Human embryonic stem cells

BR1 lineage of human embryonic stem cells (hESCs)78 was cultured in mTeSR1 media (Stemcell Technologies) on Matrigel (BD Biosciences) - coated surface. The colonies were manually passaged every seven days and maintained at 37 °C in humidified air with 5% CO 2 .

Human neural progenitor cells

To induce hESCs towards neural differentiation, we adapted Baharvand and coworkers’ protocol28,79. Briefly, 70% confluent human embryonic stem cells were differentiated to the neural lineage in defined adherent culture by retinoic acid and basic fibroblast growth factor (bFGF) within 18 days of culture. On the 18th day, neural tube-like structures were collected and plated on dishes coated with 10 μg/mL of poly-L-ornithine and 2.5 μg/mL of laminin (Thermo Fisher Scientific). The population of human neural progenitor cells (hNPCs) that migrated from neural tube-like structures was tested for the expression of neuronal markers and expanded. Expansion was done in N2B27 medium supplemented with 25 ng/mL bFGF and 20 ng/mL EGF (Thermo Fisher Scientific). N2B27 medium consisted of DMEM/F-12 supplemented with 1X N2, 1X B27, 1% penicillin/streptomycin (Thermo Fisher Scientific). Cells were incubated at 37 °C and 5% CO 2 . Medium was replaced every other day. hNPCs were expanded for no more than 5 passages. Basic characterization of this culture was published in28.

High Content Screening

Cell proliferation, cell death and arborization experiments were performed in a High Content Screening (HCS) format. hNPCs (1,500 cells per well) were plated on a 384-multiwell μClear plate (Greiner Bio-One, Kremsmünster, Austria) coated with 100 μg/mL poly-L-ornithine and 10 μg/mL laminin (Thermo Fisher Scientific). After 24h, cells were treated for 4 days in quintuplicate (five wells per condition) with 5-MeO-DMT (Sigma-Aldrich) in N2B27 medium supplemented with bFGF and EGF. Cells were labeled with 10 μM EdU for 2h min prior to fixation and image acquisition. For the cell death assessment, cells were labeled with LIVE/DEAD® viability/cytotoxicity kit (Thermo Fisher Scientific). This kit contains two probes: calcein AM and ethidium homodimer (EthD-1). The former allows measuring of intracellular esterase activity and the latter, plasma membrane integrity. Mixing of probes was done in DMEM/F-12 (without phenol red, Life Technologies), together with the cell-permeant nuclear dye Hoechst. After incubation for 30 min at 37 °C and 5% CO 2 , the dye cocktail was replaced by new medium and images were acquired. For arborization experiments, neural differentiation was induced 24h after plating by removal of bFGF and EGF from N2B27 medium. Treatment with 5-MeO-DMT was done concomitantly with neural differentiation. Medium was changed after 4 days of treatment and cells were allowed to differentiate for 3 more days. On day 7 cells were fixed for immunocytochemistry.

High Content Analysis

All images were acquired on Operetta high-content imaging system (Perkin Elmer, USA). For proliferation, incorporated EdU was detected with Alexa Fluor 488 using Click-iT EdU kit (C10351, Invitrogen, Carlsbad, USA) following the manufacturer’s instructions. Total number of cells was calculated by nuclei stained with 1 mg/mL of DAPI (4’,6-diamidino-2-phenylindole). S phase was determined by percentage of total cells labeled with EdU. Images were acquired with a 10x objective with high numerical aperture (NA).

Live cell imaging was performed with LIVE/DEAD® viability/cytotoxicity kit, using temperature and CO 2 control option (TCO) of Operetta, set to 37 °C and 5% CO 2 at 10x magnification. Quantification analyses were normalized to the number of cells in the well segmented by nucleus dyes.

Neuronal arborization was evaluated of fixed cells stained for MAP2 after 7 days of differentiation. The images were analyzed using the Neurite Outgrowth script of the Harmony software. Briefly, neurites were detected on MAP2 positive cells using the Find Neurite building block, which provides a dedicated algorithm for segmenting neurites. Morphological characteristics of neuronal arborization, such as total neurite length (sum of the length of all neurites attached to the cell), number of extremities, number of segments, and number of nodes (type I) were defined based on selected threshold parameters of the Find Neurite building block.

All analysis sequences were designated by combining segmentation steps with morphological- and fluorescence-based object characterizations using the image analysis software Harmony 3.5.1 (Perkin Elmer, Waltham, MA, USA).

Differentiation into cerebral organoids

Differentiation of hESCs into cerebral organoids was based on a previously described protocol26,29. Briefly, hESC cells were inoculated into a spinner flask, and to enable embryoid body formation, after six days the medium was changed to neural induction media (DMEM/F12, 1X N2 supplement (Gibco), 2 mM Glutamax (Invitrogen), 1% MEM-NEAA, and 1 μg/mL heparin (Sigma)) and the aggregates were cultured for five more days. After being embedded in matrigel, differentiation media composed of 1:1 DMEM/F12:Neurobasal (Gibco), 0.5X N2, 1X B27 minus vitamin A (Gibco), 2 mM Glutamax, 0.5% MEM-NEAA, 0.2 μM 2-mercaptoethanol and 2.5 μg/mL insulin was used. After 4 days, cell aggregates were grown in neuronal differentiation media, composed as aforementioned except by replacing with 1X B27 containing vitamin A (Gibco). The medium was changed once per week. Cerebral organoids were grown for 45 days (30 days in neuronal differentiation media).

RNA isolation and PCR analysis

Total RNA was isolated using the GeneJET RNA Purification Kit (Thermo Scientific) and digested with DNase using DNase I (Invitrogen), following the manufacturer’s instructions. Complementary DNA was generated from 1 μg total RNA using M-MLV Reverse Transcriptase (Invitrogen), according to the manufacturer’s recommendations. PCR was performed using the following primer sequences: GFAP-For: 5′-TTC GAC AGT CAG CCG CAT C-3′ GFAP-Rev: 5′-GAC TCC ACG ACG TAC TCA GC-3′, Sigma receptor 1-For: 5′-AGT AGG ACC ATG CAC TCA CAC C-3′, Sigma receptor 1-Rev: 5′-CCC CAT CCT TAA CTC TAG AAC C-3′, 5-HT 2A -For: 5′-TTG GGC TAC AGG ACG ATT-3′, 5-HT 2A -Rev: 5′-GAA GAA AGG GCA CCA CAT C-3′, 5-HT 2C -For: 5′-TGT CCC TAG CCA TTG CTG ATA TGC-3′, 5-HT 2C -Rev: 5′-GCA ATC TTC ATG ATG GCC TTA GTC-3′. Each PCR reaction was carried out for 40 cycles in a reaction mixture containing 0.25 U Taq DNA Polymerase (Invitrogen), 1x Taq DNA Polymerase Buffer containing 1.5 mM MgCl 2 (Invitrogen), 200 nM of each primer (forward and reverse), 200 μM dNTP mixture containing the four deoxyribonucleotides (dATP, dCTP, dTTP, dGTP), and 15 ng of cDNA.

Immunohistochemistry

On the 45th day of differentiation, cerebral organoids were fixed in 4% paraformaldehyde, incubated with sucrose solutions (10, 20, and 30%) in phosphate buffered saline (PBS), embedded in optimal cutting temperature compound (OCT), and frozen in liquid nitrogen. The organoids were sectioned with a cryostat into 20 μm thick sections. Immunofluorescence was performed using the primary antibodies: anti-MAP2 (M1406, Sigma-Aldrich), anti-AMPAR1 (Abcam, ab86141), anti-NMDAR1 (Abcam, ab28669), anti-sigma receptor 1 (sc-137075, Santa Cruz), and anti-5-HT 2A receptor (RA24288, Neuromics). Secondary antibodies used were as follows: Alexa Fluor 488 goat anti-mouse (A11001, Invitrogen) and Alexa Fluor 594 goat anti-mouse (A-11008, Invitrogen). DAPI was used for nucleus staining. Images were acquired using an Operetta Imaging System (Perkin Elmer) and a Leica TCS SP8 confocal microscope, when specified.

Treatment of cerebral organoids with 5-MeO-DMT

On day 45 of differentiation, four to five organoids per group were transferred from the spinner flask to a non-adherent dish and treated with either 13 µM 5-MeO-DMT (Sigma-Aldrich), 0.3% ethanol (vehicle) or only medium (control), for 24 hours. After treatment, cerebral organoids were pelleted and homogenized in buffer containing 7 M Urea, 2 M thiourea, 4% CHAPS, 70 mM DTT, and Complete Protease Inhibitor Cocktail (Roche)80. The homogenates were kept on ice for about 20 min and frozen at −80 °C until sample processing for mass spectrometry-based label-free shotgun proteomics. The experiment was repeated three times with the three different derivations of cerebral organoids.

Sample preparation

Sample lysates were thawed and centrifuged at 10,000 × g for 10 min at 4 °C. The supernatant was collected and total protein was quantified by Qubit® 3.0 Fluorometer (Thermo Fisher Scientific). Each sample (50 µg) was subjected to a SDS-PAGE gel electrophoresis. Gel lanes were sliced and digested in gel overnight as previously described80. Generated peptides were dried in a SpeedVac concentrator and stored at −80 °C prior to shotgun mass spectrometry analyses.

Liquid chromatography-mass spectrometry

Qualitative and quantitative proteomic analyses were performed on a 2D-LC-MS/MS system with ion-mobility-enhanced, data-independent acquisitions81. Peptides were injected for two-dimensional, reverse-phase liquid chromatography using an Acquity UPLC M-Class System (Waters Corporation, Milford, MA) coupled to a Synapt G2-Si mass spectrometer (Waters Corporation, Milford, MA).

In first-dimension chromatography, peptides (5 µg) were loaded into a M-Class BEH C18 Column (130 Å, 5 µm, 300 µm × 50 mm, Waters Corporation, Milford, MA). Fractionation was performed using discontinuous steps of acetonitrile (11%, 14%, 17%, 20%, and 50%). After each step, peptide loads continued to second-dimension separation, in a nanoACQUITY UPLC HSS T3 Column (100 Å, 1.8 µm, 75 µm × 150 mm, Waters Corporation, Milford, MA). Peptide elution was achieved using an acetonitrile gradient from 7% to 40% (v/v) for 54 min at a flow rate of 0.4 µL/min directly into a Synapt G2-Si. The mass spectrometer acquired in data-independent acquisition mode (DIA) with ion-mobility separation. This approach, called high-definition data-independent mass spectrometry (HDMSE), significantly enhances the proteome coverage82. MS/MS analyses were performed by nano-electrospray ionization in positive ion mode, nanoESI (+), and used a NanoLock Spray (Waters, Manchester, UK) ionization source. The lock mass channel was sampled every 30 s. The mass spectrometer was calibrated with an MS/MS spectrum of a [Glu1]-Fibrinopeptide B human (Glu-Fib) solution that was delivered through the reference sprayer of the NanoLock Spray source. Samples were all run in technical and biological triplicates, for a total of 9 replicates per sample.

Database search and quantification

Raw data was processed with Progenesis® QI version 2.1 (Waters) and proteins were identified. Quantitative data was processed using dedicated algorithms and searched against the Uniprot human proteomics database (version 2015/09), with the default parameters for ion accounting and quantitation83. The databases used were reversed “on the fly” during the database queries and appended to the original database to assess the false-positive identification rate. The following parameters were considered in identifying peptides: 1) Digestion by trypsin with at most one missed cleavage; 2) variable modifications by oxidation (M) and fixed modification by carbamidomethyl (C); and 3) false discovery rate (FDR) less than 1%. Identifications that did not satisfy these criteria were not considered.

In silico analysis

Protein networks and canonical pathways associated with differentially expressed proteins were identified using Ingenuity Pathway Analysis software (IPA, Ingenuity Systems, Qiagen, Redwood, CA, USA; www.ingenuity.com). This software uses curated connectivity information from literature to determine interaction networks among the differentially expressed proteins and canonical pathways in which they are involved. Here, we have considered information from nervous system tissues and cells, immune cells, and stem cells. The significant biological functions were based on Fisher’s exact test. Multiple correlation hypotheses were based on Benjamini-Hochberg (B-H) approach using a 1% FDR threshold; the significance of the IPA test was expressed as p-values.