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.
Serotonin is a neurotransmitter involved in many physiological functions in the brain1,2. The serotonin 5-HT2A receptor (5-HT2AR) is a serotonin receptor subtype encoded by the 5-HT2AR gene (HTR2A). Mutations in this gene are associated with susceptibility to a variety of neurological diseases. For example, rs6313 (also called T102C3) and rs6311, which is in complete linkage disequilibrium with rs63134,5,6, are genetic polymorphisms of HTR2A. They have been associated with schizophrenia7,8, psychotic symptoms in Alzheimer’s disease9, certain features of depression10, sleep breathing disorders11, and sleep bruxism12.
Understanding the functional consequences of genetic variants is a critical first step towards appreciating their roles in disease development. While previous case–control studies have reported a significant association of genetic polymorphisms of HTR2A with a variety of neurological diseases7,8,9,10,11,12,13,14,15, the effect of the variants on disease pathogenesis has not been elucidated in detail, mainly because of the limited accessibility to the brain. In vitro modelling of human diseases using disease-specific human-induced pluripotent stem cells (hiPSCs) has the potential to provide dramatic progress in the elucidation of the pathogenic mechanisms of neurological diseases16,17,18,19,20.
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-HT2A 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.
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 protocol20 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-HT2AR 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-expressing-reporter 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 cells23 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-HT2AR 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-HT2AR (Fig. 3c). The proportion of 5-HT2AR-positive cells among the ZsGreen1-positive cells was 77%, while the proportion of ZsGreen1-positive cells among the 5-HT2AR-positive cells was 9% (Fig. S2a–c).
To evaluate the function of the reporter lentivirus, lentivirus-transfected hiPSCs were analyzed using fluorescence microscopy and flow cytometry. The control lentivirus (described as “Empty” in Fig. 3d–f) and constructed reporter lentivirus (described as “HTR2Apro” in Fig. 3d–f) were transfected into hiPSCs. The ZsGreen1-positive cells (DIV-5) were not detected by either fluorescence microscopy or flow cytometry (ZsGreen1-positive cells, 0.97 ± 0.17%) (Figs. 2a, 3d–f). The ZsGreen1-positive cells were detected in neurons derived from hiPSCs at DIV 41 and 51 by fluorescence microscopy, and the positive ratio was 25.2 ± 1.5% and 63.9 ± 2.6%, respectively (Fig. 3d–f). Consistent with this, the sorted ZsGreen1-positive cells at DIV 41 showed significantly higher expression of HTR2A mRNA than the whole-cell lysate (Fig. 3g). Furthermore, single-cell RNA-sequencing (scRNA-seq) analysis performed with hiPSCs at DIV 51 showed that the induced hiPSCs were derived from neurons and glial lineages, and 90% of these cells showed MAP2 gene expression (Fig. 4a–f). In addition, these neurons also showed the expression of synaptic markers such as SYN1, SYN2, SYP and DLG4 (Fig. 4g–j), suggesting that hiPSCs had differentiated into mature neurons. These induced iPSCs-derived neurons showed expression of glutamatergic, GABAergic, cholinergic, dopaminergic, and serotonergic neuronal markers (Fig. S3a–e). A total of 24% of MAP2-positive cells were HTR2A-positive (Fig. 4b,k). In addition, 5% of the MAP2-positive cells were ZsGreen1-positive (Fig. 4l), and the HTR2A-positive rate was 80% (4 cells/5 cells; Fig. S3f). At the same time, 18% of the HTR2A-positive cells were ZsGreen1-positive (4 cells/22 cells; Fig. S3g). These results suggested that ZsGreen1 expression reflects the gene expression of HTR2A with high specificity. This is a useful tool to identify HTR2A-positive cells among hiPSC-derived neurons. In addition, HTR2A and ZsGreen1-positive cells showed relatively high expression of glutamatergic, GABAergic and cholinergic neuron markers (Fig. 4m).
Functional properties of ZsGreen1-positive neurons
To investigate the electrophysiological properties and the responsiveness of HTR2A-positive neurons derived from lentivirus-transfected hiPSCs to a 5-HT2AR agonist, whole-cell patch-clamp recordings were performed on ZsGreen1-positive (n = 15) and negative (n = 7) neurons at DIV 42–67.
In the current-clamp configuration, all recorded cells were capable of generating repetitive action potentials (AP) in response to further depolarizing current injections (Fig. 5a,b). The mean peak AP firing frequency for ZsGreen1-positive and ZsGreen1-negative neurons were 18.7 ± 2.5 Hz and 21.4 ± 4.8 Hz respectively (Fig. 5c). The average resting membrane potential was − 60.7 ± 2.6 mV for ZsGreen1-positive and − 61 ± 1.9 mV for ZsGreen1-negative neurons (Fig. 5d). Input resistance was 741.6 ± 60.2 MΩ for ZsGreen1-positive and 774.0 ± 75.6 MΩ for ZsGreen1-negative neurons (Fig. 5e). Cell capacitance was 39.6 ± 3.3 pF for ZsGreen1-positive and 38.3 ± 5.8 pF for ZsGreen1-negative neurons (Fig. 5f). An Injection of a 300-ms depolarizing current pulse elicited action potentials with amplitude of 91.6 ± 2.5 mV for ZsGreen1-positive and 91.9 ± 3.7 mV for ZsGreen1-negative neurons (Fig. 5g) and half width 3.5 ± 0.4 ms and 2.5 ± 0.2 ms at first spike for ZsGreen1-positive and ZsGreen1-negative neurons, respectively (Fig. 5h).
We next examined the effect of TCB-2, which is a selective agonist of 5-HT2ARs, on the ZsGreen1-positive and negative neurons at a holding potential of − 60 mV under the voltage-clamp configuration. Bath application of TCB-2 (40 μM) produced an inward current (14.1 ± 1.2 pA of amplitude) in 70% of the ZsGreen1-positive neurons (n = 10) (Fig. 5i,j). In contrast, ZsGreen1-negative neurons exhibited no response to TCB-2 (n = 6). These data suggested that ZsGreen1-positive neurons derived from hiPSCs are electrophysiologically active and functionally express 5-HT2AR.
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 themselves24,25,26,27. This is a notable feature given that such cells have been technologically and ethically difficult to obtain in the past28. 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 axes20,29. In this study, using the same method with slight modifications, we further investigated the time course of the maturation of HTR2A-expressing 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 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 interacted22. 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 neurons30,31,32,33, 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 studies34,35. Regarding the 5-HT2AR-specific cell responses, application of TCB-2 generated inward currents in HTR2A-positive neurons, which was also consistent with a previous report36. These results strongly suggested that the dynamics of the neurons induced from hiPSCs was similar to that of 5-HT2AR-expressing neurons in vivo, because 5-HT2AR agonists are supposed to cause cell membrane depolarization in these neurons1,37.
In conclusion, our in vitro monitoring system allowed the identification and functional analysis of HTR2A-positive neurons induced from hiPSCs, which could be used to elucidate the pathophysiological mechanism of various neurological diseases associated with genetic variations of the HTR2A gene7,8,9,10,11,12,38,39.
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.
All the experiments were performed with the hiPSC line (C2)20. hiPSCs were generated from monocytes in peripheral blood samples of the healthy adult20 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% CO2. The medium was changed every other day.
Neuronal differentiation of hiPSCs
hiPSCs were pretreated for 5 days with 3 μM SB431542 (Tocris, Bristol, UK), 3 μM dorsomorphin (Sigma Aldrich), and 3 μM CHIR99021 (ReproCELL, Kanagawa, Japan). They were then dissociated and seeded at a density of 10 cells/μl39 in KBM Neural Stem Cell medium (Kohjin-Bio, Saitama, Japan) with selected growth factors and inhibitors under conditions of 4% O2/5% CO2. The growth factors and inhibitors included 1 × B-27 supplement (Thermo Fisher Scientific), 0.5% penicillin/streptomycin, 20 ng/ml FGF-2, 2 μM SB431542, and 3 μM CHIR99021. The neurosphere culture was started at day in vitro (DIV) 0, and neurospheres were passaged at a density of 50 cells/μl on DIV 7 and 14. The following additives were included in the neurosphere culture medium: 10 μM Y-27632 (Fujifilm, Tokyo, Japan) on DIV 0–7 and 100 ng/ml Shh-C24II (R&D Systems, Minneapolis, MN) and 1 μM purmorphamine (Millipore, Burlington, MA) on DIV 1–21. On DIV 21, neurospheres were replated on dishes coated with poly-l-lysine solution and fibronectin and cultured under conditions of 5% CO2 at a density of 40 × 104 cells/well in a 48-well plate, 150 × 104 cells/well in a 12-well plate, or 300 × 104 cells/well in a 6-well plate. The medium was changed to Neurobasal Plus Medium (Thermo Fisher Scientific) supplemented with 1 × B-27 Plus supplement (Thermo Fisher Scientific), 0.5% penicillin/streptomycin, 20 ng/ml brain-derived neurotrophic factor (BDNF; BioLegend, San Diego, CA), 20 ng/ml glial cell-derived neurotrophic factor (GDNF; Alomone Labs, Jerusalem BioPark, Israel), 0.2 mM ascorbic acid (Sigma Aldrich), 0.5 mM dbcAMP (Nacalai Tesque, Kyoto, Japan), 1 ng/ml transforming growth factor-β (TGF-β; BioLegend), 10 μM DAPT (Sigma Aldrich), and 3 μM CHIR99021. Half of the volume of medium was replaced with new medium (including all supplements except CHIR99021) every 3 or 4 days.
Reverse transcription and quantitative polymerase chain reaction (RT-qPCR)
Total RNA was isolated with the RNeasy mini kit (QIAGEN, Hilden, Germany) and the RNeasy micro kit (QIAGEN) with DNase I treatment. cDNA was prepared by using SuperScript First-Standard (Thermo Fisher Scientific) or the TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific). qRT-PCR analysis was performed on a QuantStudio 7 Flex (Thermo Fisher Scientific). Values were normalized to ACTB expression. Reactions were carried out in duplicate, and data were analyzed by using the comparative (ddCt) method. Relative expression levels are presented as geometric means ± geometric standard error of the mean (s.e.m.). The primer sets used in the SYBR Green assay are listed in Table 1. Custom primers for the GAPDH gene (PN4453320; Thermo Fisher Scientific) and HTR2A gene (PN4448892; Thermo Fisher Scientific) were used in the TaqMan assay.
Cells were subsequently fixed with 4% paraformaldehyde for 30 min at room temperature and then washed three times with phosphate-buffered solution (PBS). After incubating with blocking buffer (PBS containing 5% normal fetal bovine serum, 0.01% sodium azide, and 0.3% Triton X-100) for 15 min at room temperature, the cells were incubated overnight at 4 °C with primary antibodies diluted with PBS containing 0.05% Triton X-100. As primary antibodies, anti-βIII-tubulin (1:500; Biolegend), anti-5-HT2AR (1:200; Millipore), and anti-MAP2 (1:5000; Abcam, Cambridge, UK) antibodies were used. The cells were again washed three times with PBS and incubated with secondary antibodies conjugated with Alexa Fluor 488 (1:500; Thermo Fisher Scientific), Alexa Fluor 594 (1:500; Thermo Fisher Scientific) for 1 h and Hoechst 33342 (1:1000; Sigma Aldrich) for 10 min at room temperature. After washing three times with PBS, samples were mounted on slides and examined by using an all-in-one fluorescence microscope (Keyence, Osaka, Japan).
Transduction and detection of HTR2A promoter activity-induced ZsGreen1 expression in hiPSCs
For lentiviral packaging, the pLV-hHTR2Apro-ZsGreen1 plasmid containing the HTR2A promoter sequence was co-transfected with the Lentiviral High Titer Packaging Mix (Takara, Shiga, Japan) using Fugene HD (Promega, Madison, WI) into LentiX-293 T cells (Z2180N; Takara). The 48-h–cultured supernatant was filtrated through 0.45-μm pore cellulose acetate filters (Millipore) and concentrated using Centriprep Centrifugal Filters (Millipore). Viral titration was determined by flow cytometric quantification of ZsGreen1-positive cells. Lenti-HTR2A-ZsGreen1 particles were transduced into hiPSCs at a multiplicity of infection (MOI) of 10 (Fig. S1). Flow cytometry was performed to measure HTR2A promoter activity that induced ZsGreen1 expression in hiPSCs. The lentivirus was transduced into hiPSCs at DIV 22 and hiPSCs were collected at DIV 41 and 51. As a negative control of HTR2A, the lentivirus was transduced into hiPSCs at DIV-10 and hiPSCs were collected at DIV-5. ZsGreen1-positive cells were sorted using the SH800 cell sorter (Sony, Tokyo, Japan). Dead cells were excluded by staining with Fixable Viability Dye eFlour 450 (Thermo Fisher Scientific) before fluorescence-activated cell sorting analysis.
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.
HTR2A transfected hiPSCs at DIV 51 were used for single-cell RNA-seq. The transcriptome library was prepared using Chromium Single Cell 3′ Reagent Kits v3 (10× Genomics) according to the manufacturer’s instructions. The single-cell RNA-seq libraries were sequenced using the DNBSEQ-G400 platforms (MGI Tech Co., Ltd.). Data analysis was performed with Cell Ranger (v4.0.0) and Seurat (v4.0.0).
Whole-cell patch-clamp recordings were performed at room temperature under continuous perfusion with an extracellular solution containing (in mM): NaCl (130), NaHCO3 (26), glucose (10), KCl (3), CaCl2 (2), MgCl2 (2), and NaH2PO4 (1.25). The solution was oxygenated with 95% O2 and 5% CO2, to establish a pH of 7.4. HTR2A-positive hiPSCs were identified under fluorescence and infrared differential interference contrast microscopy on an upright microscope (BX51WI; Olympus, Tokyo, Japan) using a 40× water immersion objective, and selected for recordings based on a spherical and bright cell body. ZsGreen1 reporter fluorescence was detected with a U-MNIBA3 filter cube (excitation: 460–495 nm, dichroic mirror: 505 nm, emission: 510–550 nm; Olympus, Tokyo, Japan) and processed with MetaMorph software (MetaMorph; Molecular Devices, Sunnyvale, CA). Patch pipettes were pulled from borosilicate glass (GD-1.5; Narishige, Tokyo, Japan) with a P-97 puller (Sutter, Atlanta, GA). Pipettes were filled with an internal solution containing (in mM): KCl (10), K-gluconate (130), HEPES (10), EGTA (0.4), MgCl2 (2), Na2-GTP (0.3), and Mg-ATP (2), pH 7.25, 285–300 mOsm. The pipette resistance ranged from 2.5 to 5.0 MΩ when the electrodes were filled with the internal solution. Membrane potentials and currents were recorded with a MultiClamp 700B amplifier and a Digidata 1440A (Molecular Devices) and the data were filtered at 10 kHz, digitized at 20 kHz, and analysed using pCLAMP 10.7 software (Molecular Devices) and Origin 2019 (OriginLab). The measured liquid junction potential of 10 mV was subtracted from all membrane potentials. The cell capacitance and input resistance were calculated under voltage clamp mode from the current to a 10-mV hyperpolarizing step pulse at a holding potential of − 60 mV. TCB-2 induced inward currents were defined as downward deflections of more than 2 standard deviations above baseline.
Statistical analysis was performed with Student’s t-test or one-way analysis of variance (ANOVA) with post hoc Dunnett’s test. The data are presented as the mean ± s.e.m.
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The authors thank Dr. Hideyuki Okano, Professor of Department of Physiology, Keio University School of Medicine, Tokyo, Japan, for providing iPSCs. This work was supported in part by JSPS KAKENHI Grant Numbers 17H04395, 17K17191.
The authors declare no competing interests.
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Nakai, K., Shiga, T., Yasuhara, R. et al. In vitro monitoring of HTR2A-positive neurons derived from human-induced pluripotent stem cells. Sci Rep 11, 15437 (2021). https://doi.org/10.1038/s41598-021-95041-3