31+CD34+生血内皮细胞。
作为本发明的进一步改进，步骤S1的具体方法为：使用消化液Accutase消化对数期生长的hCiPSCs，37℃，5min，使用培养液3倍稀释中止酶反应，收集细胞离心300g，3min；用传代培养基重悬细胞，计数，以30万每板的密度接种到包被好玻连蛋白的培养板。
作为本发明的进一步改进，步骤S2的具体方法为：按照第一阶段培养基组成配制培养基，分别在0天和2天换液，连续培养3天。
作为本发明的进一步改进，步骤S3的具体方法为：按照第二阶段培养基组成配制培养基，分别在3天和5天换液，连续培养3天，第6天检测生血内皮的分化效率。
本发明具有如下有益效果：本发明诱导培养体系中不含有血清、异源成分和基质细胞且成分明确、诱导周期短、方便进行调控和标准化，促进了其分化下游细胞的临床研究和应用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1为多潜能干细胞向生血内皮分化流程图；
图2为hCiPSCs向生血内皮分化6天的效率图；
图3为EB和spin EB法分化体系对比图；
图4为小分子CHIR99021的使用浓度对分化效率的影响对比图；
图5为不同接种密度对分化效率的影响对比图；
图6为流式分析分化最终分化产物的纯度图。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
如图1，方法包括以下步骤：
S1.准备分化用单层化学诱导多能干细胞，培养两天；具体方法为：使用消化液Accutase消化对数期生长的hCiPSCs，37℃，5min，使用培养液3倍稀释中止酶反应，收集细胞离心300g，3min；用传代培养基重悬细胞，计数，以30万每板的密度接种到包被好Vitronectin（玻连蛋白）的培养板。
S2.将细胞完全换液，换成第一阶段培养基（配方见表1），得到中胚层样细胞,培养3天；具体方法为，按照培养基组成配制培养基，分别在0天和2天换液。
表1. 第一阶段培养基配方
S3.将细胞完全换液，将所有培养基替换成第二阶段培养基（配方见表2），得到CD31+ CD34+生血内皮细胞，培养3天。具体为，按照培养基组成配制培养基，分别在3天和5天换液，第6天检测生血内皮的分化效率。
表2. 第二阶段培养基配方
测试例1
1、WNT通路抑制剂与ROCK通路抑制剂协同促进生血内皮分化：
对于成分确定的培养体系的研究结果以及我们之前的专利，表明胰岛素、转铁蛋白、***钠、乙醇胺的组合是细胞存活和增殖的最基本的成分，通过在分化培养基中进一步添加维生素C，能够促进细胞的存活和增殖，而脂质混合物和人白蛋白的添加能够进一步促进分化体系的稳定，此7种成分构成了分化体系中的添加物组合。在2D无饲养层分化体系中，WNT通路抑制剂CHIR99021与ROCK通路抑制剂Y27632协同，6天即能有效诱导hCiPSCs向CD34+CD31+生血内皮的分化（图2）。
相同条件下，3D的EB培养体系和spin EB法培养体系，只能诱导低比例的生血内皮细胞；而商售的常用于分化的B27添加物在相同条件的生血内皮的比例也低于7种添加物组合添加CHIR99021和Y27632的条件（图3）。上述实验结果表明，在生血内皮的分化过程中，通过简单的7中添加物与两个小分子抑制剂的组合即可诱导高效、短时诱导生血内皮。
2、小分子抑制剂的浓度对生血内皮分化的影响
WNT信号通路抑制剂CHIR99021的剂量对CD31+CD34+生血内皮细胞效率的影响。流式细胞检测结果表明，相比于DMSO组，分化前3天添加5-10 μM CHIR99021与2 μMY27632组合均可高效促进生血内皮产生，呈一定范围的浓度依赖性，大于10 μM 会影响细胞的存活（图4）。
3、hCiPSCs细胞起始密度对生血内皮分化的影响
为了探究起始细胞密度对于生血内皮的影响，在铺板的时候采用不同接种密度的方式来摸索。实验结果显示，细胞密度影响CD31+CD34+生血内皮的数量，30-40万每细胞培养板的密度是适宜的（图5）。
4、分化的生血内皮具有向造血干祖细胞的分化能力
通过基础培养基中添加各种成分和信号通路抑制剂的组合，我们确定了成分明确的、无异源的、高效、6天即可诱导生血内皮的培养基和方法。还需确认生血内皮通过EHT(内皮-造血转换)过程，能够有效分化为造血干祖细胞。实验结果显示，在分化D8天即可观察到，非贴壁的、圆形的血细胞，随着分化进程，数量也越来越多。流式结果表明，上层的血细胞90%以上均是CD34+CD45+（图6）。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (8)
1.一种诱导化学诱导多能干细胞向生血内皮细胞分化的培养基，其特征在于，包括第一阶段培养基和第二阶段培养基，所述第一阶段培养基由以下成分组成：基础培养基DMEM/F12、非必须氨基酸、谷氨酰胺、硫代甘油、胰岛素、转铁蛋白、***钠、乙醇胺、维生素C、脂质混合物、人白蛋白、WNT通路抑制剂、ROCK通路抑制剂，所述第二阶段培养基由以下成分组成：基础培养基DMEM/F12、非必须氨基酸、谷氨酰胺、硫代甘油、胰岛素、转铁蛋白、***钠、乙醇胺、维生素C、脂质混合物、人白蛋白、重组人骨形态发生蛋白-4、碱性纤维生长因子、血管内皮生长因子。
2.根据权利要求1所述诱导化学诱导多能干细胞向生血内皮细胞分化的培养基，其特征在于，所述WNT通路抑制剂为CHIR99021；所述ROCK通路抑制剂为Y27632。
3.根据权利要求1所述诱导化学诱导多能干细胞向生血内皮细胞分化的培养基，其特征在于，所述第一阶段培养基的配方如下：
。
4.根据权利要求1所述诱导化学诱导多能干细胞向生血内皮细胞分化的培养基，其特征在于，所述第二阶段培养基的配方如下：
。
5.一种诱导化学诱导多能干细胞向生血内皮细胞分化的方法，其特征在于，包括以下步骤：
S1.准备分化用单层化学诱导多能干细胞，培养；
S2.细胞完全换液，换成权利要求1中所述第一阶段培养基，培养，得到中胚层样细胞；
S3.细胞完全换液，将所有培养基替换成权利要求1中所述第二阶段培养基，培养，得到CD31+ CD34+生血内皮细胞。
6.根据权利要求5所述诱导化学诱导多能干细胞向生血内皮细胞分化的方法，其特征在于，步骤S1的具体方法为：使用消化液Accutase消化对数期生长的hCiPSCs，37℃，5min，使用培养液3倍稀释中止酶反应，收集细胞离心300g，3min；用传代培养基重悬细胞，计数，以30万每板的密度接种到包被好玻连蛋白的培养板。
7.根据权利要求5所述诱导化学诱导多能干细胞向生血内皮细胞分化的方法，其特征在于，步骤S2的具体方法为：按照第一阶段培养基组成配制培养基，分别在0天和2天换液，连续培养3天。
8.根据权利要求5所述诱导化学诱导多能干细胞向生血内皮细胞分化的方法，其特征在于，步骤S3的具体方法为：按照第二阶段培养基组成配制培养基，分别在3天和5天换液，连续培养3天，第6天检测生血内皮的分化效率。
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