NeuCyte Inc., San Carlos, California 94070, USA.
Institute for Stem Cell Biology and Regenerative Medicine, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305, USA.
Toxicol Sci. 2021 Apr 12;180(2):295-312. doi: 10.1093/toxsci/kfab008.
Assessment of neuroactive effects of chemicals in cell-based assays remains challenging as complex functional tissue is required for biologically relevant readouts. Recent in vitro models using rodent primary neural cultures grown on multielectrode arrays allow quantitative measurements of neural network activity suitable for neurotoxicity screening. However, robust systems for testing effects on network function in human neural models are still lacking. The increasing number of differentiation protocols for generating neurons from human-induced pluripotent stem cells (hiPSCs) holds great potential to overcome the unavailability of human primary tissue and expedite cell-based assays. Yet, the variability in neuronal activity, prolonged ontogeny and rather immature stage of most neuronal cells derived by standard differentiation techniques greatly limit their utility for screening neurotoxic effects on human neural networks. Here, we used excitatory and inhibitory neurons, separately generated by direct reprogramming from hiPSCs, together with primary human astrocytes to establish highly functional cultures with defined cell ratios. Such neuron/glia cocultures exhibited pronounced neuronal activity and robust formation of synchronized network activity on multielectrode arrays, albeit with noticeable delay compared with primary rat cortical cultures. We further investigated acute changes of network activity in human neuron/glia cocultures and rat primary cortical cultures in response to compounds with known adverse neuroactive effects, including gamma amino butyric acid receptor antagonists and multiple pesticides. Importantly, we observed largely corresponding concentration-dependent effects on multiple neural network activity metrics using both neural culture types. These results demonstrate the utility of directly converted neuronal cells from hiPSCs for functional neurotoxicity screening of environmental chemicals.
基于细胞的检测方法评估化学物质的神经活性仍然具有挑战性，因为需要具有复杂功能的组织才能获得与生物学相关的检测结果。最近使用多电极阵列培养的啮齿动物原代神经培养物的体外模型允许对适合神经毒性筛选的神经网络活性进行定量测量。然而，用于测试人类神经模型中网络功能影响的稳健系统仍然缺乏。从人诱导多能干细胞 (hiPSC) 生成神经元的分化方案数量不断增加，为克服人类原代组织不可用和加快基于细胞的检测提供了巨大潜力。然而，大多数通过标准分化技术衍生的神经元细胞的活性变异性、较长的个体发生和相当不成熟的阶段极大地限制了它们在筛选对人类神经网络的神经毒性影响方面的效用。在这里，我们使用通过 hiPSC 直接重编程分别生成的兴奋性和抑制性神经元，以及原代人星形胶质细胞，以建立具有明确定义细胞比例的高度功能性培养物。这种神经元/神经胶质共培养物在多电极阵列上表现出明显的神经元活性和稳健的同步网络活性形成，尽管与原代大鼠皮质培养物相比存在明显的延迟。我们进一步研究了在人神经元/神经胶质共培养物和大鼠原代皮质培养物中，对具有已知不良神经活性作用的化合物（包括γ-氨基丁酸受体拮抗剂和多种农药）的网络活性的急性变化。重要的是，我们使用两种神经培养物观察到对多个神经网络活性指标的浓度依赖性影响，这些影响大致相同。这些结果表明，hiPSC 直接转化的神经元细胞可用于环境化学物质的功能神经毒性筛选。
