Project description:The healthy human brain is a mosaic of varied genomes. Long interspersed element-1 (LINE-1 or L1) retrotransposition is known to create mosaicism by inserting L1 sequences into new locations of somatic cell genomes. Using a machine learning-based, single-cell sequencing approach, we discovered that somatic L1-associated variants (SLAVs) are composed of two classes: L1 retrotransposition insertions and retrotransposition-independent L1-associated variants. We demonstrate that a subset of SLAVs comprises somatic deletions generated by L1 endonuclease cutting activity. Retrotransposition-independent rearrangements in inherited L1s resulted in the deletion of proximal genomic regions. These rearrangements were resolved by microhomology-mediated repair, which suggests that L1-associated genomic regions are hotspots for somatic copy number variants in the brain and therefore a heritable genetic contributor to somatic mosaicism. We demonstrate that SLAVs are present in crucial neural genes, such as DLG2 (also called PSD93), and affect 44-63% of cells of the cells in the healthy brain.
Project description:Analysis of dopaminergic neuronal gene expression changes by Nurr1 and/or Foxa2 overexpression. Result provides that Foxa2 potentiates Nurr1-induced DA neuronal phenotype gene expression. To identify the syergism of Nurr1 and Foxa2 for developing DA neural precursors, neural precusor cells (NPCs) isolated from embryonic brain were treated control, Nurr1, Foxa2 and Nurr1-Foxa2 retrovirus. After treatment of retroviruses, NPCs were cultrued in N2 media withdrawn mitogen (bFGF, EGF) for differetiation of DA neuron. Total RNA was obtained from NPCs in differentiation day 2.
Project description:Neural precursor cells (NPCs) are multipotent cells that can generate neurons, astrocytes, and oligodendrocytes in the mammalian central nervous system. Although high mobility group nucleosomal binding domain 1 (HMGN1) was highly expressed in NPCs, its functions in neural development are not fully understood. We performed microarray analysis to examine changes in gene expression between control and HMGN1-overexpressed NPCs. NPCs derived from E11.5 mouse forebrains were infected with control or HMGN1 retrovirus in the presence of fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF). Three days after infection, the virus-infected NPCs were dissociated and seeded on poly-D-lysine -coated dishes, and subsequently the cells were cultured for 1 day without FGF2 and EGF.
Project description:Neural stem cells (NSCs) and progenitor cells (NPCs) are increasingly appreciated to hold great promise for regenerative medicine to treat CNS injuries and neurodegenerative diseases. However, evidence for effective stimulation of neuronal production from endogenous or transplanted NPCs for cell replacement with small molecules remains limited. To identify novel chemical entities/targets for neurogenesis, we had established a NPC phenotypic screen assay and validated it using known small-molecule neurogenesis inducers. Through screening small molecule libraries with annotated targets, we identified BET bromodomain inhibition as a novel mechanism for enhancing neurogenesis. BET bromodomain proteins, Brd2, Brd3, and Brd4 were found to be downregulated in NPCs upon differentiation, while their levels remain unaltered in proliferating NPCs. Consistent with the pharmacological study using bromodomain selective inhibitor (+)-JQ-1, knockdown of each BET protein resulted in an increase in the number of neurons with simultaneous reduction in both astrocytes and oligodendrocytes. Gene expression profiling analysis demonstrated that BET bromodomain inhibition induced a broad but specific transcription program enhancing directed differentiation of NPCs into neurons while suppressing cell cycle progression and gliogenesis. Together, these results highlight a crucial role of BET proteins as promising epigenetic regulators in NPC development and suggest a therapeutic potential of BET inhibitors in treating CNS injury and neurodegenerative diseases. NPCs were treated with (+)-JQ-1 or inactive enantiomer (-)-JQ-1 at 0.2 μM or 0.5 μM for 12 and 24 hrs in differentiation medium. The experiments were carried out in 3 independent preparations of NPCs (triplicates)
Project description:The idea that stem cell therapies work only via cell replacement is challenged by the observation of consistent intercellular molecule exchange between the graft and the host. Here we defined a mechanism of cellular signaling by which neural stem/precursor cells (NPCs) communicate with the microenvironment via extracellular vesicles (EVs), and we elucidated its molecular signature and function. We observed cytokine-regulated pathways that sort proteins and mRNAs into EVs. We described induction of interferon gamma (IFN-?) pathway in NPCs exposed to proinflammatory cytokines that is mirrored in EVs. We showed that IFN-? bound to EVs through Ifngr1 activates Stat1 in target cells. Finally, we demonstrated that endogenous Stat1 and Ifngr1 in target cells are indispensable to sustain the activation of Stat1 signaling by EV-associated IFN-?/Ifngr1 complexes. Our study identifies a mechanism of cellular signaling regulated by EV-associated IFN-?/Ifngr1 complexes, which grafted stem cells may use to communicate with the host immune system. polyA RNA profiling of Neural Stem/Progenitor cells (NPCs) cultured in basal/Th1/Th2 conditions, of Exosomes derived from NPCs cultured in basal/Th1/Th2 conditions and of EVs derived from NPCs cultured in Basal/Th1/Th2 conditions. Total RNA was purified using Trizol. Purity and integrity were confirmed by BioAnalyser (Agilent). Paired End library construction and poly-A selection were performed by EASIH (The Eastern Sequence and Informatics Hub, University of Cambridge, Cambridge) according to the Illumina standard protocol. Sequencing was performed by EASIH using Illumina GAII.
Project description:Congenital human cytomegalovirus (HCMV) infection is one of the leading prenatal causes of mental retardation and congenital deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV has been limited by difficulties in sustaining primary cultures. Neuronal cells derived from human induced pluripotent stem (iPS) cells now provide a novel opportunity to investigate HCMV pathogenesis. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their permissiveness to infection with HCMV strain Ad169. Analysis of iPS cells and nearly pure populations of iPS-derived neural stem cells (NSCs), neuroprogenitor cells (NPCs) and neurons suggests that (i) iPS cells are not permissive to HCMV infection; (ii) Neural stem cells have impaired differentiation when infected by HCMV; (iii) NPCs are fully permissive for HCMV infection; the supernatant from infected neural stem cells and NPCs (but not mock infected cells) induced cytopathic effects in human fibroblasts; (iv) most iPS-derived neurons are not permissive to HCMV infection; and (v) infected neurons have impaired calcium influx in response to glutamate. Our approach offers powerful cellular models to investigate the effect of neurotropic viral agents on human neurodevelopment. Adherent monolayer culture of neural progenitor cells (NPCs) were either infected with HCMV Ad169 in triplicate, with each individual sample harvested separately to provide biological replicates for expression analysis. Infected and mock-infected cells were harvested 24 h p.i. RNA. NPCs were 70-80% confluence.
Project description:To isolate neuronal progenitor cells (NPCs), forebrains of E13.5 Miz1+/+ or Miz1-delta-POZ embryos were cut in small pieces, digested with trypsin and filtered through sterile gauze. Cells were cultivated in 2:1 DMEM/F12 supplemented with 1xB27 (Life technologies), 20 ng/ul EGF (Biomol), 20 ng/?l basic FGF (Biomol), 1 ug/ml fungizone (Gibco) and Penicillin/Streptomycin (PAA). NPCs were passaged every seven days. RNA expression of different genotypes was compared in sec. and quart. neurospheres.
Project description:To follow-up findings that miR-9 was abundantly expressed in control NPCs, significantly down-regulated in a subset of SZ NPCs, and that miR-9 levels/activity, neural migration and diagnosis were strongly correlated, we tested the effect of manipulating miR-9 at cellular, proteomic and transcriptomic levels. Unexpectedly, proteomic- and RNAseq-based analysis revealed that these effects were mediated primarily by small changes in expression of indirect miR-9 targets, rather than large changes in direct miR-9 targets; these indirect targets are enriched for migration-associated genes. Together these data indicate that aberrant levels and activity of miR-9 may be one of the many factors that contribute to SZ risk, at least in a subset of patients. Methods: We compared global transcription of forebrain NPCs from two control and two SZ patients with manipulated miR-9 levels by RNAseq. Results: Although RNAseq analysis revealed large inter-individual heterogeneity, we were able to resolve several functional consistencies in the effects of our miR-9 perturbations: i) the change in miR-9 activity was consistent with the inhibitory role of miR-9, ii) the gene expression fold-change of miR-9 target genes (between each perturbation and its corresponding control, summarized by the first principal component) was correlated (r=0.95, p=3.92e-04) with miR-9 fold change and iii) the differentially expressed (DE; p <0.01) gene list resulting from miR-9 perturbation (paired t-test) was enriched for miR-9 targets (1.53-fold, p=1.2e-5). Conclusions: We integrated the miR-9 perturbation RNAseq data with our existing RNAseq datasets contrasting control and SZ hiPSC NPC expression from our cohort 1 (six controls, four patients), to ask whether there was any relationship between the âSZ NPC signatureâ and âmiR-9 perturbationâ datasets; we observed that the DE (p-value <0.01) in âSZ NPC signatureâ is enriched for DE (fdr<0.01) in âmiR-9 perturbationâ (the overall enrichment is 2.31-fold (p=9.39e-09)); there is significant correlation between DE fold-change in these two datasets (overall genes r=0.188; p<10e-50). Effects were mediated primarily by small changes in expression of indirect miR-9 targets, rather than large changes in direct miR-9 targets; these indirect targets are enriched for migration-associated genes Biological duplicates of passage-matched NPCs from 1 control (female) and 1 SZ patient (female) were transduced with either RV-GFP or RV-miR-9-GFP; GFP-positive NPCs were purified by fluorescent activated cell sorting (FACS) and expanded for two passages. In parallel, passage-matched NPCs from 2 controls (1 male, 1 female) and 2 SZ patients (1 male, 1 female) were transiently transfected with either scrambled or miR-9 LNA probes. In both instances, miR-9 perturbation was confirmed by qPCR.