Garcia-Diaz Alejandro, Efe Gizem, Kabra Khushbu, Patel Achchhe, Lowry Emily R, Shneider Neil A, Corneo Barbara, Wichterle Hynek
Departments of Pathology and Cell Biology, Neuroscience, Rehabilitation and Regenerative Medicine (in Neurology), Columbia University Irving Medical Center, New York, NY 10032, USA; The Stem Cell Core Facility, Columbia University Irving Medical Center, New York, NY 10032, USA; Center for Motor Neuron Biology and Disease, Columbia University Irving Medical Center, New York, NY 10032, USA; Columbia Stem Cell Initiative, Columbia University Irving Medical Center, New York, NY 10032, USA; Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Departments of Pathology and Cell Biology, Neuroscience, Rehabilitation and Regenerative Medicine (in Neurology), Columbia University Irving Medical Center, New York, NY 10032, USA; The Stem Cell Core Facility, Columbia University Irving Medical Center, New York, NY 10032, USA.
Neuroscience. 2020 Dec 1;450:48-56. doi: 10.1016/j.neuroscience.2020.06.028. Epub 2020 Jun 30.
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.
可靠且一致的多能干细胞报告系统对于高效纯化和可视化运动神经元至关重要，是研究正常运动神经元生物学以及有效进行疾病建模的必备试剂。为克服基于转基因的报告基因固有的噪音问题，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并对运动神经元标志物HB9和ISL1进行免疫染色，对新细胞系进行了验证。为便于高效纯化脊髓运动神经元，我们进一步用经过验证的条件性CD14-GFP构建体对VACHT-Cre细胞系进行工程改造，该构建体既能基于荧光识别运动神经元，又能通过磁激活细胞分选（MACS）大规模分离分化的运动神经元。此处开发的靶向策略为跨独立衍生的多能细胞系对运动神经元进行可重复比较提供了一个标准化平台。
