Fjodorova Marija, Li Meng
Neuroscience and Mental Health Research Institute, School of Bioscience, Cardiff University, Cardiff, UK.
Methods Mol Biol. 2018;1780:585-605. doi: 10.1007/978-1-4939-7825-0_27.
Efficient generation of disease relevant neuronal subtypes from human pluripotent stem cells (PSCs) is fundamental for realizing their promise in disease modeling, pharmaceutical drug screening and cell therapy. Here we describe a step-by-step protocol for directing the differentiation of human embryonic and induced PSCs (hESCs and hiPSCs, respectively) toward medium spiny neurons, the type of cells that are preferentially lost in Huntington's disease patients. This method is based on a novel concept of Activin A-dependent induction of the lateral ganglionic/striatal fate using a simple monolayer culture paradigm under chemically defined conditions. Transplantable medium spiny neuron progenitors amenable for cryopreservation are produced in less than 20 days, which differentiate and mature into a high yield of dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa (DARPP32) expressing gamma-aminobutyric acid (GABA)-ergic neurons in vitro and in the adult rat brain after transplantation. This method has been validated in multiple hESC and hiPSC lines, and is independent of the regime for PSC maintenance.
从人多能干细胞（PSC）高效生成与疾病相关的神经元亚型，对于实现其在疾病建模、药物筛选和细胞治疗方面的前景至关重要。在此，我们描述了一个逐步方案，用于将人胚胎干细胞和诱导多能干细胞（分别为hESC和hiPSC）定向分化为中等棘状神经元，这是亨廷顿病患者中优先丢失的细胞类型。该方法基于一个新的概念，即在化学成分明确的条件下，使用简单的单层培养模式，通过激活素A依赖性诱导外侧神经节/纹状体命运。在不到20天的时间内即可产生适合冷冻保存的可移植中等棘状神经元祖细胞，这些祖细胞在体外和移植到成年大鼠大脑后可分化并成熟为高产量的表达多巴胺和cAMP调节磷蛋白（分子量32 kDa，DARPP32）的γ-氨基丁酸（GABA）能神经元。该方法已在多个hESC和hiPSC系中得到验证，且与PSC维持方案无关。
