Barretto Natalie, Zhang Hanwen, Powell Samuel K, Fernando Michael B, Zhang Siwei, Flaherty Erin K, Ho Seok-Man, Slesinger Paul A, Duan Jubao, Brennand Kristen J
Graduate School of Biomedical Science, Icahn School of Medicine at Mount Sinai, New York, NY, USA; Nash Family Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, USA; Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
Center for Psychiatric Genetics, NorthShore University HealthSystem, Evanston, IL, USA; Department of Psychiatry and Behavioral Neuroscience, University of Chicago, Chicago, IL, USA.
J Neurosci Methods. 2020 Feb 14;334:108548. doi: 10.1016/j.jneumeth.2019.108548.
Somatic cell reprogramming is routinely used to generate donor-specific human induced pluripotent stem cells (hiPSCs) to facilitate studies of disease in a human context. The directed differentiation of hiPSCs can generate large quantities of patient-derived cells; however, such methodologies frequently yield heterogeneous populations of neurons and glia that require extended timelines to achieve electrophysiological maturity. More recently, transcription factor-based induction protocols have been show to rapidly generate defined neuronal populations from hiPSCs.
In a manner similar to our previous adaption of NGN2-glutamatergic neuronal induction from hiPSC-derived neural progenitor cells (NPCs), we now adapt an established protocol of lentiviral overexpression of ASCL1 and DLX2 to hiPSC-NPCs.
We demonstrate induction of a robust and highly pure population of functional GABAergic neurons (iGANs). Importantly, we successfully applied this technique to hiPSC-NPCs derived from ten donors across two independent laboratories, finding it to be an efficient and highly reproducible approach to generate induced GABAergic neurons. Our results show that, like hiPSC-iGANs, NPC-iGANs exhibit increased GABAergic marker expression, electrophysiological maturity, and have distinct transcriptional profiles that distinguish them from other cell-types of the brain. Nonetheless, until donor-matched hiPSCs-iGANs and NPC-iGANs are directly compared, we cannot rule out the possibility that subtle differences in patterning or maturity may exist between these populations; one should always control for cell source in all iGAN experiments.
This methodology, relying upon an easily cultured starting population of hiPSC-NPCs, makes possible the generation of large-scale defined co-cultures of induced glutamatergic and GABAergic neurons for hiPSC-based disease models and precision drug screening.
体细胞重编程常用于生成供体特异性的人类诱导多能干细胞（hiPSC），以促进在人体环境中进行疾病研究。hiPSC的定向分化可产生大量患者来源的细胞；然而，此类方法常常产生神经元和神经胶质细胞的异质群体，需要较长时间才能达到电生理成熟。最近，基于转录因子的诱导方案已被证明能从hiPSC快速生成特定的神经元群体。
类似于我们之前将hiPSC来源的神经祖细胞（NPC）诱导为NGN2谷氨酸能神经元的方法，我们现在将ASCL1和DLX2慢病毒过表达的既定方案应用于hiPSC-NPC。
我们证明了可诱导出大量功能强大且高度纯化的GABA能神经元群体（iGAN）。重要的是，我们成功地将此技术应用于来自两个独立实验室的十名供体的hiPSC-NPC，发现它是一种高效且高度可重复的方法来生成诱导性GABA能神经元。我们的结果表明，与hiPSC-iGAN一样，NPC-iGAN表现出GABA能标记物表达增加、电生理成熟，并且具有将它们与大脑其他细胞类型区分开来的独特转录谱。尽管如此，在直接比较供体匹配的hiPSC-iGAN和NPC-iGAN之前，我们不能排除这些群体之间可能存在模式或成熟度细微差异的可能性；在所有iGAN实验中都应始终控制细胞来源。
这种方法依赖于易于培养的hiPSC-NPC起始群体，使得为基于hiPSC的疾病模型和精准药物筛选生成大规模特定的诱导性谷氨酸能和GABA能神经元共培养成为可能。
