Human induced pluripotent stem cells as a model for neurodevelopmental disease

Abstract Autism spectrum disorders (ASD) are neurodevelopmental disorders which encompass a wide range of symptoms, including intellectual disability and delayed development, while the phenotype can range from mild to severe. Despite the identification of over 100 candidate genes associated with increased susceptibility to autism, no underlying mechanism has been revealed for ASD. In addition to this, access to patient material and primary brain cells is limited, and animal models cannot precisely recapitulate the complex pathways which result in ASD pathology. Consequently, the aims of this research project are two-fold. Firstly, to establish an efficient method for the generation of induced pluripotent stem cells from a bank of patient-derived and non-affected control fibroblasts cells. In total 20 skin biopsies were collected, cultured and banked for the project. The second aim of this project was to differentiate iPSCs from a cohort of NRXN1-α deletion patients (one exon 6-13 deletion and two exon 1-5 deletions) and non-affected controls to examine the ability of the lines to differentiate to neuronal subtypes, identify any phenotypic differences and attempt to establish the functional impact of the deletion on neurodevelopmental pathways. iPSC lines from the deletion cohort and non-affected controls were differentiated in parallel in order to generate a population of GABA interneurons, neuronal subtypes found in the hypothalamus and a mixed population of astrocytes and neurons found in the cortex using different protocols. The principle aim of generating these neuronal subtypes was to establish whether or not there were any differences in the ability of the xiv patient and control samples to differentiate into neurons and investigate the functional impact of the deletion on the underlying biological pathways during differentiation. Firstly, it was found that there was no significant difference in the ability of the NRXN1 deletion patient lines to differentiate compared to the non-affected controls. No obvious morphological differences were identified between the samples and qPCR analysis of markers for different stages of the differentiation process demonstrated that the lines differentiated at the same rate. Following analysis of the proteins using mass spectrometry, it was found that a considerable number of proteins were found to be significantly differentially expressed affecting a number of pathways, including mitochondrial function and the cytoskeleton. Taken together, this work has led to the establishment of a bank of fibroblasts from a number of patient and control cohorts enabling the generation and validation of iPS patient-derived cell lines, and enables the investigation of the functional impact of NRXN1 on neurodevelopment in health and disease.2019-02-0