Allen Discovery Center at Tufts University, Medford, MA 02155, USA.
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA.
Cells. 2022 Aug 9;11(16):2470. doi: 10.3390/cells11162470.
All living cells maintain a charge distribution across their cell membrane (membrane potential) by carefully controlled ion fluxes. These bioelectric signals regulate cell behavior (such as migration, proliferation, differentiation) as well as higher-level tissue and organ patterning. Thus, voltage gradients represent an important parameter for diagnostics as well as a promising target for therapeutic interventions in birth defects, injury, and cancer. However, despite much progress in cell and molecular biology, little is known about bioelectric states in human stem cells. Here, we present simple methods to simultaneously track ion dynamics, membrane voltage, cell morphology, and cell activity (pH and ROS), using fluorescent reporter dyes in living human neurons derived from induced neural stem cells (hiNSC). We developed and tested functional protocols for manipulating ion fluxes, membrane potential, and cell activity, and tracking neural responses to injury and reinnervation in vitro. Finally, using morphology sensor, we tested and quantified the ability of physiological actuators (neurotransmitters and pH) to manipulate nerve repair and reinnervation. These methods are not specific to a particular cell type and should be broadly applicable to the study of bioelectrical controls across a wide range of combinations of models and endpoints.
所有活细胞通过严格控制的离子流来维持细胞膜两侧的电荷分布（膜电位）。这些生物电信号调节细胞行为（如迁移、增殖、分化）以及更高层次的组织和器官模式形成。因此，电压梯度不仅是诊断的重要参数，也是治疗出生缺陷、损伤和癌症的有前途的靶点。然而，尽管在细胞和分子生物学方面取得了很大进展，但对于人类干细胞中的生物电状态知之甚少。在这里，我们使用荧光报告染料，在体外展示了从诱导多能干细胞（hiPSC）分化而来的人源神经元中，同时跟踪离子动力学、膜电压、细胞形态和细胞活性（pH 值和 ROS）的简单方法。我们开发并测试了用于操纵离子流、膜电位和细胞活性的功能方案，并跟踪了神经元对损伤和再神经支配的反应。最后，我们使用形态传感器，测试和量化了生理调节剂（神经递质和 pH 值）调节神经修复和再神经支配的能力。这些方法不仅特定于特定的细胞类型，而且应该广泛适用于研究广泛的模型和终点组合的生物电控制。
