Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells.
Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells.
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DOI：
10.1186/s13041-015-0172-4
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发表时间：
2015-12-01
期刊：
影响因子：
3.6
通讯作者：
Okada Y
中科院分区：
文献类型：
--
作者：
Shimojo D;Onodera K;Doi-Torii Y;Ishihara Y;Hattori C;Miwa Y;Tanaka S;Okada R;Ohyama M;Shoji M;Nakanishi A;Doyu M;Okano H;Okada Y
人类多能干细胞（hPSC）正在应用于再生医学和人类难治性疾病的体外建模。特别地，来源于从神经障碍患者建立的疾病特异性人诱导多能干细胞（hiPSC）的神经细胞已被用作体外疾病模型以概括体内发病机制，因为神经细胞通常不能从患者自身获得。在这项研究中，我们建立了一种快速，高效，简单的方法，有效地从hPSC中获得运动神经元，这对病理生理学分析和治疗运动神经元疾病的药物开发是有用的。在分化的初始阶段用GSK 3 β抑制剂联合双重SMAD抑制剂处理足以在1周内诱导PAX 6+和SOX 1+神经祖细胞，随后用维甲酸（RA）和purmorphamine（其激活sonic hedgehog（SHH）信号传导）处理，导致在2周内高效诱导HB 9+和ISL-1+运动神经元。在运动神经元成熟培养基中单层分化4周后，显示hPSC衍生的运动神经元成熟，显示出更大的胞体和成熟运动神经元标志物胆碱乙酰转移酶（ChAT）的更清晰染色。此外，hPSC衍生的运动神经元能够在体外与人肌管形成神经肌肉接头，并诱导乙酰胆碱受体（AChR）聚集，如Alexa 555缀合的α-银环蛇毒素（α-BTX）所检测到的，表明这些hPSC衍生的运动神经元与骨骼肌形成功能性接触。该分化系统是简单的，并且在几个hiPSC克隆中是可再现的，从而使hPSC克隆之间的克隆变异最小化。我们还建立了一个系统，用于可视化运动神经元与HB 9（HB 9 e438：：Venus）的慢病毒报告。通过免疫细胞化学和定量RT-PCR分析，通过荧光激活细胞分选术（FACS）获得的高阳性部分，这表明其适用于运动神经元特异性分析，该报告的特异性得到证实。我们的运动神经元分化系统和基于慢病毒的运动神经元报告系统有助于分析运动神经元疾病的疾病特异性hiPSC。本文的在线版本（doi：10.1186/s13041-015-0172-4）包含补充材料，可供授权用户使用。
Human pluripotent stem cells (hPSCs) are being applied in regenerative medicine and for the in vitro modeling of human intractable disorders. In particular, neural cells derived from disease-specific human induced pluripotent stem cells (hiPSCs) established from patients with neurological disorders have been used as in vitro disease models to recapitulate in vivo pathogenesis because neural cells cannot be usually obtained from patients themselves. In this study, we established a rapid, efficient, and simple method for efficiently deriving motor neurons from hPSCs that is useful for pathophysiological analysis and the development of drugs to treat motor neuron diseases. Treatment with GSK3β inhibitors during the initial phase of differentiation in combination with dual SMAD inhibition was sufficient to induce PAX6+ and SOX1+ neural progenitors within 1 week, and subsequent treatment with retinoic acid (RA) and purmorphamine, which activates sonic hedgehog (SHH) signaling, resulted in the highly efficient induction of HB9+ and ISL-1+ motor neurons within 2 weeks. After 4 weeks of monolayer differentiation in motor neuron maturation medium, hPSC-derived motor neurons were shown to mature, displaying larger somas and clearer staining for the mature motor neuron marker choline acetyltransferase (ChAT). Moreover, hPSC-derived motor neurons were able to form neuromuscular junctions with human myotubes in vitro and induced acetylcholine receptor (AChR) clustering, as detected by Alexa 555-conjugated α-Bungarotoxin (α-BTX), suggesting that these hPSC-derived motor neurons formed functional contacts with skeletal muscles. This differentiation system is simple and is reproducible in several hiPSC clones, thereby minimizing clonal variation among hPSC clones. We also established a system for visualizing motor neurons with a lentiviral reporter for HB9 (HB9e438::Venus). The specificity of this reporter was confirmed through immunocytochemistry and quantitative RT-PCR analysis of high-positive fractions obtained via fluorescence-activated cell sorting (FACS), suggesting its applicability for motor neuron-specific analysis. Our motor neuron differentiation system and lentivirus-based reporter system for motor neurons facilitate the analysis of disease-specific hiPSCs for motor neuron diseases. The online version of this article (doi:10.1186/s13041-015-0172-4) contains supplementary material, which is available to authorized users.