Screening Biophysical Sensors and Neurite Outgrowth Actuators in Human Induced-Pluripotent-Stem-Cell-Derived Neurons

Abstract

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.

Advances in high throughput cell culture technologies for therapeutic screening and biological discovery applications

“…Using fluorescent reporters in situ and post‐culture processing (such as supernatant analysis), researchers can capture information on the impact of the compounds on cellular responses in a HT manner. These readouts can include organelle morphology and function, 19 , 20 , 21 membrane voltage and ion dynamics, 22 , 23 and cell‐specific markers including maturation markers, 19 neurite outgrowth, 23 , 24 intracellular triglycerides and lipid accumulation, 25 and IL‐1B secretion and inflammasome activation. 26 Grimm et al established multiple 384‐well toxicology screening platforms to identify toxic compounds to iPSC‐derived cardiomyocytes' and hepatocytes' cellular activity.…”