Project description:There are a total of four samples each for this analysis. Each sample consists of the cells grown on three 10 cm culture plates. Each plate should have 2x106 cells for a total of 6x106 cells per sample when all three plates are combined. The first sample is undifferentiated human embryonic stem cells, the second sample is human glutamatergic neurons derived from those human embryonic stem cells, the third sample is undifferentiated human induced pluripotent stem cells and the fourth sample is human glutamatergic neurons derived from those human induced pluripotent stem cells.
Project description:Biallelic mutations in the gene that encodes the enzyme N-glycanase 1 (NGLY1) cause a rare disease with multi-symptomatic features including developmental delay, intellectual disability, neuropathy and seizures. NGLY1’s activity in human neural cells is currently not well understood. To understand how NGLY1 gene loss leads to the specific phenotypes of NGLY1 deficiency, we employed direct conversion of NGLY1 patient-derived induced pluripotent stem cells (iPSCs) to functional cortical neurons. Transcriptomic, proteomic, and functional studies of iPSC-derived neurons lacking NGLY1 function revealed several major cellular processes that were altered, including protein aggregate-clearing functionality, mitochondrial homeostasis, and synaptic dysfunctions. These phenotypes were rescued by introduction of a functional NGLY1 gene and were observed in iPSC-derived mature neurons, but not astrocytes. Finally, laser capture microscopy followed by mass spectrometry provided detailed characterization of the composition of protein aggregates specific to NGLY1-deficient neurons. Future studies will harness this knowledge for therapeutic development.
Project description:In this study, we used human cortical organoids (hCOs) derived from induced pluripotent stem cells (iPSCs) to examine how hypoxia exposure interferes with astrocyte synapse engulfment at 6 months in culture and 10 months in culture, equivalent to mid fetal- or neonatal-equivalent stages of development, respectively. We identified that hypoxia inhibits the synaptosome engulfment by human astrocytes at both developmental stages, with a more pronounced phenotype in 10 months astrocytes. Transcriptional analyses revealed disruptions in circadian rhythm pathways in hypoxic astrocytes, but not neurons, and PER2 luciferase assays (PER2::LUC) validated the presence of circadian rhythms and their disruption by hypoxia in intact hCOs, with more pronounced rhythms in 10 months hCOs.
Project description:Astrocytes regulate the functional maturation of neurons by providing trophic support, regulating membrane properties and coordinating synapse formation. However, it is unclear to what degree astrocytes use activity-dependent mechanisms in these intercellular signalling processes. Using an induced pluripotent stem cell system and long-term optogenetic stimulation of human astrocytes, we reveal that activity-dependent astrocytic signals enhance the functional maturation of human cortical neurons, through increases in synaptic connectivity and excitability. Transcriptomic analyses determine that this involves the activity-dependent up-regulation of cholesterol synthesis – a process ascribed to astrocytes, which regulates neuronal maturation. Up-regulated astrocyte genes encode enzymes and transcription factors that control the levels of cholesterol synthesis. Biochemical assays confirm an activity-dependent upregulation of cholesterol synthesis in astrocytes, which is required for the maturational effects upon neurons. Thus, we reveal a novel mechanism that may dynamically match astrocyte function to neuronal needs, and identify targets for modulating cholesterol synthesis in the CNS.
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:FIP200 (also known as RB1CC1) has been implicated in a number of psychiatric disorders. The current project aims to characterize the membranomic changes in neurons due to FIP200 loss of function. To this end, two isogenic human pluripotent stem cell (hPSC) lines with FIP200 KO mutations in exon 4 were generated using CRISPR-Cas9-mediated genome editing. The resulting KO clones, together with control lines, were then forward programmed into neurons by overexpression of NGN2, followed by co-culture on mouse astrocytes for 3 weeks before harvesting the cells for MS analysis.
Project description:Cerebral organoids (CerOrgs) derived from human induced pluripotent stem cells (iPSCs) are a valuable tool to study human astrocytes and their interaction with neurons and microglia. The timeline of astrocyte development and maturation in this model is currently unknown and this limits the value and applicability of the model. Therefore, we generated CerOrgs from three healthy individuals and assessed astrocyte maturation after 5, 11, 19, and 37 weeks in culture. At these four time points, the astrocyte lineage was isolated based on the expression of integrin subunit alpha 6 (ITGA6). Based on the transcriptome of the isolated ITGA6-positive cells, astrocyte development started between 5 and 11 weeks in culture and astrocyte maturation commenced after 11 weeks in culture. After 19 weeks in culture, the ITGA6-positive astrocytes had the highest expression of human mature astrocyte genes, and the predicted functional properties were related to brain homeostasis. After 37 weeks in culture, a subpopulation of ITGA6-negative astrocytes appeared highlighting the heterogeneity within the astrocytes. The morphology shifted from an elongated progenitor-like morphology to the typical bushy astrocyte morphology. Based on the morphological properties, predicted functional properties, and the similarities with the human mature astrocyte transcriptome, we concluded that ITGA6+ astrocytes have developed optimally in 19-week-old CerOrgs.
Project description:Cellular plasticity is a prerequisite to adapt to ever-changing stimuli. Therefore, cells constantly reshape their translatome and, in turn, their proteome. The control of translational activity has been extensively studied at the stage of translation initiation. How cells regulate polysome speed is, however, widely unknown. Here, we exploited a kinetic approach to investigate global translation kinetics in cells. We found that polysome speeds differ between different cell types including astrocytes, induced pluripotent human stem cells, human neural stem cells, and human and rat neurons. Among these cells, we observed that mature cortical neurons translate the fastest. This finding was even more striking as these cells express the elongation factor 2 (eEF2) at lowest levels compared to other cell types. We found that neurons resolve this obstacle by inactivating a fraction of their ribosomes. This process is regulated by the levels and phosphorylation of eEF2 as well as by NMDA mediated neuronal stimulation. Our data strongly suggest a novel regulation mechanism in which nerve cells inactivate ribosomes to allow for translational remodeling. This finding has important implications for developmental brain disorders that are hallmarked by altered translation.