Lin Lin, Yuan Juan, Sander Bjoern, Golas Monika M
Department of Biomedicine, Stereology and Electron Microscopy Laboratory, Department of Clinical Medicine, and Center for Stochastic Geometry and Advanced Bioimaging, Aarhus University, Aarhus, Denmark.
Department of Biomedicine, Stereology and Electron Microscopy Laboratory, Department of Clinical Medicine, and Center for Stochastic Geometry and Advanced Bioimaging, Aarhus University, Aarhus, Denmark
Stem Cells Transl Med. 2015 Jul;4(7):775-88. doi: 10.5966/sctm.2014-0083. Epub 2015 May 13.
: Huntington's disease (HD) results from a CAG repeat expansion in the gene encoding the huntingtin protein. This inherited disorder is characterized by progressive neurodegeneration. In particular, HD progression involves the loss of striatal projection neurons. The limited availability of reliable sources of human striatal projection neurons currently hampers our understanding of HD mechanisms and hinders the development of novel HD treatments. In this paper, we described two- and three-step methods for differentiating human neural progenitor cells toward striatal projection neurons. In the two-step differentiation protocol, 90%, 54%, and 6% of MAP2-positive cells were immunopositive for GABA, calbindin (CALB1), and DARPP-32/PPP1R1B, respectively. In the three-step differentiation protocol, 96%, 84%, and 21% of MAP2-positive cells were immunopositive for GABA, calbindin, and DARPP-32/PPP1R1B, respectively. In line with a striatal projection neuron phenotype, cells differentiated with our protocols displayed significantly increased expression of MAP2, CALB1, DARPP-32/PPP1R1B, ARPP21, and CTIP2. Application of glutamate receptor agonists induced calcium influx; accordingly, the cells also expressed various ionotropic glutamate receptor subunits. Differentiated cells also released GABA on stimulation. We suggest that our three-step differentiation protocol presents a reliable and simplified method for the generation of striatal projection neurons, yielding a critical resource for neuronal physiology and neurodegenerative disorder studies.
The earliest changes in the neurodegenerative disorder Huntington's disease affect a specific type of brain neurons, the so-called medium spiny neurons of the striatum. In this study, two protocols were developed for the differentiation of neural progenitor cells into striatal medium spiny neurons, and the differentiated neurons were extensively characterized. The data indicate that the three-step differentiation protocol presents a reliable and simplified method for the generation of striatal medium spiny neurons. The generated striatal medium spiny neurons could represent a critical resource for the study of neurodegenerative disorders, a model system for drug discovery, and a step toward cell-based regeneration therapies.
亨廷顿舞蹈症（HD）是由编码亨廷顿蛋白的基因中CAG重复序列扩增所致。这种遗传性疾病的特征是进行性神经退行性变。特别是，HD的进展涉及纹状体投射神经元的丧失。目前，可靠的人类纹状体投射神经元来源有限，这阻碍了我们对HD机制的理解，并阻碍了新型HD治疗方法的开发。在本文中，我们描述了将人类神经祖细胞分化为纹状体投射神经元的两步法和三步法。在两步分化方案中,分别有90%、54%和6%的微管相关蛋白2（MAP2）阳性细胞对γ-氨基丁酸（GABA）、钙结合蛋白（CALB1）和多巴胺和3′,5′-环磷腺苷调节磷酸蛋白-32（DARPP-32）/蛋白磷酸酶1调节亚基B（PPP1R1B）呈免疫阳性。在三步分化方案中，分别有96%、84%和21%的MAP2阳性细胞对GABA、钙结合蛋白和DARPP-32/PPP1R1B呈免疫阳性。与纹状体投射神经元表型一致，用我们的方案分化的细胞显示微管相关蛋白2、钙结合蛋白、DARPP-32/PPP1R1B、活化调节正常T细胞表达和分泌因子21（ARPP21）和脑尾侧神经上皮转录抑制因子2（CTIP2）的表达显著增加。谷氨酸受体激动剂的应用诱导钙内流；因此，这些细胞还表达了各种离子型谷氨酸受体亚基。分化后的细胞在受到刺激时也会释放GABA。我们认为，我们的三步分化方案为生成纹状体投射神经元提供了一种可靠且简化的方法，为神经元生理学和神经退行性疾病研究提供了关键资源。
神经退行性疾病亨廷顿舞蹈症最早的变化影响一种特定类型的脑神经元，即所谓的纹状体中等棘状神经元。在本研究中，开发了两种将神经祖细胞分化为纹状体中等棘状神经元的方案，并对分化后的神经元进行了广泛表征。数据表明，三步分化方案为生成纹状体中等棘状神经元提供了一种可靠且简化的方法。生成的纹状体中等棘状神经元可能是神经退行性疾病研究的关键资源、药物发现的模型系统以及基于细胞的再生疗法的重要一步。
