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. 2023 Oct 5;14(20):3761–3771. doi: 10.1021/acschemneuro.3c00246

Quantitative and Qualitative Analysis of Neurotransmitter and Neurosteroid Production in Cerebral Organoids during Differentiation

Sung Bum Park †, Byumseok Koh †, Hyun Soo Kwon ‡,§, Young Eun Kim ‡,§, Seong Soon Kim †, Sung-Hee Cho ∥, Tae-Young Kim §, Myung Ae Bae †,*, Dukjin Kang ‡,*, Chul Hoon Kim ⊥,*, Ki Young Kim †,*
PMCID: PMC10587864  PMID: 37796021

Abstract

graphic file with name cn3c00246_0006.jpg

In the human brain, neurophysiological activity is modulated by the movement of neurotransmitters and neurosteroids. To date, the similarity between cerebral organoids and actual human brains has been evaluated using comprehensive multiomics approaches. However, a systematic analysis of both neurotransmitters and neurosteroids from cerebral organoids has not yet been reported. Here, we performed quantitative and qualitative assessments of neurotransmitters and neurosteroids over the course of cerebral organoid differentiation. Our multiomics approaches revealed that the expression levels of neurotransmitter-related proteins and RNA, including neurosteroids, increase as cerebral organoids mature. We also found that the electrophysiological activity of human cerebral organoids increases in tandem with the expression levels of both neurotransmitters and neurosteroids. Our study demonstrates that the expression levels of neurotransmitters and neurosteroids can serve as key factors in evaluating the maturity and functionality of human cerebral organoids.

Keywords: cerebral organoids, neurotransmitters, neurosteroids, lipids, proteomics, neurophysiological activity

Introduction

The cerebral cortex is a part of the central nervous system (CNS) that controls motions, feelings, and emotions through neural activity.1,2 This activity is mediated by the exchange of neurotransmitters and neurosteroids. For centuries, scientists have been studying the brain to understand its mechanisms and functions.3–5 However, the brain is a complex and vulnerable organ, making it difficult to access and study.6,7 Additionally, there are ethical concerns about using animals in research.8,9 As a result, there is still a large gap in our understanding of the human brain.10

Human cerebral organoids are miniature organs that resemble the brain and are artificially grown in vitro.11,12 They are derived from stem cells and closely mimic the complex neural structure of the actual human brain.13,14 The first human cerebral organoids were reported by Knoblich et al. in 2013.15 Since then, numerous studies have been reported on culturing and differentiating cerebral organoids using embryonic stem cells and induced pluripotent stem cells (iPSCs).16–20 In addition to studies of the organoids that resemble the human cerebral cortex, several other studies have reported the formation of organoids that resemble other brain parts. Some studies have even combined multiple brain regions that differentiate from different cell lineages, which are called assembloids.21–23 Numerous culturing and differentiation methods for cerebral organoids have been reported. Most recently, a study by Pasca et al. showed that the organoids they created resembled the early stage of human cortical layers.13 The similarity of cerebral organoids to the human brain is usually assessed by comparing gene expression patterns and the degree of differentiation with total RNA sequencing results and proteomics results from human brains.13,14

A neurotransmitter is a chemical messenger released by neurons that affects other cells across a synapse. The cell receiving the signal, also known as the target cell, can be another neuron, a gland, or a muscle cell.24–27 Neurotransmitters are released from synaptic vesicles into the synaptic cleft, where they bind to the neurotransmitter receptors on the target cell.28–30 The effect of the neurotransmitter on the target cell is determined by the type of receptor it binds to.31,32 Many neurotransmitters are synthesized from simple and plentiful precursors, such as amino acids, which are readily available and can be converted with only a few steps.33,34

Neurosteroids are endogenous or exogenous steroids that can rapidly alter neuronal excitability by interacting with ligand-gated ion channels and other cell surface receptors.35,36 They have a wide range of potential clinical applications, including sedation, the treatment of epilepsy,37,38 and the treatment of traumatic brain injury.39,40 Ganaxolone, a synthetic analog of the endogenous neurosteroid allopregnanolone, is under investigation for the treatment of epilepsy.41,42

Although single-cell RNA sequencing comparison can be used as a basis for comparing the similarity of human brains and organoids, neural activity in the human brain begins with the movement of neurotransmitters and neurosteroids between neurons. The production of neurotransmitters and neurosteroids in cerebral organoids has not yet been measured, but this could be direct evidence of their resemblance to actual functioning brains. Therefore, it is important to systematically analyze their production and compare it to their differentiation stage and functionality.

Here, we carefully analyzed neurotransmitter and neurosteroid and lipid production in human cerebral organoids. The amount of neurotransmitter and neurosteroid produced from cerebral organoids was analyzed with ultrasensitive liquid chromatography, and its corresponding proteomic analysis was conducted using a two-dimensional nanoflow liquid chromatography-tandem mass spectrometry (2D-nLC-MS/MS) in order to produce accurate data.

Results

Growth and Electrophysiological Activity of Cerebral Organoids

Cerebral organoid size and neuronal marker changes were monitored. Cerebral organoids grew rapidly during the neural induction and neural stem cell expansion phases and reached a plateau at approximately 90 days of culture (Figure 1A,B). During this period, the organoid diameter increased from approximately 50 nm to 3 × 104 μm and maintained a spherical shape (Figure 1B). Microtubule-associated protein 2 (MAP2) appeared at approximately 20 days of culture, and the expression level of SRY (sex-determining region Y)-box 2 (SOX2) decreased as the cerebral organoids matured (Figure 1C). To evaluate the electrophysiological activity of the cerebral organoids, multielectrode array (MEA) signal recording was conducted. No noticeable MEA signal was observed at approximately 43 days of organoid culture, and electric activity appeared at approximately day 85 of culture (Figure 1D). The mean firing rate and synchronized electric burst were increased during this period (Figure 1E,F). The MEA signal from the cerebral organoids peaked at approximately 120 days of culture.

Figure 1.

Figure 1

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Cerebral organoid culture scheme and electrophysiological signaling from mature cerebral organoids. (A) Cerebral organoid culture scheme, (B) diameter changes of cerebral organoids, and (C) immunofluorescence of 120-day cultured organoids. Electrophysiological activity of (D) mean firing rate, (E) spike and waveform, (F) number of bursts and number of network bursts from cerebral organoids with MEA recording. Error bars represent the standard error of the mean from six independent experiments. Statistical significance is denoted as # for P < 0.05, ## for P < 0.01, and ### for P < 0.001.

Neuronal Maturation of Cerebral Organoids

To examine the neural maturation of cerebral organoids during culture, immunohistochemistry was conducted. The data suggest that the expression levels of B-cell lymphoma/leukemia 11B (CTIP2), glial fibrillary acidic protein (GFAP), nestin, β3-tubulin, and hexaribonucleotide-binding protein-3 (NeuN) were increased as cerebral organoids matured, while the expression levels of paired box protein Pax-6 (PAX6), t-box, brain-1 (TBR1), t-box, brain-2 (TBR2), and epithelial cadherin (E-cadherin) gradually decreased (Figure 2A). Special AT-rich sequence-binding protein 2 (SATB2) appeared after 75 days of cerebral organoid culture (Figure 2A). Cerebral organoids were further collected, and total RNA sequencing analysis was conducted (Figure 2B, Table S1). The expression levels of key cell growth-related and differentiation-related transcriptomes BDNF, NRG2, and IL6R determined by qPCR were increased as cerebral organoids matured (Figure 2C). Transcriptomes related to neurogenesis, including cerebellin 1 precursor (CBLN1) and neural egfl-like 1 (NELL1) (Figure 2D), and 10 transcriptomes related to neuronal cell differentiation [including neurogenic differentiation 1 and 2 (NEUROD1 and NEUROD2), (Figure 2E)] were also increased during cerebral organoid differentiation.

Figure 2.

Figure 2

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Differentiation profile of cerebral organoids. (A) Immunofluorescence of 38, 60, 70, and 120 days cultured cerebral organoids. (B) Total RNA sequencing results of neurogenesis and cell growth-related RNA expression levels from the cerebral organoids. RNA expression levels of (C) cell growth and differentiation, (D) neurogenesis, and (E) neuronal cell differentiation-related genes in the cerebral organoids determined by qPCR. Error bars represent the standard error of the mean from three independent experiments.

Neurotransmitter Production in Cerebral Organoids

After confirming cerebral organoid growth and neuronal differentiation during >120 days of culture, we quantitatively analyzed the expression levels of neurotransmitters using LC–MS/MS in cerebral organoids. Our results showed that the expression levels of six neurotransmitters we detected in cerebral organoids were significantly increased during the maturation of cerebral organoids (Figure 3A). We further assessed the expression levels of adrenergic-, cholinergic-, dopaminergic-, serotonergic-, gamma-aminobutyric acid (GABA)-ergic-, and glutamatergic-receptor-related transcriptomes during cerebral organoid maturation, and our data confirmed that the expression levels of neurotransmitter-related transcriptomes increased (Figure 3B–H). The expression levels of pan-GABAergic- and glutamatergic-differentiation-related markers were assessed with the immunofluorescence (IF) of mature cerebral organoids. IF of cerebral organoids were cultured for 120 days, and the results suggested that the expression levels of glutamate decarboxylase 67 (GAD67), which catalyzes the decarboxylation of GABA, and vesicular glutamate transporter 1 (vGLUT1), which is associated with glutamate transport, were increased during cerebral organoid culture (Figure 3I).

Figure 3.

Figure 3

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Neurotransmitter production from cerebral organoids. (A) Detected levels of eight neurotransmitters from cerebral organoids cultured for 25, 70, and 120 days. (B) Total RNA sequencing results of neurotransmitter-related RNA expression levels from the cerebral organoids. RNA expression levels of (C) adrenergic-, (D) cholinergic-, (E) dopaminergic-, (F) serotonergic-, (G) GABAergic-, and (H) glutamatergic-receptor-related genes in the cerebral organoids determined by qPCR. (I) Immunofluorescence of cerebral organoids cultured for 43 days (left) and 120 days (right). Error bars represent the standard error of the mean from three independent experiments.

Metabolic Profiling in Cerebral Organoids

To investigate the metabolic alteration effects on the maturation of cerebral organoids, nontargeted metabolic profiling was performed by LC–MS/MS after sample preparation steps. In the metabolic profile analysis, 5613 positive ion pattern features (ESI+ mode) and 19 features (ESI– mode) were found. After data processing by MarkerLynx XS, the CVS files were imported to Metaboanalyst 5.0, and the metabolic characteristics with significant differences were screened based on the statistical analysis (one factor) function. Data from principal component analysis (PCA) of metabolites from cerebral organoids suggest that cerebral organoids reach a mature state at approximately 70 days of culture (Figure 4A). Nine metabolites, including lipids [PC34:2, PC35:3, LPE24:6, and Cer (18:1/24:0)], neurosteroids (20α-OH progesterone, progesterone, and pregnenolone), and others (butenylcarnitine and hexadecylamine), were significantly altered (Figure 4B–E).

Figure 4.

Figure 4

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Neurosteroid and metabolite production from cerebral organoids. (A) PCA of metabolites produced by the cerebral organoids, (B) glycerophospholipids, (C) sphingolipids, (D) progestins, and (E) amine production levels from the cerebral organoids. Error bars represent the standard error of the mean from three independent experiments.

Quantitative Proteomic Profiling of Cerebral Organoids

To investigate the temporal proteomic changes of cerebral organoids during neuronal differentiation, we performed tandem mass tag (TMT)-based quantitative proteomic analysis of cerebral organoids at multiple time points (days 0, 25, 43, 70, and 120) using 2D-nLC-MS/MS analysis. A total of 4657 proteins with at least two peptide hits were commonly quantified across five different time points (Table S2). By performing an ANOVA test adjusted with a Benjamini–Hochberg correction, 286 significant proteins were identified (Table S3). To classify the groups of proteins with distinct expression patterns during neuronal differentiation of organoids, we performed a hierarchical clustering analysis of ANOVA significant proteins, followed by a functional enrichment analysis including Gene Ontology (GO) biological process and reactome pathway. Hierarchical clustering analysis revealed the segregation of proteins into two main clusters (Figure 5A and Table S4). Cluster #1 contains 154 proteins that were progressively downregulated from day 25 to day 120 of culture (Figure 5B). The results of GO and reactome pathway analysis showed that proteins in cluster #1 were related to mRNA processing, DNA replication, and ribosome biogenesis. In contrast, cluster #2 comprised 128 proteins whose expression level gradually increased from day 25 to day 120 of culture (Figure 5B). Many proteins involved in neuronal functions including synapse organization, synaptic vesicle cycle, regulation of neurotransmitter levels, and neurotransmitter release cycle were significantly enriched in cluster #2. Heat maps of proteins related to synapse organization (n = 16) and regulation of neurotransmitters (n = 13) are presented in Figure 5C. In addition, we showed the temporal expression levels of representative presynaptic proteins (SYT1 and SYN1), postsynaptic proteins (HOMER1 and CAMK2B), and cell-adhesion proteins (NEGR1 and CNTN1) (Figure 5D). These neuronal proteins showed a rapid increase in expression from day 43 to day 70 of culture.

Figure 5.

Figure 5

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Quantitative proteomic profiling of human cerebral organoids during neuronal differentiation. (A) Heat map and hierarchical clustering analysis of 286 ANOVA significant proteins. Red and green colors indicate high and low expression levels, respectively. (B) Average expression profiles and functional annotations (GO biological process and reactome pathway) of proteins in clusters #1 and #2. (C) Heat maps of proteins involved in the synapse organization (left) and regulation of neurotransmitters (right). Red and green colors indicate high and low expression levels, respectively. (C) Temporal expression profiles of presynaptic proteins (SYT1 and SYN1), postsynaptic proteins (HOMER1 and CAMK2B) and cell-adhesion proteins (NEGR1 and CNTN1). Protein expression levels were normalized to day 0. Error bars represent the standard error of the mean.

Discussion

Immunohistochemistry staining results suggest that expression levels of key neuronal genes in cerebral organoids matched with previously reported data.14 Specifically, expression levels of SATB2, CTIP2, and GFAP gradually increased during cerebral organoid maturation, while expression levels of PAX6 decreased. In addition, neuronal differentiation and glial cell population increased from approximately 43 to 120 days of culture, in contrast to cerebral organoid size. We deduce that vitamin A, BDNF, cAMP, and ascorbic acid in neural maturation media account for the neural maturation of the cerebral organoid. Overall, our data suggest that cerebral organoids not only grow in size but also mature over the course of time of the culturing periods.

Quantitative analysis of neurotransmitters produced by cerebral organoids suggests that neuronal enzymes responsible for the production of neurotransmitters were elevated during cerebral organoid differentiation, suggesting that the histaminergic, cholinergic, serotonergic, dopaminergic, and GABAergic nervous systems were well formed in a manner similar to those of the human brain. In particular, our cerebral organoids showed a dramatic increase in the level of GABA, an inhibitory neurotransmitter, by 9- and 23-fold on days 70 and 120, respectively, compared to day 25, suggesting that GABAergic differentiation or proliferation is rapidly increasing in the early maturation of the cerebral organoid.

Neurosteroids, as steroids synthesized within the brain, produce rapid actions on neuronal substrates in the CNS. They are endogenous modulators of brain development and function by both genomic (classical intracellular steroid receptors)50,51 and nongenomic rapid actions (ion channels and membrane receptors).52,53 In this study, the levels of progesterone and its metabolites, which modulate the GABAa receptor, were increased with the maturation of organoids. The lipids in the brain are both structural components of cell membranes and act as regulators. Lipids account for approximately half of the brain tissue dry weight, and their quantity in the brain varies temporally with age.54 Phospholipids (PC34:2, PC35:3, LPE24:6) are the main lipids in the brain, and ceramides [Cer(18:1/24:0)] associated with brain aging are increased with the maturation of organoids. These results indicate that an increase in neurosteroids and lipids as regulators in brain development can induce organoid maturation. Studies suggest that neurotransmitter production and release increase during brain development. This is essential for forming new neural connections and refining existing neurons.55,56 Neurosteroids also play a role in brain development by regulating the expression of genes that code for neurotransmitters.57,58 The exact relationship between neurotransmitter and neurosteroid levels and brain development is still being investigated. However, it is clear that these two factors are essential for shaping a developing brain.

The expression levels of transcriptomes related to neurosteroids and neurotransmitters were analyzed. The RNA expression levels of neurosteroid- and neurotransmitter-related receptors showed a strong correlation with the actual neurotransmitter and neurosteroid expression levels. The expression of neurosteroids as well as neurotransmitters was also related to the organoid’s growth phase and differentiation phase, as their levels increased rapidly during 25 and 70 days of organoid culture.

In the quantitative profiling of the cerebral organoid proteome, we found that the expression levels of neuronal proteins were significantly upregulated during differentiation of cerebral organoids. Furthermore, it showed that diverse neuronal-function-related proteins implicated in synapse organization, synaptic vesicle cycle, and regulation of neurotransmitters cerebral organoids are dramatically changed during development. Most neuronal proteins showed a gradual increase in expression from day 25 to day 120 of culture and especially a rapid change in expression between day 43 and day 70 of culture. Notably, synaptic proteins that play a role in synaptic vesicle cycle and neurotransmitter release, such as synapsins (SYN1, SYN2, and SYN3) and synaptotagmin-1 (SYT1), also showed a continuous increase in expression from day 25 to day 120 of culture. In addition, we observed that expression of proteins related to mRNA processing, DNA replication, and ribosome biogenesis were continuously downregulated from day 25 to day 120 of culture. These proteomic results suggest that neuronal maturation of cerebral organoids occurred approximately between days 70 and 120 of culture at protein levels, whereas proliferation activity of cerebral organoids was decreased during maturation of organoids.

Our data suggest that proteomic and RNA expression levels of neuronal markers, synaptic organization, and neurotransmitter receptors are reasonably corroborated. However, not all proteomic and transcriptomic expression levels of genes were correlated, probably due to discrepancies between the timing of genetic changes and their translation into proteins.

The functionality of cerebral organoids was monitored using MEA. Our data showed that the electrophysiological activity of cerebral organoids increased rapidly on approximately day 85 of culture and reached a plateau on approximately day 90. This correlates well with the expression level of neurosteroids and neurotransmitters as well as their related RNA and proteins. As the protein expression level, RNA expression level, and functionality of cerebral organoids were correlated, our data suggest that neurotransmitter and neurosteroid levels in the cerebral organoids are strong indicators of their maturity and differentiation.

The findings of this study could have significant implications for our understanding of brain-related diseases such as Alzheimer’s, Parkinson’s, and schizophrenia. By understanding the maturation and differentiation of cerebral organoids better, we may be able to develop new diagnostic and treatment strategies for these conditions. Additionally, the identification of neurosteroids and neurotransmitters as potential indicators of organoid maturity and function could lead to more accurate and reliable assessments of organoid quality, which could have important implications for drug discovery and development. Although long-term cultured and fully differentiated human cortical organoids can only mimic postnatal stages between 200 and 300 days,59 we are currently working to overcome this limitation by finding new molecules to accelerate and fully differentiate cerebral organoids. Additionally, the spatial distribution of neurotransmitters and neurosteroids within cerebral organoids is an interesting research topic that we are currently planning to investigate. Overall, the results of this study have the potential to significantly advance our understanding of the brain and its functions and could ultimately lead to new treatments for a range of neurological and psychiatric disorders.

Methods

Cerebral Organoid Culture

Cerebral organoids were cultured and differentiated for >120 days according to a previous protocol with slight modification.23 iPSCs IMR90-4 (iPSCs, WiCell, Madison, WI, USA) were seeded to a 6-well cell culture plate (Corning, Corning, NY, USA) coated with 120× diluted matrigel (Corning) with Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F-12, Invitrogen, Carlsbad, CA, USA). On day 0, IMR90-4 cells were maintained in the presence of mTeSR1 (Stem Cell Technologies, Vancouver, Canada) supplemented with 1 μM Y-27632 (Tocris, Bristol, UK). Media were replaced every day with mTeSR1 without Y-27632 from day 1. For cerebral organoid formation, 2 × 104 IMR90-4 were seeded into each well of a U-bottom ultralow-attachment 96-well plate (Corning) in the presence of neural induction media. The media consisted of DMEM/F-12 containing 1% glutanax (Invitrogen), 1% minimum essential medium-non-essential amino acids (MEM-NEAA, Invitrogen), 15% knockout serum (Invitrogen), 0.1 nM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), 100 nM LDN-193189 (Sigma-Aldrich), 10 μM SB431542 (Sigma-Aldrich), and 2 μM XAV939 (Sigma-Aldrich). The cells were cultured for 10 days. The media were changed every other day. After 10 days of static culture, the cerebral organoids were transferred into ultralow-attachment 6-well plates (Corning) in the presence of neural differentiation media. The media consisted of DMEM/F12 mixed with an equal volume of neurobasal media (Corning) containing an N2 supplement (Invitrogen), a B27 supplement without vitamin A (Invitrogen), 1% MEM-NEAA, 1% GlutaMAX, and human insulin solution (Sigma-Aldrich). The cells were cultured for 8 days with orbital shaking at a speed of 80 rpm/min. For neural maturation of the cerebral organoids, neural differentiation media containing B27 supplemented with vitamin A (Invitrogen), brain-derived neurotrophic factor (BDNF, Sigma-Aldrich), cyclic adenosine monophosphate (cAMP, Sigma-Aldrich), and ascorbic acid (Sigma-Aldrich) were used and replaced every 4 days from day 18.

Immunohistochemistry

The cerebral organoids were washed with 500 μL of Dulbecco’s phosphate buffered saline (DPBS, Thermo Fisher Scientific, Waltham, MA, USA). Organoids were then incubated with 4% paraformaldehyde (PFA, Sigma-Aldrich) overnight to fix the cells. Fixed organoids were washed twice with DPBS and sequentially incubated with 10, 20, and 30% sucrose solution to increase the density of the organoids and make them easier to cut. Cryoblocks of organoids were prepared with an optimal cutting temperature (OCT, Sakura Finetech USA, Inc., Torrance, CA, USA) compound. Cryoblocks were frozen before cutting and after placing it in the OCT compound to ensure that the organoids were frozen in a uniform manner. Cryosections were washed with DPBS to remove excess OCT and blocked in 1% bovine serum albumin (BSA, Sigma-Aldrich) and 0.1% Triton X-100 diluted in DPBS for 1 h at room temperature. This blocking step helps to prevent nonspecific binding of the primary antibodies. The sections were then incubated overnight at 4 °C with primary antibodies diluted in a solution containing 1% BSA and 0.1% Triton X-100. The following primary antibodies were used for IF labeling: SOX2 (Abcam, Cambridge, UK), MAP2 (Abcam), CTIP2 (Cell Signaling Technology, CST, Danvers, MA, USA), GFAP (CST), NeuN (CST), TBR1 (CST), TBR2 (CST), SATB2 (CST), β3-tubulin (CST), nestin (CST), PAX6 (CST), and e-cadherin (Thermo Fisher Scientific, Inc.). DPBS was used to wash away any unbound primary antibodies, and the cryosections were incubated with secondary antibodies containing 2% NGS and 0.1% Triton X-100 for 1 h. The secondary antibodies were conjugated to Alexa Fluor dyes (488, 568) to amplify the signal. Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific, Inc.) to visualize the nuclei.

MEA Recording

A CytoView MEA 24 plate with 16 electrodes (Axion Biosystems, Atlanta, GA, USA) was used to record neuronal activity. In brief, the MEA plate was coated with 10 μL of 0.01% polyethylenimine (PEI, Sigma-Aldrich) solution in borate buffer and incubated for 1 h at 37 °C in a 5% CO2 incubator. Ten microliters of laminin (10 μg/mL) was added to the organoid culture media. The cerebral organoids were placed on the center of the electrode. First, 10, 30, 50, 100, and 200 μL of media were added dropwise over the course of time for 4 h. The organoids were then incubated for 3–7 days at 37 °C in a 5% CO2 incubator. Next, 500 μL of organoid culture media was added to the well 4-h post-cell seeding. Immediately before the activity was measured, 400 μL of the organoid culture media was removed to ensure close intact between the organoid and the electrode. The electrophysiological activity was monitored with a Maestro Edge MEA system (Axion Biosystems). MEA signaling was recorded over the course of time for 20 min, and AxIS metric plotting tool and AxIS navigator (Axion Biosystems) software were used to analyze mean firing rate, spike waveform, number of bursts, and number of network bursts.

Total RNA Sequencing of Cerebral Organoids

The total RNA concentration was determined using the Quant-IT RiboGreen assay kit (Invitrogen, #R11490). The integrity of the total RNA was assessed by running the samples on the TapeStation RNA screentape (Agilent Technologies, Waldbronn, Germany). RNA was isolated from each sample and used to construct sequencing libraries using the SMARTer Universal Low Input RNA Kit for Sequencing, following the manufacturer’s protocol. After the rRNA depletion, first-strand cDNA synthesis was primed by the SMART N6 CDS Primer and the SMARTer II An Oligonucleotide was used for template switching at the 5′ end of the transcript. The first-strand cDNA selectively bound to the SPRI beads, leaving contaminants in the solution, which were removed by magnetic separation. The beads were then directly used for PCR amplification. The Advantage 2 Polymerase Mix was used to amplify the cDNA templates by long-distance PCR. PCR-amplified cDNA was purified by immobilization on AMPure XP beads. The beads were then washed with 80% ethanol, and cDNA was eluted with an elution buffer. Prior to the generation of the final library for Illumina sequencing, amplified cDNA was digested with RsaI to remove the SMART adapter. These cDNA fragments then underwent an end repair process, addition of a single “A” base, and ligation of the indexing adapters. The products were then purified and enriched with PCR to create the final cDNA library. The libraries were quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantification kits for Illumina Sequencing platforms) and qualified using an Agilent Technologies 4200 TapeStation (Agilent Technologies). Indexed libraries were then sequenced using the NovaSeq platform (Illumina, San Diego, CA, USA) and Macrogen Incorporated.

mRNA-Seq Data Processing

The raw reads from the sequencer were preprocessed to remove low-quality and adapter sequences before analysis. The processed reads were aligned to Homo sapiens (GRCh37) using HISAT v2.1.0 (1). HISAT uses two types of indexes for alignment: a global whole-genome index and tens of thousands of small local indexes. These two types of indexes are constructed using the same BWT (Burrows–Wheeler transform) and graph FM index as Bowtie2. Because of its use of these efficient data structures and algorithms, HISAT generates spliced alignments several times faster than the widely used Bowtie and BWA. The reference genome sequence of H. sapiens (GRCh37) and annotation data were downloaded from NCBI. After alignment, StringTie v2.1.3b was used to assemble aligned reads into transcripts and to estimate their abundance. It provided the relative abundance estimates as read count values of transcripts and genes expressed in each sample. Library preparation was performed by using the SMARTer universal low RNA library (Ribo-Zero) kit. Paired-end sequencing reads (101 bp) generated by the Illumina instruments were verified for sequence quality with FastQC (version 0.11.7). Before starting the analysis, Trimmomatic (version 0.38)43 was used to remove adapter sequences and bases with base quality lower than 3 from the end reads. Additionally, using the sliding window trim method, bases that did not qualify for window size = 4 and mean quality = 15 were removed. Afterward, reads with a minimum length of 36 bp were removed to produce cleaned data.

Quantification of Neurotransmitter Production

The quantitative analysis of 8 {histamine, acetylcholine, serotonin (5-HT), dopamine (DA), norepinephrine, GABA, glutamate (GLU), and glutamine (GLN) neurotransmitters was performed using ultraperformance liquid chromatography (UPLC) coupled with a Xevo TQ-S triple quadrupole mass spectrometer (MS) according to a previously reported method.44 Cerebral organoids were washed three times with 0.1 M phosphate buffered saline (pH 7.4) and then added to distilled water containing 1% formic acid (FA). The organoids were homogenized using a probe-type sonicator, and the homogenate was centrifuged at 15,000 rpm for 20 min at 4 °C. The supernatant was extracted by adding an equal volume of methanol containing 1% FA, vortexing, and centrifuging at 15,000 rpm for 10 min at 4 °C. The clear supernatant was transferred to LC vials for analysis. The concentrations of the 8 neurotransmitters were normalized by the protein content of the organoid homogenate following LC–MS/MS analysis.

Metabolic Profiling in Cerebral Organoids

Metabolic profiling in cerebral organoids was performed using ultrahigh performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) analysis. The Waters ACQUITY UPLC system (Waters Corporation) was coupled to a Waters Xevo Q-TOF MS (Waters Corporation) in both electrospray ionization (ESI) positive (ESI+) and negative (ESI−) modes. Sample preparation for metabolite extraction from the cerebral organoids was carried out with modifications based on a previously published protocol.45 Briefly, the medium was removed with a Kimtech Science Wiper. Metabolite extraction was performed using ice-cold methanol. Methanol was added to the sample tube and sonicated for 10 min. The sample was then incubated for 1 h at room temperature. After vortex mixing, the sample was centrifuged at 13,500 rpm for 10 min. The supernatant was separated from the sample pellet and transferred to another tube. The extract was evaporated under nitrogen gas. The extracts were reconstituted in 200 μL of 10% methanol. The sample was transferred to an LC vial for UPLC-Q-TOF-MS analysis. PCA was performed using Metaboanalyst 5.0 software, which has PCA chemometrics analysis embedded.

Sample Preparation for Proteomic Analysis

Cerebral organoid pellets were resuspended with lysis buffer (75 mM NaCl, 8 M urea, 50 mM Tris–HCl [pH 8.0], and protease inhibitors) followed by being homogenized via an ultrasonic homogenizer (Omni, Kennesaw, GA, USA). The lysate was centrifuged at 12,000 rpm for 15 min to remove cell debris, and the resulting supernatant was collected for further proteomic analysis. Protein concentration was determined by a bicinchoninic acid assay. One-hundred microgram from each sample was used for tryptic digestion. In brief, proteins were reduced with 10 mM dithiothreitol for 2 h at 37 °C and subsequently alkylated with 20 mM iodoacetamide for 30 min at room temperature in the dark. The alkylation reaction was quenched with 20 mM l-cysteine for 30 min at room temperature. Samples were diluted with 50 mM ammonium bicarbonate buffer to a final urea concentration of 1 M, and then incubated with trypsin (1:50 enzyme to protein ratio) for 18 h at 37 °C. After adding the 1% FA to stop the reaction, the resulting peptides were cleaned up with a 10-mg OASIS HLB cartridge (Waters, USA), and dried in a vacuum concentrator (Eppendorf, UK).

The TMT labeling of peptides was performed using TMT 10-plex reagents (Thermo Fisher Scientific). Briefly, dried peptides were dissolved in 100 μL of 100 mM triethylammonium bicarbonate buffer and labeled with TMT 10-plex reagents as follows: TMT-126/-127N for day 0; TMT-127C/TMT-128N for day 25; TMT-128C/129N for day 43; TMT-129C/130N for day 70; and TMT-130C/-131 for day 120. Following incubation for 1 h at room temperature, the reactions for labeling were quenched with hydroxylamine. TMT-labeled peptides were pooled, desalted with OASIS HLB cartridge, and dried in a vacuum concentrator. The resulting peptides were reconstituted in 0.1% FA for liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis. Quantitative analyses of cerebral organoid proteomes were carried out in triplicate with two technical replicates (two consecutive LC–MS/MS runs of the same sample).

Two-Dimensional Nanoflow LC–MS/MS (2D-nLC-MS/MS) Analysis

Online 2D-nLC-MS/MS was performed using an Agilent 1290 infinity capillary LC system (Agilent Technologies, Santa Clara, CA, USA) coupled with a Q-Exactive Hybrid-Quadrupole-Orbitrap-MS (Thermo Fisher Scientific, Inc.). The TMT-labeled peptides were loaded to the biphasic reverse phase (RP)/strong cation exchange (SCX) trap column (360 μm-O.D., 200 μm-I.D., 40 mm in length) packed in-house with 5 mm of C18 resin (5 μm-200 Å) followed by 15 mm of SCX resin (5 μm-200 Å). For 2D-nLC separation, the isobarically labeled digests were first loaded onto SCX resin and then followed by 12-stepwise elution (0, 15, 20, 22, 25, 28, 30, 50, 80, 100, 200, and 1000 mM ammonium bicarbonate), enabling the migration of the fractionated peptides toward next RP resin of a biphasic trap column. Finally, the resulting peptides were subsequently separated on an RP column (360 μm-O.D., 75 μm-I.D., 150 mm in length) packed in-house with C18 resin (3 μm-100 Å) at a column flow rate of 200 nL/min. A 120-min RP gradient was generated by mobile phases A (0.1% FA in water) and B (2% water/0.1% FA in acetonitrile). The MS was operated in a data-dependent mode. Full-scan MS spectra were obtained at a resolution of 70,000 with automatic gain control target value of 3 × 106, and within scan mass range of 300 to 1800 m/z. The 12 most abundant precursor ions were isolated with exclusion of a single or unassigned charge state and fragmented by high-energy collision dissociation with a normalized collision energy of 27%. MS/MS spectra were acquired at a resolution of 35,000 with a first fixed mass of 100 m/z. Dynamic exclusion was set to 30 s.

Proteomic Data Analysis

MS raw files were processed with MaxQuant software (version 1.6.6.0) using the Andromeda search engine,46 and the spectra were searched against UniProt H. sapiens database (release version of Mar 2021 containing 78,120 entries). The search type was set to “Reporter ion MS2” with 10-plex TMT for isobaric label quantification. Enzyme specificity was set to trypsin, allowing for up to two missed cleavages. The mass tolerance for precursor ions was 4.5 ppm, and that for fragment ions was 20 ppm. Acetylation (N-term) and oxidation (methionine) were selected as variable modifications, and carbamidomethylation (cysteine) was set as a fixed modification. Protein identifications were filtered with at least two unique peptides per protein and a 1% false discovery rate (FDR) on the protein and peptide levels. Statistical analyses and hierarchical clustering analysis were performed using the Perseus software (version 1.6.14.0).47 Potential contaminants, reverse hits, and proteins only identified by site were excluded from the protein list before analyses. The TMT ratios were log2-transformed and normalized by median subtraction. Only proteins quantified at least three times out of six replicates were considered for statistical analysis. Multiple-sample test (one-way ANOVA) was applied with Benjamini–Hochberg correction (FDR < 0.05) for time-resolved analysis of proteome. The functional enrichment analyses including GO and reactome pathway were conducted in R (version 4.3.1) using the Bioconductor (version 3.17).48,49

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA) and Origin 8.5 (OriginLab Corporation, Northampton, MA, USA). The values are expressed as the mean ± standard error of means. The statistical significance was determined by Student’s t-test for independent samples or one-way ANOVA with Tukey’s multiple comparisons. Statistical significance was considered at P < 0.05.

Data Availability Statement

The MS-based proteomic data sets generated during the current study are available in PRIDE, accession number: http://www.ebi.ac.uk/pride.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.3c00246.

  • Neurogenesis- and neurotransmitter-related markers for total RNA sequencing (PDF)

  • List of proteins quantified through proteomics analysis, hierarchical clustering of cerebral organoid proteome, list of ANOVA significant protein identified, list of ANOVA significant protein filtered based on threshold of twofold changes compared to day 0 (XLSX)

Author Contributions

S.B.B., B.K., H.S.K., Y.E.K., M.A.B., D.K., C.H.K., and K.Y.K. equally contributed to the study. M.A.B., D.K., C.H.K., and K.Y.K. conceived the project and were responsible for the overall experimental design. S.B.P., B.K., H.S.K., and Y.E.K. performed experiments and analyzed all data presented. S.S.K. and S–H.C. performed the neurotransmitter and neurosteroid/lipid analysis experiments. T.Y.K. supervised proteomics study. S.B.P. and B.K. wrote the manuscript with input from all the authors.

The authors greatly acknowledge financial support from the Ministry of Science and ICT (2021R1A2C2011195), the Ministry of Trade, Industry & Energy (20009774), grants from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT) (NRF-2019R1A2C3002354 to C.H.K.) and the Korea Research Institute of Chemical Technology (SI2231-40, KK2252-10) of Republic of Korea.

The authors declare no competing financial interest.

Supplementary Material

cn3c00246_si_001.pdf (30.9KB, pdf)
cn3c00246_si_002.xlsx (1.3MB, xlsx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

cn3c00246_si_001.pdf (30.9KB, pdf)
cn3c00246_si_002.xlsx (1.3MB, xlsx)

Data Availability Statement

The MS-based proteomic data sets generated during the current study are available in PRIDE, accession number: http://www.ebi.ac.uk/pride.

Articles from ACS Chemical Neuroscience are provided here courtesy of American Chemical Society

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