Direct Conversion of hESCs to Neurons in the Study of Neurodevelopmental Disorders

Introduction – Research of neurodevelopmental disorders has mainly been carried out with animal models. Neurons derived from human embryonic stem cells (hESCs) through in vitro neural differentiation using small molecules, are relatively heterogeneous and functionally immature.

Aim – To implement the transcription factor direct differentiation system to generate induced neurons (iNs) and mature neuronal networks from hESCs for the study of different neurodevelopmental disorders.

Methods – hESCs were induced to overexpress the neurogenic transcription factor NEUROG-1 under the control of doxycycline by either infection with lenti-virus vectors, or by transfection with PiggyBac transposon vectors. Neuronal identity of the iNs was determined by immunofluorescence and qRT-PCR for the expression of neuronal markers (Tuj1, MAP2, GABA, V-Glut1). Functionality of the derived neural networks was analyzed by patch clamp electrophysiology and Ca²⁺ imaging.

Results – Fast conversion of hESCs into iNs with distinct neuronal morphology was observed within 4 days following induction. Both methods for expressing NEUROG-1 were very efficient and robust in generating iNs that were positive for all neuronal markers. However, repetitive spontaneous Ca²⁺ transients which are a good indicator of neuronal maturity were much more pronounced following the viral infection method. Additionally, electrophysiological analysis demonstrated that the response to glutamate, spikes number in response to depolarizing pulses, and spontaneous synaptic currents were more prominent following viral infection.

Conclusions – Overexpression of NEUROG-1 by viral infection drives neuronal differentiation of hESCs more efficiently than the PiggyBac vectors. The derived iNs offer a novel source of functional human neurons to model neurodevelopmental and neurodegenerative disease.