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.

www.nature.com/scientificreports/ To construct an in vitro HTR2A-related disease model using hiPSCs, we have previously established a protocol to induce HTR2A-expressing neurons from hiPSCs. Here, we investigated the maturation stages and electrophysiological properties of HTR2A-positive neurons induced from hiPSCs using our previously established protocol. We also developed reporter lentiviruses, which contain a HTR2A promoter-driven fluorescent protein (ZsGreen1) expression construct, to identify and enrich for HTR2A-positive neurons. Furthermore, we confirmed the electrophysiological responses of the fluorescently labelled neurons derived from hiPSCs to application of the 5-HT 2A R agonist, TCB-2. Our newly established monitoring system of HTR2A-positive neurons derived from hiPSCs is expected to be a promising method to elucidate the pathological mechanism of neurological diseases associated with genetic variations of HTR2A. To the best of our knowledge, this is the first report of an in vitro, cell-level monitoring system specific to HTR2A-positive neurons.

Results
Generation and characterization of neurons derived from hiPSCs. The hiPSC line (C2) 20 was cultured in D-MEM medium to induce a chemically provoked transitional embryoid-body-like state (CtraS) 21 . Neuronal differentiation from hiPSCs was conducted according to a previously reported protocol 20 with slight modification (Fig. 1a). The day when the medium was changed to KBM medium, was defined as day in vitro (DIV) 0. To investigate the neuronal nature of hiPSC-derived neurons, the expression of Nestin (a neural progenitor cell marker), βIII-tubulin (a neuron marker), and MAP2 (a mature neuron marker) was analyzed in hiPSCs at DIV 10, 31, 41, and 51 (Fig. 1b). In early stages of the protocol, cells expressed Nestin (DIV 10) and βIII-tubulin (DIV 31), suggesting the successful neuronal differentiation of hiPSCs. The gene expression of Nestin was decreased from DIV 41 onwards. The levels of gene expression of MAP2 increased in a time-dependent manner until DIV 41. Extensive immunocytochemical analyses of MAP2 protein in hiPSCs at DIV 33 showed that 54.4 ± 10.3% (mean ± s.e.m., n = 3) of the stained cells were MAP2-positive (Fig. 1c).
We next investigated the gene expression of HTR2A. The expression of HTR2A mRNA was first detected after DIV 31. Thereafter, it increased in a time-dependent manner until DIV 51 (Fig. 2a). 5-HT 2A R protein immunofluorescence was found in 32.2 ± 7.0% of the induced neurons at DIV 33 (Fig. 2b,c).
Live-cell labelling of hiPSC-derived neurons dependent on HTR2A promoter activity. To distinguish HTR2A-positive neurons from HTR2A-negative ones, we generated a lentiviral ZsGreen1-expressingreporter construct dependent on HTR2A promoter activity for the live-cell labelling of neurons (Fig. S1). We amplified two different promoter sequences form human genomic DNA: Prom I and Prom II (Fig. 3a). These sequences were inserted upstream of the luciferase gene of the dual-reporter system. The promoter sequence of Prom I, but not that of Prom II, contains the promoter regulatory sequences 22 . Human neuroblastoma SK-N-SH cells 23 were used as HTR2A-positive cells, while 293 cells were used as HTR2A-negative cells (Fig. S1b). The HTR2A promoter activity of the reporter lentiviruses containing Prom I was much higher than that of the reporter containing Prom II in SK-N-SH. However, the HTR2A promoter activity of the reporter containing Prom I was not detected in 293 cells (Fig. 3b).
Extensive immunocytochemical analyses of 5-HT 2A R protein in hiPSCs at DIV 28 transfected with the constructed reporter lentiviruses showed that ZsGreen1-positive cells were co-localized with those that stained positive for 5-HT 2A R (Fig. 3c). The proportion of 5-HT 2A R-positive cells among the ZsGreen1-positive cells was 77%, while the proportion of ZsGreen1-positive cells among the 5-HT 2A R-positive cells was 9% (Fig. S2a-c).

Discussion
The use of hiPSCs derived from patients with a certain neurological disease allows the preparation of brain cells that contain the actual genetic information of the patients themselves [24][25][26][27] . This is a notable feature given that such cells have been technologically and ethically difficult to obtain in the past 28 . We have already reported the successful induction of HTR2A-expressing neurons of ventral hindbrain region derived from hiPSCs by controlling the regional identity along the anteroposterior axes and the dorsoventral axes 20,29 . In this study, using the  www.nature.com/scientificreports/ same method with slight modifications, we further investigated the time course of the maturation of HTR2Aexpressing neurons induced from hiPSCs to determine the optimal culture duration for the functional analyses. In these HTR2A-expressing neurons, the expression of neuronal markers and the HTR2A gene increased over  www.nature.com/scientificreports/ time up to DIV 41 and 51, respectively. Based on this result, we conducted the functional analyses using induced neurons at DIV 41 or later.
To identify the adequate target cells to conduct functional analyses, we developed a reporter lentivirus, which was capable of labelling HTR2A. Prom I, the adopted HTR2A promoter, showed higher activity than that of Prom II in HTR2A-positive cells, but no activity in HTR2A-negative cells. This suggested that multiple transcription factors encompassed in Prom I, like Sp1, PEA3, E-box, CRE binding proteins, CACCC box and CCAAT box were interacted 22 . This could be applied not only to electrophysiological analysis of the target cells but also to quantify the proportion of cells expressing HTR2A among all the induced cells in the hiPSC culture. The proportion of sorted ZsGreen1-positive cells increased in a time-dependent manner, reaching 64% at DIV 51. This result was consistent with the increase in the expression level of HTR2A mRNA. Besides, in scRNA-seq analysis, 80% of the ZsGreen1-positive cells were HTR2A-positive, which indicated that ZsGreen1 expression reflects HTR2A gene expression with high specificity. We believe that this result is due to the low sensitivity of scRNA-seq. In the flow cytometry experiment, a relatively high percentage (64%) of ZsGreen1-positive cells were MAP2-positive, and we believe that we are able to detect neurons with high sensitivity. In fact, since we used flow cytometry for detection, we expected to be able to detect cells with higher sensitivity than that observed in the scRNA-seq experimental results (18%).
Furthermore, HTR2A and ZsGreen1-positive cells were likely to be glutamatergic, or GABAergic or cholinergic neurons. Since several neurological diseases might be associated with specific neuronal populations, such as GABAergic neurons [30][31][32][33] , it is desirable to develop a tool that can label not only HTR2A but also specific types of neurons. Such a strategy should be developed in the future.
Additionally, we successfully showed that HTR2A-positive neurons derived from hiPSCs exhibited repetitive action potentials in response to depolarizing current injection, which was consistent with the activity pattern recorded from the neurons induced from hiPSCs in previous studies 34,35 . Regarding the 5-HT 2A R-specific cell responses, application of TCB-2 generated inward currents in HTR2A-positive neurons, which was also consistent with a previous report 36 . These results strongly suggested that the dynamics of the neurons induced from hiPSCs was similar to that of 5-HT 2A R-expressing neurons in vivo, because 5-HT 2A R agonists are supposed to cause cell membrane depolarization in these neurons 1,37 .
In conclusion, our in vitro monitoring system allowed the identification and functional analysis of HTR2Apositive neurons induced from hiPSCs, which could be used to elucidate the pathophysiological mechanism of various neurological diseases associated with genetic variations of the HTR2A gene 7-12,38,39 .

Methods
All experimental procedures using hiPSCs were approved by the Showa University Ethics Committee for Genome Research (approval no. 179) and the Juntendo University School of Medicine Ethics Committee (approval no. 2016117). The study protocol was in accordance with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. hiPSC culture. All the experiments were performed with the hiPSC line (C2) 20 . hiPSCs were generated from monocytes in peripheral blood samples of the healthy adult 20 and formal informed consent was obtained from the subject. hiPSCs were cultured on mitomycin C-treated SNL murine fibroblast feeder cells in standard hESC medium (DMEM/F12, Sigma Aldrich, St. Louis, MO) containing 20% KnockOut Serum Replacement (KSR; Thermo Fisher Scientific, Waltham, MA), 2 mM l-glutamine, 0.1 mM non-essential amino acids (Sigma Aldrich), 0.1 mM 2-mercaptoethanol (Sigma Aldrich), 0.5% penicillin/streptomycin, and 4 ng/ml fibroblast growth factor 2 (FGF-2; PeproTech, Rocky Hill, NJ) in an atmosphere containing 3% CO 2 . The medium was changed every other day.

Reverse transcription and quantitative polymerase chain reaction (RT-qPCR).
Transfection and luciferase assay. HTR2A reporter plasmids were co-transfected with a Renilla luciferase-expressing plasmid (E2241, pRL-TK-luc; Promega) to normalize transfection efficiency in neuroblastoma SK-N-SH cells (RCB0426; RIKEN BRC, Saitama, Japan through the National BioResource Project of the MEXT/ AMED, Japan) or 293 cells. Transfection was performed using Fugene HD (Promega) according to the manufacturer's instructions. pGL4.13-SV40 (E6681; Promega) was used as a positive control. Luciferase activities were measured using the Dual-Glo Luciferase Assay System (E2920; Promega), and the ratio of firefly luminescence to Renilla luminescence was used to express reporter activity.
Single-cell RNA-sequencing. HTR2A transfected hiPSCs at DIV 51 were used for single-cell RNA-seq.