Gianfilippo Coppola

FOXG1-Dependent Dysregulation of GABA/Glutamate Neuron Differentiation in Autism Spectrum Disorders

Autism spectrum disorder (ASD) is a disorder of brain development. Most cases lack a clear etiology or genetic basis, and the difficulty of re-enacting human brain development has precluded understanding of ASD pathophysiology. Here we use three-dimensional neural cultures (organoids) derived from induced pluripotent stem cells (iPSCs) to investigate neurodevelopmental alterations in individuals with severe idiopathic ASD. While no known underlying genomic mutation could be identified, transcriptome and gene network analyses revealed upregulation of genes involved in cell proliferation, neuronal differentiation, and synaptic assembly. ASD-derived organoids exhibit an accelerated cell cycle and overproduction of GABAergic inhibitory neurons. Using RNA interference, we show that overexpression of the transcription factor FOXG1 is responsible for the overproduction of GABAergic neurons. Altered expression of gene network modules and FOXG1 are positively correlated with symptom severity. Our data suggest that a shift toward GABAergic neuron fate caused by FOXG1 is a developmental precursor of ASD.

Modeling human cortical development in vitro using induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) are emerging as a tool for understanding human brain development at cellular, molecular, and genomic levels. Here we show that hiPSCs grown in suspension in the presence of rostral neuralizing factors can generate 3D structures containing polarized radial glia, intermediate progenitors, and a spectrum of layer-specific cortical neurons reminiscent of their organization in vivo. The hiPSC-derived multilayered structures express a gene expression profile typical of the embryonic telencephalon but not that of other CNS regions. Their transcriptome is highly enriched in transcription factors controlling the specification, growth, and patterning of the dorsal telencephalon and displays highest correlation with that of the early human cerebral cortical wall at 8–10 wk after conception. Thus, hiPSC are capable of enacting a transcriptional program specifying human telencephalic (pallial) development. This model will allow the study of human brain development as well as disorders of the human cerebral cortex.

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.

Human induced pluripotent stem cells for modelling neurodevelopmental disorders

We currently have a poor understanding of the pathogenesis of neurodevelopmental disorders, owing to the fact that postmortem and imaging studies can only measure the postnatal status quo and offer little insight into the processes that give rise to the observed outcomes. Human induced pluripotent stem cells (hiPSCs) should, in principle, prove powerful for elucidating the pathways that give rise to neurodevelopmental disorders. hiPSCs are embryonic-stem-cell-like cells that can be derived from somatic cells. They retain the unique genetic signature of the individual from whom they were derived, and thus enable researchers to recapitulate that individuals idiosyncratic neural development in a dish. In the case of individuals with disease, we can re-enact the disease-altered trajectory of brain development and examine how and why phenotypic and molecular abnormalities arise in these diseased brains. Here, we review hiPSC biology and possible experimental designs when using hiPSCs to model disease. We then discuss existing hiPSC models of neurodevelopmental disorders. Our hope is that, as some studies have already shown, hiPSCs will illuminate the pathophysiology of developmental disorders of the CNS and lead to therapeutic options for the millions that are affected by these conditions.

One thousand somatic SNVs per skin fibroblast cell set baseline of mosaic mutational load with patterns that suggest proliferative origin

Few studies have been conducted to understand post-zygotic accumulation of mutations in cells of the healthy human body. We reprogrammed 32 skin fibroblast cells from families of donors into human induced pluripotent stem cell (hiPSC) lines. The clonal nature of hiPSC lines allows a high-resolution analysis of the genomes of the founder fibroblast cells without being confounded by the artifacts of single-cell whole-genome amplification. We estimate that on average a fibroblast cell in children has 1035 mostly benign mosaic SNVs. On average, 235 SNVs could be directly confirmed in the original fibroblast population by ultradeep sequencing, down to an allele frequency (AF) of 0.1%. More sensitive droplet digital PCR experiments confirmed more SNVs as mosaic with AF as low as 0.01%, suggesting that 1035 mosaic SNVs per fibroblast cell is the true average. Similar analyses in adults revealed no significant increase in the number of SNVs per cell, suggesting that a major fraction of mosaic SNVs in fibroblasts arises during development. Mosaic SNVs were distributed uniformly across the genome and were enriched in a mutational signature previously observed in cancers and in de novo variants and which, we hypothesize, is a hallmark of normal cell proliferation. Finally, AF distribution of mosaic SNVs had distinct narrow peaks, which could be a characteristic of clonal cell selection, clonal expansion, or both. These findings reveal a large degree of somatic mosaicism in healthy human tissues, link de novo and cancer mutations to somatic mosaicism, and couple somatic mosaicism with cell proliferation.

Experimental study of highly turbulent isothermal opposed-jet flows

Opposed-jet flows have been shown to provide a valuable means to study a variety of combustion problems, but have been limited to either laminar or modestly turbulent conditions. With the ultimate goal of developing a burner for laboratory flames reaching turbulence regimes of relevance to practical systems, we characterized highly turbulent, strained, isothermal, opposed-jet flows using particle image velocimetry (PIV). The bulk strain rate was kept at 1250 s−1 and specially designed and properly positioned turbulence generation plates in the incoming streams boosted the turbulence intensity to well above 20%, under conditions that are amenable to flame stabilization. The data were analyzed with proper orthogonal decomposition (POD) and a novel statistical analysis conditioned to the instantaneous position of the stagnation surface. Both POD and the conditional analysis were found to be valuable tools allowing for the separation of the truly turbulent fluctuations from potential artifacts introduced by r...

Neurobiology meets genomic science: the promise of human-induced pluripotent stem cells.

The recent introduction of the induced pluripotent stem cell technology has made possible the derivation of neuronal cells from somatic cells obtained from human individuals. This in turn has opened new areas of investigation that can potentially bridge the gap between neuroscience and psychopathology. For the first time we can study the cell biology and genetics of neurons derived from any individual. Furthermore, by recapitulating in vitro the developmental steps whereby stem cells give rise to neuronal cells, we can now hope to understand factors that control typical and atypical development. We can begin to explore how human genes and their variants are transcribed into messenger RNAs within developing neurons and how these gene transcripts control the biology of developing cells. Thus, human-induced pluripotent stem cells have the potential to uncover not only what aspects of development are uniquely human but also variations in the series of events necessary for normal human brain development that predispose to psychopathology.

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). PNNs inhibit neuroplasticity and astroglia contribute to the formation, organization, and maintenance of PNNs. To validate our TRAPseq findings, we showed an increase in PNNs following CVS. Degradation of PNNs in the prefrontal cortex of mice exposed to CVS reversed the CVS-induced behavioral phenotype in the forced swim test. These data lend further support to the neuroplasticity hypothesis of depressive behaviors and, in particular, extend this hypothesis beyond neuronal plasticity to include an overall decrease in genes associated with cortical astroglial plasticity following CVS. Further studies will be needed to assess the antidepressant potential of directly targeting astroglial cell function in models of depression.

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.

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.