Project description:For decades, technical and cost hurdles have prevented the systematic investigation of non-coding sequences in complex human diseases, and thus our knowledge about autism spectrum disorders (ASD) has been primarily obtained from analysis of protein-coding sequences. We have combined the analysis of whole genome sequencing with global studies of regulatory sequences of human cortical neurons to reveal the regulatory architecture of ASD. Analysis of de novo mutations from whole genome sequencing of 261 autism families revealed the physical proximity of ASD de novo mutations specifically to the cortical expression quantitative loci (eQTLs) of synaptic genes. We performed ATAC-Seq, ChIP-Seq, RNA-Seq and Hi-C experiments on human cortical neurons, which for the first time provided a paranormal view of the regulatory landscape in these cells. We found that ASD de novo mutations preferentially affect regulatory elements, and the associated genes are shared targets of two ASD syndromic factors, CHD8 and PTEN. Analyzing 15 chromatin states across 127 human tissue/cell types revealed a significant enrichment of ASD de novo mutations in active transcription start sites and the perturbed genes implicated in neuron functions; this distribution enabled us to develop a machine-learning algorithm to assess potential ASD risk for a given individual. Taken together, our study for the first time revealed the regulatory landscape in human neurons, demonstrated the importance of the non-coding genome in ASD and provides a general framework for analyzing regulatory mutations for other complex human diseases.
Project description:We aim to profile the transcriptomic changes in neurons when responding to cooling, in order to understand the neuroprotective effects of hypothermia. polyA selected RNA-seq were performed from human iPSC-derived cortical neurons under control (37 ºC), cooled (72 h at 32 ºC, day 15-18) and rewarmed (72 h at 32 ºC, day 15-18, followed by 72 h at 37 ºC, day 18-21) conditions.
Project description:Induced pluripotent stem cell (iPSC)-derived cortical neurons present a powerful new model of neurological disease. Previous work has established that differentiation protocols produce cortical neurons but little has been done to characterise these at cellular resolution. In particular, it is unclear to what extent in vitro two-dimensional, relatively disordered culture conditions recapitulate the development of in vivo cortical layer identity. Single cell multiplex RT-qPCR was used to interrogate the expression of genes previously implicated in cortical layer or phenotypic identity in individual cells. Unexpectedly, 22.7% of neurons analysed frequently co-expressed canonical fetal deep and upper cortical layer markers, and this co-expression was also present at the level of translated protein. By comparing our results to available single cell RNA-seq data from human fetal and adult brain, we observed that this co-expression of layer markers was also seen in primary tissue. These results suggest that establishing neuronal layer identity in iPSC-derived or primary cortical neurons using canonical marker genes transcripts is unlikely to be informative. Single cell RNA-seq of 16 iPSC-derived cortical neurons. This dataset was used for normalization purposes for GSE67835.
Project description:We have assessed the importance of SQSTM1 in human induced pluripotent stem cell (iPSC)-derived cortical neurons with and without SQSTM1. By combining high-content imaging, RNA-Seq, and functional mitochondrial readouts, we showed that SQSTM1 depletion causes aberrations in mitochondrial gene expression and functionality in iPSC-derived neurons.
Project description:Huntington's disease (HD) is a neurodegenerative disease caused by an expanded CAG repeat in the Huntingtin (HTT) gene. Induced pluripotent stem cell (iPSC) models of HD provide an opportunity to study the mechanisms underlying disease pathology in patient tissues relevant to disease. Murine studies have demonstrated that HTT is intricately involved in corticogenesis, and mutant (mt) HTT cannot compensate for the loss of non-CAG-expanded HTT. However, the critical effect of mtHTT in human corticogenesis has not yet been specifically explored and due to inherent differences in cortical development and timing between humans and mice. We therefore differentiated HD and non-diseased iPSCs into functional cortical neurons. While HD patient iPSCs can be successfully differentiated towards a cortical fate in culture, the resulting neurons display transcriptomic, morphological and functional phenotypes indicative of altered neurodevelopment. This is the first demonstration of altered corticogenesis from HD human patient cells, further supporting the potential neurodevelopmental aspect of HD.
Project description:This SuperSeries is composed of the following subset Series: GSE24440: Sprouting transcriptome in cortical neurons: young GSE24441: Sprouting transcriptome in cortical neurons: aged Refer to individual Series
Project description:To determine direct targets of PTBP2-dependent alternative splicing, we performed CLIP-seq analysis of PTBP2 binding in both human cortical tissue and human neurons derived from induced-pluripotent stem cells (iPSC-neurons), and we combine this with splicing analysis following PTBP2 depletion in iPSC-neurons.
Project description:Single-cell RNA-seq: We used single-cell RNAseq to investigate the maturation of astrocytes within human cortical spheroids Bulk RNA-seq: Bulk sequencing from astrocytes and neurons purified (via immunopanning) from iPSC-derived coritical spheroids at varying in vitro differentiation states
Project description:It has been unclear whether ischemic stroke induces neurogenesis or neuronal DNA-rearrangements in the human neocortex. We show here that neither is the case, using immunohistochemistry, transcriptome-, genome- and ploidy-analyses, and determination of nuclear bomb test-derived 14C-concentration in neuronal DNA. A large proportion of cortical neurons display DNA-fragmentation and DNA-repair short time after stroke, whereas neurons at chronic stages after stroke show DNA-integrity, demonstrating the relevance of an intact genome for survival. Analyze of potential fusion transcripts in 13 samples, seven cortical ischemic stroke tissue and six control cortex, by deep sequencing using Illumina HiSeq 2000