Morphological and transcriptomic analyses of stem cell-derived cortical neurons reveal mechanisms underlying synaptic dysfunction in schizophrenia.

BACKGROUND: Postmortem studies in schizophrenia consistently show reduced dendritic spines in the cerebral cortex but the mechanistic underpinnings of these deficits remain unknown. Recent genome-wide association studies and exome sequencing investigations implicate synaptic genes and processes in the disease biology of schizophrenia. METHODS: We generated human cortical pyramidal neurons by differentiating iPSCs of seven schizophrenia patients and seven healthy subjects, quantified dendritic spines and synapses in different cortical neuron subtypes, and carried out transcriptomic studies to identify differentially regulated genes and aberrant cellular processes in schizophrenia. RESULTS: Cortical neurons expressing layer III marker CUX1, but not those expressing layer V marker CTIP2, showed significant reduction in dendritic spine density in schizophrenia, mirroring findings in postmortem studies. Transcriptomic experiments in iPSC-derived cortical neurons showed that differentially expressed genes in schizophrenia were enriched for genes implicated in schizophrenia in genome-wide association and exome sequencing studies. Moreover, most of the differentially expressed genes implicated in schizophrenia genetic studies had lower expression levels in schizophrenia cortical neurons. Network analysis of differentially expressed genes led to identification of NRXN3 as a hub gene, and follow-up experiments showed specific reduction of the NRXN3 204 isoform in schizophrenia neurons. Furthermore, overexpression of the NRXN3 204 isoform in schizophrenia neurons rescued the spine and synapse deficits in the cortical neurons while knockdown of NRXN3 204 in healthy neurons phenocopied spine and synapse deficits seen in schizophrenia cortical neurons. The antipsychotic clozapine increased expression of the NRXN3 204 isoform in schizophrenia cortical neurons and rescued the spine and synapse density deficits. CONCLUSIONS: Taken together, our findings in iPSC-derived cortical neurons recapitulate cell type-specific findings in postmortem studies in schizophrenia and have led to the identification of a specific isoform of NRXN3 that modulates synaptic deficits in schizophrenia neurons.

Disease-specific differences in gene expression, mitochondrial function and mitochondria-endoplasmic reticulum interactions in iPSC-derived cerebral organoids and cortical neurons in schizophrenia and bipolar disorder.

We compared transcriptomic profiles of cerebral organoids differentiated from induced pluripotent stem cells of eight schizophrenia and eight bipolar disorder patients to identify genes that were differentially expressed in cerebral organoids between two disorders. Gene ontology analysis showed relative up-regulation in schizophrenia organoids of genes related to response to cytokines, antigen binding and clathrin-coated vesicles, while showing up-regulation in bipolar disorder of genes involved in calcium binding. Gene set enrichment analysis revealed enrichment in schizophrenia of genes involved in mitochondrial and oxidative phosphorylation while showing enrichment in bipolar disorder of genes involved in long term potentiation and neuro-transporters. We compared mitochondrial function in cerebral organoids from schizophrenia and bipolar disorder subjects and found that while schizophrenia organoids showed deficits in basal oxygen consumption rate and ATP production when compared to healthy control organoids, while bipolar disorder organoids did not show these deficits. Gene ontology analyses also revealed enrichment in bipolar disorder of genes in ion binding and regulation of transport. Experiments examining the interaction between mitochondria and endoplasmic reticulum in cortical neurons from bipolar disorder subjects showed a significantly lower number of contact sites between mitochondria and endoplasmic reticulum when compared to cortical neurons from schizophrenia patients. These results point to disease-specific deficits in mitochondrial respiration in schizophrenia and in mitochondrial-endoplasmic reticulum interactions in bipolar disorder. Supplementary Information: The online version contains supplementary material available at 10.1007/s44192-023-00031-8.

Distinct effects of interleukin-6 and interferon-γ on differentiating human cortical neurons.

Translational evidence suggests that cytokines involved in maternal immune activation (MIA), such as interleukin-6 (IL-6) and interferon-γ (IFN-γ), can cross the placenta, injure fetal brain, and predispose to neuropsychiatric disorders. To elaborate developmental neuronal sequelae of MIA, we differentiated human pluripotent stem cells to cortical neurons over a two-month period, exposing them to IL-6 or IFN-γ. IL-6 impacted expression of genes regulating extracellular matrix, actin cytoskeleton and TGF-ß signaling while IFN-γ impacted genes regulating antigen processing, major histocompatibility complex and endoplasmic reticulum biology. IL-6, but not IFN-γ, altered mitochondrial respiration while IFN-γ, but not IL-6, induced reduction in dendritic spine density. Pre-treatment with folic acid, which has known neuroprotective and anti-inflammatory properties, ameliorated IL-6 effects on mitochondrial respiration and IFN-γ effects on dendritic spine density. These findings suggest distinct mechanisms for how fetal IL-6 and IFN-γ exposure influence risk for neuropsychiatric disorders, and how folic acid can mitigate such risk.

Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells.

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia in health and disease. Furthermore, iPSC-derived microglia can be maintained in co-culture with iPSC-derived cortical neurons, which enable investigations of microglia-neuron interactions that are hypothesized to be dysregulated in a number of neuropsychiatric disorders. Human iPSCs were differentiated to generate microglia using an adapted version of a protocol developed by the Fossati group, and the iPSC-derived microglia were validated with marker analysis and real-time PCR. Human microglia generated using this protocol were positive for the markers CD11C, IBA1, P2RY12, and TMEM119, and expressed the microglial-related genes AIF1, CX3CR1, ITGAM, ITGAX, P2RY12, and TMEM119. Human iPSC-derived cortical neurons that had been differentiated for 30 days were plated with microglia and maintained in co-culture until day 60, when experiments were undertaken. The density of dendritic spines in cortical neurons in co-culture with microglia was quantified under baseline conditions and in the presence of pro-inflammatory cytokines. In order to examine how microglia modulate neuronal function, calcium imaging experiments of the cortical neurons were undertaken using the calcium indicator Fluo-4 AM. Live calcium activity of cortical neurons was obtained using a confocal microscope, and fluorescence intensity was quantified using ImageJ. This report describes how co-culturing human iPSC-derived microglia and cortical neurons provide new approaches to interrogate the effects of microglia on cortical neurons.

Comparative Transcriptomic Analysis of Cerebral Organoids and Cortical Neuron Cultures Derived from Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) can be differentiated along various neuronal lineages to generate two-dimensional neuronal cultures as well as three-dimensional brain organoids. Such iPSC-derived cellular models are being utilized to study the basic biology of human neuronal function and to interrogate the molecular underpinnings of disease biology. The different cellular models generated from iPSCs have varying properties in terms of the diversity and organization of the cells as well as the cellular functions that are present. To understand transcriptomic differences in iPSC-derived monolayer neuronal cultures and three-dimensional brain organoids, we differentiated eight human iPSC lines from healthy control subjects to generate cerebral organoids and cortical neuron monolayer cultures from the same set of iPSC lines. We undertook RNA-seq experiments in these model systems and analyzed the gene expression data to identify genes that are differentially expressed in cerebral organoids and two-dimensional cortical neuron cultures. In cerebral organoids, gene ontology analysis showed enrichment of genes involved in tissue development, response to stimuli, and the interferon-γ pathway, while two-dimensional cortical neuron cultures showed enrichment of genes involved in nervous system development and neurogenesis. We also undertook comparative analysis of these gene expression profiles with transcriptomic data from the human fetal prefrontal cortex (PFC). This analysis showed greater overlap of the fetal PFC transcriptome with cerebral organoid gene expression profiles compared to monolayer cortical neuron culture profiles. Our studies delineate the transcriptomic differences between cortical neuron monolayer cultures and three-dimensional cerebral organoids and can help inform the appropriate use of these model systems to address specific scientific questions.

Transcriptome analysis and functional characterization of cerebral organoids in bipolar disorder.

BACKGROUND: Reprogramming human induced pluripotent stem cells (iPSCs) from somatic cells and generating three-dimensional brain organoids from these iPSCs provide access to live human neuronal tissue with disease-specific genetic backgrounds. METHODS: Cerebral organoids were generated from iPSCs of eight bipolar disorder (BPI) patients and eight healthy control individuals. RNA-seq experiments were undertaken using RNA isolated from the cerebral organoids. Functional activity in the cerebral organoids was studied using microelectrode arrays. RESULTS: RNA-seq data comparing gene expression profiles in the cerebral organoids showed downregulation of pathways involved in cell adhesion, neurodevelopment, and synaptic biology in bipolar disorder along with upregulation of genes involved in immune signaling. The central hub in the network analysis was neurocan (NCAN), which is located in a locus with evidence for genome-wide significant association in BPI. Gene ontology analyses suggested deficits related to endoplasmic reticulum biology in BPI, which was supported by cellular characterization of ER-mitochondria interactions. Functional studies with microelectrode arrays revealed specific deficits in response to stimulation and depolarization in BPI cerebral organoids. CONCLUSIONS: Our studies in cerebral organoids from bipolar disorder showed dysregulation in genes involved in cell adhesion, immune signaling, and endoplasmic reticulum biology; implicated a central role for the GWAS hit NCAN in the biology of BPI; and showed evidence of deficits in neurotransmission.

Transcriptomic Landscape and Functional Characterization of Induced Pluripotent Stem Cell-Derived Cerebral Organoids in Schizophrenia.

Importance: Three-dimensional cerebral organoids generated from patient-derived induced pluripotent stem cells (iPSCs) may be used to interrogate cellular-molecular underpinnings of schizophrenia. Objective: To determine transcriptomic profiles and functional characteristics of cerebral organoids from patients with schizophrenia using gene expression studies, complemented with investigations of mitochondrial function through measurement of real-time oxygen consumption rate, and functional studies of neuronal firing with microelectrode arrays. Design, Setting, and Participants: This case-control study was conducted at Massachusetts General Hospital between 2017 and 2019. Transcriptomic profiling of iPSC-derived cerebral organoids from 8 patients with schizophrenia and 8 healthy control individuals was undertaken to identify cellular pathways that are aberrant in schizophrenia. Induced pluripotent stem cells and cerebral organoids were generated from patients who had been diagnosed as having schizophrenia and from heathy control individuals. Main Outcomes and Measures: Transcriptomic analysis of iPSC-derived cerebral organoids from patients with schizophrenia show differences in expression of genes involved in synaptic biology and neurodevelopment and are enriched for genes implicated in schizophrenia genome-wide association studies (GWAS). Results: The study included iPSC lines generated from 11 male and 5 female white participants, with a mean age of 38.8 years. RNA sequencing data from iPSC-derived cerebral organoids in schizophrenia showed differential expression of genes involved in synapses, in nervous system development, and in antigen processing. The differentially expressed genes were enriched for genes implicated in schizophrenia, with 23% of GWAS genes showing differential expression in schizophrenia and control organoids: 10 GWAS genes were upregulated in schizophrenia organoids while 15 GWAS genes were downregulated. Analysis of the gene expression profiles suggested dysregulation of genes involved in mitochondrial function and those involved in modulation of excitatory and inhibitory pathways. Studies of mitochondrial respiration showed lower basal consumption rate, adenosine triphosphate production, proton leak, and nonmitochondrial oxygen consumption in schizophrenia cerebral organoids, without any differences in the extracellular acidification rate. Microelectrode array studies of cerebral organoids showed no differences in baseline electrical activity in schizophrenia but revealed a diminished response to stimulation and depolarization. Conclusions and Relevance: Investigations of patient-derived cerebral organoids in schizophrenia revealed gene expression patterns suggesting dysregulation of a number of pathways in schizophrenia, delineated differences in mitochondrial function, and showed deficits in response to stimulation and depolarization in schizophrenia.

Synaptic deficits in iPSC-derived cortical interneurons in schizophrenia are mediated by NLGN2 and rescued by N-acetylcysteine.

Human postmortem studies suggest a major role for abnormalities in GABAergic interneurons in the prefrontal cortex in schizophrenia. Cortical interneurons differentiated from induced pluripotent stem cells (iPSCs) of schizophrenia subjects showed significantly lower levels of glutamate decarboxylase 67 (GAD67), replicating findings from multiple postmortem studies, as well as reduced levels of synaptic proteins gehpyrin and NLGN2. Co-cultures of the interneurons with excitatory cortical pyramidal neurons from schizophrenia iPSCs showed reduced synaptic puncta density and lower action potential frequency. NLGN2 overexpression in schizophrenia neurons rescued synaptic puncta deficits while NLGN2 knockdown in healthy neurons resulted in reduced synaptic puncta density. Schizophrenia interneurons also had significantly smaller nuclear area, suggesting an innate oxidative stressed state. The antioxidant N-acetylcysteine increased the nuclear area in schizophrenia interneurons, increased NLGN2 expression and rescued synaptic deficits. These results implicate specific deficiencies in the synaptic machinery in cortical interneurons as critical regulators of synaptic connections in schizophrenia and point to a nexus between oxidative stress and NLGN2 expression in mediating synaptic deficits in schizophrenia.

Identifying Extrinsic versus Intrinsic Drivers of Variation in Cell Behavior in Human iPSC Lines from Healthy Donors.

Large cohorts of human induced pluripotent stem cells (iPSCs) from healthy donors are a potentially powerful tool for investigating the relationship between genetic variants and cellular behavior. Here, we integrate high content imaging of cell shape, proliferation, and other phenotypes with gene expression and DNA sequence datasets from over 100 human iPSC lines. By applying a dimensionality reduction approach, Probabilistic Estimation of Expression Residuals (PEER), we extracted factors that captured the effects of intrinsic (genetic concordance between different cell lines from the same donor) and extrinsic (cell responses to different fibronectin concentrations) conditions. We identify genes that correlate in expression with intrinsic and extrinsic PEER factors and associate outlier cell behavior with genes containing rare deleterious non-synonymous SNVs. Our study, thus, establishes a strategy for examining the genetic basis of inter-individual variability in cell behavior.

Corrigendum: Common genetic variation drives molecular heterogeneity in human iPSCs.

This corrects the article DOI: 10.1038/nature22403.

Modelling Autistic Neurons with Induced Pluripotent Stem Cells.

Autism spectrum disorder (ASD) is a neurodevelopmental condition that affects more than 1% of children per current estimates. It has been characterised by the following two core behavioural phenotypes: (1) deficits in social interaction and communication and (2) repetitive behaviours, restricted interests and activities. Due to the complex nature of ASD, there are currently no effective treatments. The reason behind this is the clinical and genetic heterogeneity between affected individuals on the one hand and the lack of understanding of the underpinning pathophysiological mechanisms on the other hand. Induced pluripotent stem cells (iPSCs) are reprogrammed stem cells from adult cells. These have the capacity to self-renew and differentiate into any type of cells in the body. Therefore, human iPSCs provide a unique opportunity to study the human cellular and molecular phenotypes associated with ASD. Here, we systematically review various ASD variants and co-morbid diseases modelled using human iPSCs.

Common genetic variation drives molecular heterogeneity in human iPSCs.

Technology utilizing human induced pluripotent stem cells (iPS cells) has enormous potential to provide improved cellular models of human disease. However, variable genetic and phenotypic characterization of many existing iPS cell lines limits their potential use for research and therapy. Here we describe the systematic generation, genotyping and phenotyping of 711 iPS cell lines derived from 301 healthy individuals by the Human Induced Pluripotent Stem Cells Initiative. Our study outlines the major sources of genetic and phenotypic variation in iPS cells and establishes their suitability as models of complex human traits and cancer. Through genome-wide profiling we find that 5-46% of the variation in different iPS cell phenotypes, including differentiation capacity and cellular morphology, arises from differences between individuals. Additionally, we assess the phenotypic consequences of genomic copy-number alterations that are repeatedly observed in iPS cells. In addition, we present a comprehensive map of common regulatory variants affecting the transcriptome of human pluripotent cells.

The utility of patient specific induced pluripotent stem cells for the modelling of Autistic Spectrum Disorders.

Until now, models of psychiatric diseases have typically been animal models. Whether they were to be used to further understand the pathophysiology of the disorder, or as drug discovery tools, animal models have been the choice of preference in mimicking psychiatric disorders in an experimental setting. While there have been cellular models, they have generally been lacking in validity. This situation is changing with the advent of patient-specific induced pluripotent stem cells (iPSCs). In this article, we give a methodological evaluation of the current state of the iPS technology with reference to our own work in generating patient-specific iPSCs for the study of autistic spectrum disorder (ASD). In addition, we will give a broader perspective on the validity of this technology and to what extent it can be expected to complement animal models of ASD in the coming years.