31889
Development of Human Stem Cell Pre-Clinical Models to Understand Novel ASD Genes Identified in Multigenerational Families
In the last five years there have been exciting developments regarding the genetic causes of ASD, however mechanistic analysis of ASD pathophysiology has been impaired by the inaccessibility of disease-relevant brain tissue. Patient-derived induced pluripotent stem cells (iPSC) enable modelling of genetic diseases ex vivo, providing an outstanding opportunity to investigate disease mechanisms and ultimately develop and test novel therapies for ASD. Here, we have applied a novel approach to gene discovery in ASD by studying large families and then used iPSC models to determine how the mutation may be linked to ASD.
Objectives:
To develop pre-clinical models to test the role of the genetic variants and more broadly demonstrate the utility of iPSCs in ASD research.
Methods:
iPSC lines from 9 individuals were generated from the family that the variant linked to ASD was identified. Isogenic controls were generated using to CRISPR-Cas9 technology to use alongside the iPSC that carry the variant. A glial/neuron co-culture differentiation protocol was used to generate neuronal networks and visualised using confocal microscopy. Real time PCR, confocal microscopy, calcium signalling assays, multi-electrode arrays (MEAs) and synaptogenesis assays were used for characterisation studies.
Results:
We have successfully generated a neuron/glia co-culture system which have active and functional neuronal networks. We have recently generated CRISPR-Cas9-corrected isogenic control iPSC lines in two members from one of our ASD families where we identified the novel variant linked to ASD. Our preliminary data in our neuron/glial cultures, demonstrates a reduction in neuronal network activity in our mutant iPSCs compared to our isogenic control cells. More specifically we have shown a decreased firing rate, bursting rate and synchrony metrics of the neuronal networks in the cells containing the mutation compared to the isogenic control cells. We are currently establishing a calcium signalling assays to determine functionality of these networks as well as synaptogenesis assays to determine whether there are any defects in synapse function in mutant vs isogenic control cells.
Conclusions:
We have generated a pre-clinical pipeline using human iPSCs to determine how novel genes identified in multi-generational pedigrees may be contributing to ASD. This approach will make an important contribution to our understanding of the aetiology and pathogenesis of ASD and will establish the pipelines to use more broadly with other ASD genes.