T3 enhances neuronal activity in an induced pluripotent stem cell derived model of early human brain development

Thyroid hormones are key modulators of brain development, including neuronal activity. Since most studies have been carried out in animal models, information on thyroid hormone signaling in human neurons is scarce. Therefore, human neuronal models that allow electrophysiological assessments are highly warranted.

We generated human induced pluripotent stem cell (hiPSC)-derived excitatory neurons through Neurogenin-2 (NGN2) overexpression (iNeurons). Astrocytes were added to the cultures to enhance neuron survival. Co-cultures were treated with or without 1 nM triiodothyronine (T3). We assessed thyroid hormone metabolism and transcriptional response in iNeurons with metabolism assays and RT-qPCR of the T3-responsive genes KLF9 and hairless (HR). We investigated the effects of 1 nM T3 on neuronal electrophysiology using multi-electrode arrays (MEAs).

iNeurons presented a dose-dependent induction of KLF9 (up to nine-fold change) and HR (up to eight-fold change). Cultures showed substantial type 3 deiodinase (D3) activity in lysates and intact neurons. MEA recordings showed that T3 treatment increased the overall neuronal activity of the cultures with a ∼2-fold increase in firing rate, ∼3-fold increase in total number of spikes and ∼3.5-fold increase in the number of bursts. Moreover, T3 increased synchronicity by strongly promoting the formation of network bursts (∼14-fold increase), being most prominent between ∼3 and 4 weeks of culture.

Our results indicate that T3 regulates multiple features of neuronal activity in NGN2-differentiated neurons, illustrating its potential to study normal and disordered thyroid hormone signaling in a human model for early brain development.