Characterizing the Molecular Mechanisms Underlying Autism Using iPSC-Based Models of Neurodevelopment

Background: Although studies using animal models or autopsy samples have increased our understanding of ASD pathophysiology, many questions still remain. A major constraint in ASD research has been the paucity of disease-relevant human tissues and cells with which to study the molecular mechanisms of ASD.  Since ASD is a neurodevelopmental disorder, approaches are needed that facilitate characterization of neurons during development.

Objectives: We have used patient-specific induced pluripotent stem cells (iPSCs) to begin to dissect the molecular mechanisms underlying ASD during neuronal development. IPSCs permit the analysis of neuronal cells as they differentiate from pluripotent stem cells into functionally mature inhibitory and excitatory cortical-like neurons. The development of these neurons is essential to establishing proper circuitry in many regions of the brain, particularly those that have been identified as abnormal in ASD.  Using our established ASD-specific iPSC lines, we are investigating several key neurobiological mechanisms that govern GABAergic and glutamatergic synapse formation at multiple time points during in vitro neurogenesis.

Methods: iPSC lines were developed from peripheral blood mononuclear cells (PBMCs) isolated from individuals with autism and healthy control individuals. These iPSC lines were validated for their pluripotency and self-renewal characteristics through RNA profile analysis and ICC. Once validated, the ASD-specific and control iPSC lines were differentiated into GABAergic or glutamatergic neurons through the serial treatment with cytokines and morphogens designed to induce neurogenesis mimicking in vivo neurodevelopment. To reduce potential heterogeneity, specific neuronal cell types (eg. GABAergic neurons) were isolated and analyzed to identify key molecular mechanisms and functions that are altered in ASD.

Results: These ASD-specific iPSC lines are able to differentiate into neural stem cells and progenitors that give rise to electrophysiologically active cortical-like GABAergic and glutamatergic neurons. One key challenge working with stem cell derived neuronal cultures has been the heterogeneity associated with the differentiation process. This can often mask subtle changes in key cell types that may have significant effects on the disease mechanism. To reduce this inherent heterogeneity, we have developed fluorescent reporter constructs that permit the identification and isolation of specific neuronal cell types from the mixed culture. We have been able to achieve over 95% purity of GABAergic neurons by flow cytometry that can be re-plated and are viable for at least two weeks post FACS. These sorted neurons had a significantly different gene expression profile than the original unsorted neuronal culture providing a more accurate picture of the transcriptional make-up of the ASD-specific neurons. Furthermore, neurons derived from the ASD-specific iPSCs exhibit changes in transcriptional profiles compared to iPSC-derived neurons from unaffected individuals, including changes in key genes involved in synaptic functionality such as RELN, NLGN3 and NLGN4X.

 Conclusions: iPSCs provide a valuable resource for understanding the molecular mechanisms that govern ASDs and facilitate analysis of the impact that specific genetic variations have on neuronal development and functionality. The identification of disease-associated mechanisms will open up new avenues for the discovery of novel therapeutic strategies, as well as, the identification of biomarkers to improve diagnosis and early intervention.