Compromised Neurite Morphology of Induced Pluripotent Stem Cell-Derived Neurons: Similar Patterns from Independent Non-Syndromic Autism Cases

Background:  Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental condition, yet without approved pharmacological treatment options for the core symptoms. This significant need for effective treatments has resulted in great efforts to find reliable preclinical models of the disorder. Human behavioral aspects of ASD seem to be overwhelmingly difficult to recapitulate in in vivo models, therefore the want for in vitro disease models with high translational value is huge.

Objectives:  The aim of the present study was to establish a human-derived in vitro disease model of ASD using the induced pluripotent stem cell (iPSC) technology.

Methods: Blood samples of three subjects on the autism spectrum and two controls were taken after ethical approval and obtaining written informed consent. The diagnosis of subjects was confirmed with the Autism Diagnostic Observation Schedule and Autism Diagnostic Interview Revised.  To exclude syndromic ASD forms, next generation sequencing was performed with TruSight Autism Rapid Capture Kit (Illumina) to analyse 103 ASD associated gene variations. Mononuclear cells were isolated from the blood samples and a non-integrating delivery system was used to overexpress the genes of reprogramming factors in the cells. Two to three clones per subject were selected for further differentiation among the reprogrammed clones. The iPSCs showed embryonic stem cell morphology, normal karyotype, expressed pluripotency markers, and were able to spontaneously differentiate into cells of the three germ layers. Then, iPSCs were differentiated into neuronal precursor cells and neurons by a dual-SMAD inhibition protocol. Neuronal differentiation was demonstrated by neuron specific immunolabelling for MAP2, detection of tetrodotoxin-sensitive sodium currents and bicuculline-sensitive chloride currents by using patch clamp, as well as live cell calcium imaging.

Results:  iPSC-derived neuronal cell cultures were investigated in order to detect substantial phenotypical differences between neurons originated from autistic and neurotypical subjects. As ASD is widely accepted as a neurodevelopmental connectivity disorder, first neurite morphology of the cells was explored. According to the morphological parameters (measured by using an Operetta® High Content Imaging System; PerkinElmer), neuronal maturation was found significantly less pronounced in autistic samples. Investigation of further cell biological parameters is in progress.

Conclusions:  Our findings support the approach that iPSC-derived neuronal cultures may serve as relevant in vitro models that can shed light on the pathophysiology of autism, help to identify novel biomarkers and/or therapeutic targets for the treatment of ASD as well as provide a platform for screening novel drug candidates.