Wallenberg Neuroscience Center, Department of Experimental Medical Sciences, Lund University, Lund, Sweden.
The Danish Stem Cell Center (DanStem), Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
Nat Protoc. 2017 Sep;12(9):1962-1979. doi: 10.1038/nprot.2017.078. Epub 2017 Aug 31.
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.
从人类多能干细胞（hPSCs）中生成精确图案化的神经细胞对于开发疾病模型和干细胞疗法至关重要。 在这里，我们提供了一个详细的 16 天协议，用于获得高纯度的腹侧中脑（VM）多巴胺（DA）祖细胞，以便进行脑内移植到动物模型中，并进行体外成熟为神经元。 我们已经成功地将这些细胞移植到大鼠中； 但是，原则上，这些细胞可用于移植到任何动物模型中，并且该方案旨在与临床移植到人类兼容。 我们展示了如何精确设置模式形成因子的平衡，以获得特定的尾部 VM 祖细胞，从而产生富含 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，该方案将促进在不同实验室和各种 hPSC 系中实施。 为了便于实验的重现性并能够在中心之间运输细胞，我们提出了一种用于冷冻保存祖细胞的方法，以便随后直接进行移植或终末分化为 DA 神经元。 该方案不含异种衍生产品，并且可以在良好生产规范（GMP）条件下进行。
