Atypical Neurogenesis and Excitatory-Inhibitory Progenitor Generation in Induced Pluripotent Stem Cell (iPSC) from Autistic Individuals

Background: Autism is a set of neurodevelopmental conditions with a complex genetic basis. Induced pluripotent stem cell (iPSC) studies with autistic individuals having macroencephaly have revealed atypical neuronal proliferation and GABA/glutamate imbalance, the latter also observed in magnetic resonance spectroscopy (MRS) studies. Functional genomics of autism post mortem brain tissue has identified convergent gene expression networks associated with autism. However, it is not clear whether the established autism phenotypes are relevant in the wider autism spectrum. It also not known whether autism-associated in vivo gene expression patterns are recapitulated during in vitro neural differentiation.

Objectives: To establish differences during early neural differentiation from autism and controls iPSCs by studying the following markers/phenotypes: 1. Neuronal markers, 2. GABA/glutamate cell markers, 3. Neuronal gene expression networks.

Methods: We generated induced pluripotent stem cells (iPSCs) from a cohort of 9 autistic individuals with heterogeneous backgrounds – including six non-syndromic autistic individuals, one individual with 3p deletion syndrome and two autistic individuals with a mutation in the NRXN1 gene, and 3 typically developing individuals, and differentiated them into early neural precursors, late neural precursors and early neural cells using an in vitro model of cortical neurogenesis. We undertook high throughput imaging to analyse cellular/molecular markers and RNA sequencing/gene expression analyses from a subset of autistic individuals and controls to analyse neuronal gene expression pathways.

Results: We observed atypical neural differentiation of autism iPSCs compared with controls, and imbalance in GABA/glutamate cell populations over time. Gene expression analysis identified altered gene co-expression networks correlated with neural maturation and GABA/glutamate imbalance networks associated with autism post-mortem brains. Gene expression analyses also identified immune pathways enriched in autism neural cells, and found CD44, an autism-associated gene, to be significant.

Conclusions: Our study demonstrates appreciable differences in neural differentiation between autism and control iPSCs including GABA/glutamate precursor imbalance, and preservation of atypical autism-associated gene networks observed in other model systems.