Identifying Underlying Disease Mechanisms in Autism Using iPSC-Based Models of Neurodevelopment

Background: Autism spectrum disorder (ASD) is a heterogeneous, neurodevelopmental disorder characterized by deficiencies in social interaction, verbal and non-verbal communication, and repetitive stereotypical behaviors. Although advances have been made in our understanding ASD pathophysiology, many questions still remain. A major constraint in ASD research has been the paucity of disease-relevant tissues and cells with which to study the molecular mechanisms of ASD. A number of studies have used post-mortem brain tissue collected from individuals affected with autism. However, any findings that are identified in post-mortem ASD brain samples are likely to only represent an end point in the pathology of autism. Since ASD is a neurodevelopmental disorder, approaches are needed that will facilitate analysis during neurogenesis.  

Objectives: The focus of this work was to examine the molecular mechanisms that underlie the cellular pathophysiology of ASD during neurodevelopment using patient-specific induced pluripotent stem cells (iPSCs) as a model. This method permits the observation 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, thus represent potentially vulnerable cell populations in this neurodevelopmental disorder.  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) derived from individuals with autism and healthy control individuals. These iPSC lines were validated for their pluripotency and self-renewal characteristics by immunocytochemical staining and quantitative real-time PCR analysis of key pluripotency genes. Once validated, the ASD-specific and control iPSC lines were differentiated into GABAergic or glutamatergic neurons through the serial treatment with cytokines and morphogens aimed at inducing neurogenesis and mimicking the in vivo temporal process. The mature, fully differentiated neurons were functionally characterized for electrophysiological activity, morphology and synapse formation.

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 in a process that imitates in vivo neurodevelopment. Neurons derived from the ASD-specific iPSCs exhibit aberrations in cellular function and structure compared to iPSC-derived neurons from unaffected individuals. This system provides an ideal opportunity for the application of high content transcriptome analysis and functional characterization to illuminate the fundamental biological processes at play in the development of autism. This strategy has proved useful at answering important questions about the pathophysiology of autism involving excitatory/inhibitory balance and could potentially be used toward the advancement of novel therapeutics and the identification of biomarkers to improve diagnosis for early intervention.

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.