Increased excitability of human iPSC-derived neurons in HTR2A variant-related sleep bruxism.

Sleep bruxism (SB) is a sleep-related movement disorder characterized by grinding and clenching of the teeth during sleep. We previously found a significant association between SB and a single nucleotide polymorphism (SNP), rs6313, in the neuronal serotonin 2A receptor gene (HTR2A), and established human induced pluripotent stem cell (iPSC)-derived neurons from SB patients with a genetic variant. To elucidate the electrophysiological characteristics of SB iPSC-derived neural cells bearing an SB-related genetic variant, we generated ventral hindbrain neurons from SB patients and unaffected controls, and explored the intrinsic membrane properties of these neurons using the patch-clamp technique. We found that the electrophysiological properties of iPSC-derived neurons mature in a time-dependent manner in long-term control cultures. SB neurons exhibited higher action potential firing frequency, higher gain, and shorter action potential half duration. This is the first in vitro modeling of SB using patient-specific iPSCs. The revealed electrophysiological characteristics may serve as a benchmark for further investigation of pathogenic mechanisms underlying SB. Moreover, our results on long-term cultures provide a strategy to define the functional maturity of human neurons in vitro, which can be implemented for stem cell research of neurogenesis, and neurodevelopmental disorders.

In vitro monitoring of HTR2A-positive neurons derived from human-induced pluripotent stem cells.

The serotonin 5-HT2A receptor (5-HT2AR) has been receiving increasing attention because its genetic variants have been associated with a variety of neurological diseases. To elucidate the pathogenesis of the neurological diseases associated with 5-HT2AR gene (HTR2A) variants, we have previously established a protocol to induce HTR2A-expressing neurons from human-induced pluripotent stem cells (hiPSCs). Here, we investigated the maturation stages and electrophysiological properties of HTR2A-positive neurons induced from hiPSCs and constructed an HTR2A promoter-specific reporter lentivirus to label the neurons. We found that neuronal maturity increased over time and that HTR2A expression was induced at the late stage of neuronal maturation. Furthermore, we demonstrated successful labelling of the HTR2A-positive neurons, which had fluorescence and generated repetitive action potentials in response to depolarizing currents and an inward current during the application of TCB-2, a selective agonist of 5-HT2ARs, respectively. These results indicated that our in vitro model mimicked the in vivo dynamics of 5-HT2AR. Therefore, in vitro monitoring of the function of HTR2A-positive neurons induced from hiPSCs could help elucidate the pathophysiological mechanisms of neurological diseases associated with genetic variations of the HTR2A gene.

Generation of neural cells using iPSCs from sleep bruxism patients with 5-HT2A polymorphism.

PURPOSE: Sleep bruxism (SB) is classified as a sleep-related movement disorder characterized by grinding and clenching of the teeth during sleep, which is responsible for a variety of clinical problems such as abnormal tooth attrition and fracture of teeth or roots. Little is known about the etiology of SB. Our previous study identified a genomic association of the serotonin 2A receptor (5-HT2A) single nucleotide polymorphism (SNP), rs6313 C>T, with SB, where the C allele carrier is associated with a 4.25-fold increased risk of SB. Based on this finding, the aim of this study was to generate of neural cells using SB patient-specific induced pluripotent stem cells (iPSCs). METHODS: Two SB patients with C/C genotype of rs6313 and two controls with T/T genotype were screened by laboratory-based polysomnographic recordings and the TaqMan genotyping assay. Four lines of iPSCs, two from SB patients and two from controls, were established from peripheral blood mononuclear cells by introduction of reprogramming factors. We performed quality control assays on iPSCs using expression of markers for undifferentiated pluripotent cells, immunostaining for pluripotency markers, a three-germ layer assay, and karyotype analysis. The established iPSCs were differentiated into neurons using the neurosphere culture system. 5-HT2A gene expression in these neurons was evaluated by quantitative real-time PCR. RESULTS: Patient-specific iPSCs were successfully differentiated into neurons expressing 5-HT2A. CONCLUSIONS: This report is the first successful generation of neural cells using iPSCs from sleep bruxism patients with 5-HT2A polymorphism, which has the potential to elucidate the etiology and underlying mechanism of SB.