Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation
Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation
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DOI：
10.1038/nprot.2017.078
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发表时间：
2017-09-01
期刊：
影响因子：
14.8
通讯作者：
Kirkeby, Agnete
中科院分区：
文献类型：
--
作者：
Nolbrant, Sara;Heuer, Andreas;Kirkeby, Agnete
从人类多能干细胞（hPSC）中产生精确图案化的神经细胞有助于开发疾病模型和干细胞疗法。在这里，我们提供了一个详细的16天协议，获得高纯度腹侧中脑（VM）多巴胺（DA）祖细胞脑内移植到动物模型和体外成熟成神经元。我们已经成功地将这些细胞移植到大鼠中;然而，原则上，这些细胞可以用于移植到任何动物模型中，并且该方案被设计为也与临床移植到人类中兼容。我们展示了如何精确地设置模式化因素的平衡，以获得特定的尾VM祖细胞，这些祖细胞产生富含DA的移植物（+）通过指定如何进行质量控制（QC），故障排除和程序的适应，该方案将有助于在不同的实验室和各种hPSC系中实施。为了促进实验的重现性，并使中心之间的细胞运输，我们提出了一种方法冷冻保存的祖细胞随后直接移植或终末分化为DA神经元。本方案不含异种衍生产品，可在良好生产规范（GMP）条件下执行。
Generation of precisely patterned neural cells from human pluripotent stem cells (hPSCs) is instrumental in developing disease models and stem cell therapies. Here, we provide a detailed 16-d protocol for obtaining high-purity ventral midbrain (VM) dopamine (DA) progenitors for intracerebral transplantation into animal models and for in vitro maturation into neurons. We have successfully transplanted such cells into the rat; however, in principle, the cells can be used for transplantation into any animal model, and the protocol is designed to also be compatible with clinical transplantation into humans. We show how to precisely set the balance of patterning factors to obtain specifically the caudal VM progenitors that give rise to DA-rich grafts(+) By specifying how to perform quality control (QC), troubleshooting and adaptation of the procedure, this protocol will facilitate implementation in different laboratories and with a variety of hPSC lines. To facilitate reproducibility of experiments and enable shipping of cells between centers, we present a method for cryopreservation of the progenitors for subsequent direct transplantation or terminal differentiation into DA neurons. This protocol is free of xeno-derived products and can be performed under good manufacturing practice (GMP) conditions.