Project description:To assess RNA regulation in FALS for gene expression and alternative processing of RNA in the motor neuron precurssors (MPCs) Amyotrophic lateral sclerosis (ALS) is a late-onset motor neuron disorder. Although its neuropathology is well understood, the cellular and molecular mechanisms that lead to the initiation and progression of this disease are yet to be elucidated due to limitations in the currently available human genetic data. In this study, we generated induced pluripotent stem cells (iPSC) from two familial ALS (FALS) patients with a missense mutation in the fused-in sarcoma (FUS) gene carrying the heterozygous FUS H517D mutation, and the isogenic iPSCs with the homozygous FUS H517D mutation obtained by genome editing from the healthy control iPSCs. These cell-derived motor neurons mimicked several neurodegenerative phenotypes. A part of the mutant FUS protein was localized outside the nucleus and co-localized with stress granules under stress conditions. Moreover, FALS motor neurons showed more apoptotic activity than did control motor neurons. Exon array analysis using motor neuron precursor cells (MPCs) combined with CLIP-seq data sets revealed aberrant gene expression and/or splicing pattern in FALS-MPCs. These results suggest that iPSC-derived motor neurons are a useful tool for analyzing the pathogenesis of human motor neuron disorders.
Project description:We performed total RNA sequencing in order to gain insights into the transcriptional differences between iPSC-derived ALS-diseased (2 patient cell lines, each harboring a C9orf72-mutation) and healthy (2 control cell lines) motor neurons at two different time points. Furthermore, we compared the transcriptomes of each genotype between the two time points to uncover alterations in neuronal maturation in ALS_C9orf72 motor neurons.
Project description:Amyotrophic Lateral Sclerosis is a fatal neurodegenerative disorder that affects motor neurons (MN). We used single cell RNA-seq of degenerating human MN derived from ALS patients to understand molecular drivers of MN degeneration. Patient-derived iPSC bearing a point mutation in the SOD1 gene (SOD1 E100G) were differentiated into MN. MN derived from CRISPR-Cas9 corrected isogenic control iPSC (SOD1 E100E) were used as control. Survival analysis indicated that at 44 days of in vitro differentiation, ALS MN dislpayed survival deficits. At this point, cells were harvested for single cell transcriptomics using the Fluidigm C1 system.
Project description:The goal of this study is to gain insight into the early biomarkers and molecular pathways affected by the SOD1+/A272C mutation in human motor neurons. Isogenic control line was created by CRISPR/Cas9 mediated targeted gene correction. Motor neurons were derived from isogenic iPSC lines, and RNA sequencing was employed to determine differentially expressed genes. This study provides an isogenic platform to study ALS disease mechanism at the early stage.
