Q-State Biosciences, 179 Sidney Street, Cambridge, MA, 02139, USA.
Takeda California, San Diego, USA.
Neurochem Res. 2019 Mar;44(3):714-725. doi: 10.1007/s11064-018-2694-5. Epub 2019 Jan 2.
Induced pluripotent stem (iPS) cells offer the exciting opportunity for modeling neurological disorders in vitro in the context of a human genetic background. While significant progress has been made in advancing the use of iPS cell-based disease models, there remains an unmet need to characterize the electrophysiological profile of individual neurons with sufficient throughput to enable statistically robust assessment of disease phenotypes and pharmacological modulation. Here, we describe the Optopatch platform technology that utilizes optogenetics to both stimulate and record action potentials (APs) from human iPS cell-derived excitatory neurons with similar information content to manual patch clamp electrophysiology, but with ~ 3 orders of magnitude greater throughput. Cortical excitatory neurons were produced using the NGN2 transcriptional programming approach and cultured in the presence of rodent glial cells. Characterization of the neuronal preparations using immunocytochemistry and qRT-PCR assays reveals an enrichment of neuronal and glutamatergic markers as well as select ion channels. We demonstrate the scale of our intrinsic cellular excitability assay using pharmacological assessment with select ion channel modulators quinidine and retigabine, by measuring changes in both spike timing and waveform properties. The Optopatch platform in human iPS cell-derived cortical excitatory neurons has the potential for detailed phenotype and pharmacology evaluation, which can serve as the basis of cellular disease model exploration for drug discovery and phenotypic screening efforts.
诱导多能干细胞（iPS 细胞）为在人类遗传背景下体外模拟神经疾病提供了令人兴奋的机会。虽然在推进基于 iPS 细胞的疾病模型的应用方面已经取得了重大进展，但仍需要对个体神经元的电生理特征进行特征描述，以实现足够的高通量，从而能够对疾病表型进行统计学上稳健的评估和药理学调节。在这里，我们描述了 Optopatch 平台技术，该技术利用光遗传学同时刺激和记录源自人类 iPS 细胞衍生的兴奋性神经元的动作电位（AP），其信息含量与手动膜片钳电生理学相似，但具有 ~3 个数量级更高的吞吐量。皮质兴奋性神经元是使用 NGN2 转录编程方法产生的，并在鼠胶质细胞存在的情况下进行培养。使用免疫细胞化学和 qRT-PCR 检测对神经元制剂进行的特征描述显示，神经元和谷氨酸能标志物以及选择性离子通道得到了富集。我们通过使用选择性离子通道调节剂奎尼丁和瑞替加滨进行药理学评估，展示了我们的内在细胞兴奋性测定的规模，通过测量尖峰定时和波形特性的变化来实现。在源自人类 iPS 细胞的皮质兴奋性神经元中的 Optopatch 平台具有进行详细表型和药理学评估的潜力，这可以作为药物发现和表型筛选工作中细胞疾病模型探索的基础。
