Fig. 3.

Towards cellular models of neuronal network dysfunction. Shown here are the principal stages of differentiation of hPSCs towards synaptically competent mature neurons. a First, neural ectoderm induction generates characteristic three-dimensional rosette-like structures expressing the transcription factor PLZF and the tight-junction protein ZO1 (upper). From these, neuroepithelial precursors expressing NPC markers Sox2 and nestin can be isolated and expanded (lower). b Further differentiation yields neurons extending MAP2a/b-positive dendrites. c Maturing neurons show colocalized synaptic markers, such as the presynaptic marker synapsin and the postsynaptic density protein SHANK3. d Mature neuronal cultures form functionally connected neuronal networks, characterized by synchronized spontaneous network activity seen in recordings from multi electrode arrays. Pathways dysregulated in different forms of ASD may converge on the cellular and molecular levels, allowing identification of early phenotypes in neuronal cultures from patient-derived hiPSCs. Such phenotypes include the identification of translational and transcriptional dysfunction which at the cellular level may be associated with abnormal proliferation and differences in extent and kinetics of early neuronal differentiation. Other pathways may converge at the synapse level, requiring mature neurons with synaptic connections. Dysregulation at the neuronal connectivity level due to synaptic dysfunction may only be identified in human neuronal cultures forming relevant neuronal circuits that resemble those of the human cortex