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Standardized Reporter Systems for Purification and Imaging of Human Pluripotent Stem Cell-derived Motor Neurons and Other Cholinergic Cells.
Neuroscience ( IF 3.3 ) Pub Date : 2020-06-30 , DOI: 10.1016/j.neuroscience.2020.06.028
Alejandro Garcia-Diaz 1 , Gizem Efe 2 , Khushbu Kabra 3 , Achchhe Patel 4 , Emily R Lowry 5 , Neil A Shneider 6 , Barbara Corneo 3 , Hynek Wichterle 5
Affiliation  

Reliable and consistent pluripotent stem cell reporter systems for efficient purification and visualization of motor neurons are essential reagents for the study of normal motor neuron biology and for effective disease modeling. To overcome the inherent noisiness of transgene-based reporters, we developed a new series of human induced pluripotent stem cell lines by knocking in tdTomato, Cre, or CreERT2 recombinase into the HB9 (MNX1) or VACHT (SLC18A3) genomic loci. The new lines were validated by directed differentiation into spinal motor neurons and immunostaining for motor neuron markers HB9 and ISL1. To facilitate efficient purification of spinal motor neurons, we further engineered the VACHT-Cre cell line with a validated, conditional CD14-GFP construct that allows for both fluorescence-based identification of motor neurons, as well as magnetic-activated cell sorting (MACS) to isolate differentiated motor neurons at scale. The targeting strategies developed here offer a standardized platform for reproducible comparison of motor neurons across independently derived pluripotent cell lines.



中文翻译:

用于人类多能干细胞衍生的运动神经元和其他胆碱能细胞的纯化和成像的标准化报告系统。

用于运动神经元有效纯化和可视化的可靠且一致的多能干细胞报告系统是研究正常运动神经元生物学和有效疾病建模的重要试剂。为了克服基于转基因的报告基因固有的噪音,我们通过将 tdTomato、Cre 或 CreERT2 重组酶敲入 HB9 (MNX1) 或 VACHT (SLC18A3) 基因组位点,开发了一系列新的人类诱导多能干细胞系。通过定向分化为脊髓运动神经元和运动神经元标记物 HB9 和 ISL1 的免疫染色,对新细胞系进行了验证。为了促进脊髓运动神经元的有效纯化,我们使用经过验证的条件性 CD14-GFP 构建体进一步设计了 VACHT-Cre 细胞系,该构建体允许基于荧光的运动神经元识别以及磁激活细胞分选 (MACS)大规模分离分化的运动神经元。这里开发的靶向策略提供了一个标准化平台,用于对独立衍生的多能细胞系的运动神经元进行可重复的比较。

更新日期:2020-06-30
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