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High-throughput screen for compounds that modulate neurite growth of human induced pluripotent stem cell-derived neurons.

ABSTRACT: Development of technology platforms to perform compound screens of human induced pluripotent stem cell (hiPSC)-derived neurons with relatively high throughput is essential to realize their potential for drug discovery. Here, we demonstrate the feasibility of high-throughput screening of hiPSC-derived neurons using a high-content, image-based approach focused on neurite growth, a process that is fundamental to formation of neural networks and nerve regeneration. From a collection of 4421 bioactive small molecules, we identified 108 hit compounds, including 37 approved drugs, that target molecules or pathways known to regulate neurite growth, as well as those not previously associated with this process. These data provide evidence that many pathways and targets known to play roles in neurite growth have similar activities in hiPSC-derived neurons that can be identified in an unbiased phenotypic screen. The data also suggest that hiPSC-derived neurons provide a useful system to study the mechanisms of action and off-target activities of the approved drugs identified as hits, leading to a better understanding of their clinical efficacy and toxicity, especially in the context of specific human genetic backgrounds. Finally, the hit set we report constitutes a sublibrary of approved drugs and tool compounds that modulate neurites. This sublibrary will be invaluable for phenotypic analyses and interrogation of hiPSC-based disease models as probes for defining phenotypic differences and cellular vulnerabilities in patient versus control cells, as well as for investigations of the molecular mechanisms underlying human neurite growth in development and maintenance of neuronal networks, and nerve regeneration.

SUBMITTER: Sherman SP

PROVIDER: S-EPMC5894944 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Due to the increasing number of drugs and untested environmental compounds introduced into commercial use, there is recognition for a need to develop reliable and efficient screening methods to identify compounds that may adversely impact the nervous system. One process that has been implicated in neurodevelopment is neurite outgrowth; the disruption of which can result in adverse outcomes that persist later in life. Here, we developed a green fluorescent protein (GFP) labeled neurite outgrowth assay in a high-content, high-throughput format using induced pluripotent stem cell (iPSC) derived human spinal motor neurons and cortical glutamatergic neurons. The assay was optimized for use in a 1536-well plate format. Then, we used this assay to screen a set of 84 unique compounds that have previously been screened in other neurite outgrowth assays. This library consists of known developmental neurotoxicants, environmental compounds with unknown toxicity, and negative controls. Neurons were cultured for 40 h and then treated with compounds at 11 concentrations ranging from 1.56 nM to 92 μM for 24 and 48 h. Effects of compounds on neurite outgrowth were evaluated by quantifying total neurite length, number of segments, and maximum neurite length per cell. Among the 84 tested compounds, neurite outgrowth in cortical neurons and motor neurons were selectively inhibited by 36 and 31 compounds, respectively. Colchicine, rotenone, and methyl mercuric (II) chloride inhibited neurite outgrowth in both cortical and motor neurons. It is interesting to note that some compounds like parathion and bisphenol AF had inhibitory effects on neurite outgrowth specifically in the cortical neurons, while other compounds, such as 2,2',4,4'-tetrabromodiphenyl ether and caffeine, inhibited neurite outgrowth in motor neurons. The data gathered from these studies show that GFP-labeled iPSC-derived human neurons are a promising tool for identifying and prioritizing compounds with developmental neurotoxicity potential for further hazard characterization.

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High-throughput screen for compounds that modulate neurite growth of human induced pluripotent stem cell-derived neurons.

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