Atypical neurogenesis and excitatory-inhibitory progenitor generation in induced pluripotent stem cell (iPSC) from autistic individuals

Autism is a set of neurodevelopmental conditions with a complex genetic basis. Previous induced pluripotent stem cell (iPSC) studies with autistic individuals having macroencephaly have revealed atypical neuronal proliferation and GABA/glutamate imbalance, the latter also being observed in magnetic resonance spectroscopy (MRS) studies. Functional genomics of autism post mortem brain tissue has identified convergent gene expression networks. However, it is not clear whether the established autism phenotypes are observed in the wider autism spectrum. It also not known whether autism-associated in vivo gene expression patterns are recapitulated during in vitro neural differentiation. To examine this we have generated induced pluripotent stem cells (iPSCs) from a cohort of autistic individuals with heterogeneous backgrounds, which were differentiated into early and late neural precursors, and early neural cells using an in vitro model of cortical neurogenesis. We observed atypical neural differentiation of autism iPSCs compared with controls, and dynamic imbalance in GABA/glutamate cell populations over time. RNA-sequencing identified altered gene coexpression networks associated with neural maturation and GABA/glutamate imbalance, and these pathways correlated with pathways in post-mortem brains. Autism neural cells also recapitulated autism post mortem immune pathways, and found CD44, an autism-associated gene, to be predicted as a highly connected gene. In conclusion, our study demonstrates significant differences in neural differentiation between autism and control iPSCs including GABA/glutamate precursor imbalance, and significant preservation of atypical autism-associated gene networks observed in other model systems. Studies using both 2D and 3D iPSC-cultures derived from autistic individuals with macrocephaly, have demonstrated atypical neural differentiation and increased cell proliferation of neural precursor cells (NPCs), and also an imbalance in excitatory (glutamate-producing) and inhibitory (GABA-producing) receptor activity 18, 19 , and these cellular effects were found to correlate with enlarged brain size of participants. These observations strengthen the hypothesis that iPSC-based systems can recapitulate cellular phenotypes relevant for disease 18 . In 3D iPSC cultures derived from autistic individuals with macrocephaly, an overproduction of GABAergic neurons has been observed 19 , while in the 2D cultures, alterations in excitatory/inhibitory (E/I) neural networks suggest decreased glutamatergic excitation 18 . Critically, there is increasing evidence from magnetic resonance spectroscopy (MRS) studies of autistic individuals, of abnormalities in levels of excitatory glutamate and inhibitory GABA metabolites 20, 21 . These reports appear to demonstrate a common trend consistent across various model systems, of a reduction of glutamate signalling versus GABA signalling 18, 19, 21 . This also opposes an existing hypothesis of GABA/glutamate signalling, which suggested increased glutamate signalling 22 , but which was based on the co-occurrence of epilepsy in autism. However, most autistic individuals do not have seizures 23 , and epilepsy cannot be explained as a simple consequence of glutamate overproduction. Imbalances in GABA-glutamate neuron markers have also been observed in autism post mortem brains 11, 24 . major neuronal autism far,

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