Cell-to-Cell Adhesion and Neurogenesis in Human Cortical Development: A Study Comparing 2D Monolayers with 3D Organoid Cultures

SUMMARY Organoids (ORGs) are increasingly used as models of cerebral cortical development. Here, we compared transcriptome and cellular phenotypes between telencephalic ORGs and monolayers (MONs) generated in parallel from three biologically distinct induced pluripotent stem cell (iPSC) lines. Multiple readouts revealed increased proliferation in MONs, which was caused by increased integrin signaling. MONs also exhibited altered radial glia (RG) polarity and suppression of Notch signaling, as well as impaired generation of intermediate progenitors, outer RG, and cortical neurons, which were all partially reversed by reaggregation of dissociated cells. Network analyses revealed co-clustering of cell adhesion, Notch-related transcripts and their transcriptional regulators in a module strongly downregulated in MONs. The data suggest that ORGs, with respect to MONs, initiate more efficient Notch signaling in ventricular RG owing to preserved cell adhesion, resulting in subsequent generation of intermediate progenitors and outer RG, in a sequence that recapitulates the cortical ontogenetic process

Differentiation of Human iPSCs Into Telencephalic Neurons Using 3D Organoids and Monolayer Culture

Human induced pluripotent stem cells (hiPSCs) are emerging as a useful tool for modelling in vitro early brain development and neurological disorders. Molecular mechanisms and cell interactions that regulate the neurodevelopment at early stages remain unclear because of human brain’s complexity and limitations of functional studies. Two major culture methodologies are used to differentiate in vitro hiPSCs into neurons: monolayer (2D) and organoid (3D) cultures. Here we investigate the effect of cell dissociation and the loss of 3D organization during the early differentiation process of neuronal progenitors. Using the same culture media, we first differentiated hiPSCs into neural progenitor cells (NPCs) and then induced their differentiation into neurons in 3 different modalities: 3D undissociated organoids, dissociated NPCs followed by immediate re-aggregation into an organoid, and dissociated NPCs cultured as monolayer. We assessed neuronal differentiation efficiency of each method by immunocytochemistry, qPCR, western blot, and RNA-Seq analysis over a time course. Our data revealed substantial differences in gene and protein expression among the three systems, including genes of the Notch pathway (e.g. NEUROD1, NEUROG2), earliest determinants of cortical region differentiation (e.g. SOX1, FEZF1) as well as later transcriptional regulators that specify cortical neuron subtypes (e.g. TBR1, CTIP2), which were all downregulated in monolayer. Moreover, we found that genes and pathways mediating cell-to-cell interactions (e.g. CNTNs, CAMs) were mostly upregulated in the 3D culture systems, whereas cell-extracellular matrix interaction molecules (e.g. ITG, LAM) were mostly upregulated in 2D, indicating that cell surface molecules may be involved in specification of neuronal cell types. Our results address the methodological question of the appropriateness of a differentiation method for a particular experimental goal, and, beyond that, reveal important early determinants that exert a decisive influence on neuronal differentiation and regional specification of human neural stem cells.

Comparative transcriptome and gene regulation in human iPSC-derived organoids and donor-identical brain tissue

Modeling human brain development in vitro is critically important to understand the pathophysiology of neuropsychiatric disorders. As part of the PsychENCODE project, we generated human induced pluripotent stem cells (hiPSCs) from skin fibroblasts of three human specimens at 15, 16 and 17 postconceptional weeks. These hiPSC were differentiated into telencephalic organoids to study early genetic programs in forebrain development. By using RNA-seq and histone chromatin immunoprecipitation (ChIP-seq), we compared transcriptomes and epigenomes of hiPSCs-derived organoids to donor-identical cortical brain tissue. Immunocytochemical characterization of the organoids over a time course (TD0, TD11 and TD30) showed expression of radial glial markers and mature cortical neurons confirming telencephalic fate. Hierarchical clustering of the organoids’ transcriptomes demonstrated stage-specific patterns of gene expression during in vitro development. Mapping organoids’ transcriptomes against the BrainSpan dataset suggested highest correlations with neocortex and showed their correspondence to post-conceptional weeks 8-16 of human fetal development. We then inferred transcriptional alterations, by differential gene expression, between organoids and the two brain regions analyzed. We found ~5000 of differentially expressed genes (DEG) between TD0 and fetal cortex and a decreasing number of DEG at TD11 and TD30 suggesting a stronger, albeit incomplete similarity of the organoids to the cortex at later time points. ChIP-seq experiments identified H3K27ac and H3K4me3 peaks (putative promoters and enhancers) differentially active at different organoids developmental stages and between organoids and fetal brain. Overall, however, hierarchical clustering of H3K27ac and H3K4me3 peaks demonstrated clustering of organoids with human fetal brain samples from various databases, whereas neonatal and adult brain samples formed separate clusters. These data suggest that organoids recapitulate in part transcriptome and epigenome features of fetal human brain

The PsychENCODE project

Recent research on disparate psychiatric disorders has implicated rare variants in genes involved in global gene regulation and chromatin modification, as well as many common variants located primarily in regulatory regions of the genome. Understanding precisely how these variants contribute to disease will require a deeper appreciation for the mechanisms of gene regulation in the developing and adult human brain. The PsychENCODE project aims to produce a public resource of multidimensional genomic data using tissue- and cell type–specific samples from approximately 1,000 phenotypically well-characterized, high-quality healthy and disease-affected human post-mortem brains, as well as functionally characterize disease-associated regulatory elements and variants in model systems. We are beginning with a focus on autism spectrum disorder, bipolar disorder and schizophrenia, and expect that this knowledge will apply to a wide variety of psychiatric disorders. This paper outlines the motivation and design of PsychENCODE

Differentiation of human iPSCs into telencephalic neurons using 3D organoids and monolayer culture.

Differentiation of Human iPSCs Into Telencephalic Neurons Using 3D Organoids and Monolayer Culture Giovanna GIuseppina Altobelli1,2, Soraya Scuderi2, Gianfilippo Coppola2, Jun Hyan Park3, Vincenzo Cimini1, Flora Maria Vaccarino2,3 1 Department of Advanced Biomedical Sciences, University of Naples “Federico II”, Naples, Italy 2 Child Study Center, Yale University, New Haven, CT, USA. 3 Neuroscience, Yale University, New Haven, CT, USA. Human induced pluripotent stem cells (hiPSCs) are emerging as a useful tool for modelling in vitro early brain development and neurological disorders. Molecular mechanisms and cell interactions that regulate the neurodevelopment at early stages remain unclear because of human brain’s complexity and limitations of functional studies. Two major culture methodologies are used to differentiate in vitro hiPSCs into neurons: monolayer (2D) and organoid (3D) cultures. Here we investigate the effect of cell dissociation and the loss of 3D organization during the early differentiation process of neuronal progenitors. Using the same culture media, we first differentiated hiPSCs into neural progenitor cells (NPCs) and then induced their differentiation into neurons in 3 different modalities: 3D undissociated organoids, dissociated NPCs followed by immediate re-aggregation into an organoid, and dissociated NPCs cultured as monolayer. We assessed neuronal differentiation efficiency of each method by immunocytochemistry, qPCR, western blot, and RNA-Seq analysis over a time course. Our data revealed substantial differences in gene and protein expression among the three systems, including genes of the Notch pathway (e.g. NEUROD1, NEUROG2), earliest determinants of cortical region differentiation (e.g. SOX1, FEZF1) as well as later transcriptional regulators that specify cortical neuron subtypes (e.g. TBR1, CTIP2), which were all downregulated in monolayer. Moreover, we found that genes and pathways mediating cell-to-cell interactions (e.g. CNTNs, CAMs) were mostly upregulated in the 3D culture systems, whereas cell-extracellular matrix interaction molecules (e.g. ITG, LAM) were mostly upregulated in 2D, indicating that cell surface molecules may be involved in specification of neuronal cell types. Our results address the methodological question of the appropriateness of a differentiation method for a particular experimental goal, and, beyond that, reveal important early determinants that exert a decisive influence on neuronal differentiation and regional specification of human neural stem cells

High-resolution molecular phenotyping of human IPSC organoids using CLARITY and 2-photon microscopy

High-resolution molecular phenotyping of human IPSC organoids using CLARITY and 2-photon microscopy Simone Tomasi, Soraya Scuderi, Anahita Amiri, Giovanna G. Altobelli, Jessica Mariani, Cheryl Dambrot, Gianfilippo Coppola, Flora M. Vaccarino To understand the role of gene regulation in human brain development and neuropsychiatric disorders, it is essential to develop cellular models of the human brain. Induced pluripotent stem cells (iPSC)-derived brain organoids can be used to investigate the role of gene regulatory elements, noncoding RNA, and in general, noncoding disease associated gene variants in brain development and function. Organoids enable gradients of morphogenes and other extracellular cues to build up in the intercellular milieu and to interact with the genetic and epigenetic background of a given progenitor cell during the course of brain development. We have developed an iPSC-derived organoid model of the early human forebrain, where differentiation of cortical excitatory and inhibitory neurons can be studied in a reproducible fashion, enabling a more precise identification of molecular events crucially involved in the specification of distinct neuronal subtypes. However, a precise assessment of protein and RNA expression in intact organoids is hampered by the limited penetration of molecular probes, therefore requiring the preparation of thin sections and greatly limiting the capacity of exploring molecular and cellular features in a 3D environment. Here, we labeled telencephalic excitatory and inhibitory lineages in using pLenti-CAMKII-GFP and pLenti-DlxI12b-BG-DsRed vectors, then used two-photon microscopy to image the genetically-encoded fluorescence at higher resolution in live forebrain organoids. Next, we used CLARITY to clear the organoids and perform immunostainings on the intact cell aggregates. Our current protocol enables a 3D reconstruction of GFP/TdTomato filled cells allowing the analysis of axonal and dendritic arborization, dendrite length, synapse and spine distribution, as well as stereological counts of structures labeled by specific markers. Using these combined approaches, we aim at comparing intra-organoid layer cytoarchitecture and its emerging connectivity with parallel data from RNA-seq and ChIP-seq experiments, and develop new tools for linking the molecular and cellular features of organoids derived from different individuals

Profiling changes in cortical astroglial cells following chronic stress

Recent studies have suggested that cortical astroglia play an important role in depressive-like behaviors. Potential astroglial contributions have been proposed based on their known neuroplastic functions, such as glutamate recycling and synaptic plasticity. However, the specific mechanisms by which astroglial cells may contribute or protect against a depressive phenotype remain unknown. To delineate astroglial changes that accompany depressive-like behavior, we used astroglial-specific bacTRAP mice exposed to chronic variable stress (CVS) and profiled the astroglial translatome using translating ribosome affinity purification (TRAP) in conjunction with RNAseq. As expected, CVS significantly increased anxiety- and depressive-like behaviors and corticosterone levels and decreased GFAP expression in astroglia, although this did not reflect a change in the total number of astroglial cells. TRAPseq results showed that CVS decreased genes associated with astroglial plasticity: RhoGTPases, growth factor signaling, and transcription regulation, and increased genes associated with the formation of extracellular matrices such as perineuronal nets (PNNs).