Human pluripotent stem cell models of autism spectrum disorder: emerging frontiers, opportunities, and challenges towards neuronal networks in a dish

Abstract

Autism spectrum disorder (ASD) is characterized by deficits in language development and social cognition and the manifestation of repetitive and restrictive behaviors. Despite recent major advances, our understanding of the pathophysiological mechanisms leading to ASD is limited. Although most ASD cases have unknown genetic underpinnings, animal and human cellular models of several rare, genetically defined syndromic forms of ASD have provided evidence for shared pathophysiological mechanisms that may extend to idiopathic cases. Here, we review our current knowledge of the genetic basis and molecular etiology of ASD and highlight how human pluripotent stem cell-based disease models have the potential to advance our understanding of molecular dysfunction. We summarize landmark studies in which neuronal cell populations generated from human embryonic stem cells and patient-derived induced pluripotent stem cells have served to model disease mechanisms, and we discuss recent technological advances that may ultimately allow in vitro modeling of specific human neuronal circuitry dysfunction in ASD. We propose that these advances now offer an unprecedented opportunity to help better understand ASD pathophysiology. This should ultimately enable the development of cellular models for ASD, allowing drug screening and the identification of molecular biomarkers for patient stratification.

Fig. 1

A roadmap towards ASD modeling in hPSCs. Genomic analysis from ASD patients identifies those who harbor genetic defects that might be causal and hiPSCs from these patients can be generated from biopsy material. Insight into shared pathophysiological trajectories comes from pathway analyses of genetic aberrations, as well as from network-based analyses of gene expression in postmortem brains of ASD patients. Guided by the identification of pathways commonly dysregulated in larger subsets of patients, this knowledge informs the selection of genes to be targeted to create additional ASD models. If present in the patient cohort, these can be generated by somatic cell reprogramming. Alternatively, additional genotypes can be obtained from normal hPSCs via targeted genetic manipulation using genome editing. Control hPSCs may also be generated from patient hPSCs by reverting the causative genetic aberration using genome editing

Fig. 2

Transcriptome analyses validate the relevance of hPSC-based models of neuronal differentiation. Principal component analysis (PCA) of the expression profiles of 4,818 genes of postmortem tissue samples from human cerebellum and frontal and temporal cortex (Voineagu et al. 2011) are compared to the expression profiles of hESC-derived neural progenitor cells (NPCs) and their differentiated progeny (our unpublished work). NPCs were generated from hPSCs by dual SMAD inhibition and differentiated for 7–35 days (D) in medium containing BDNF, glial-derived neurotrophic factor, ascorbic acid and dibutyryl-cyclic adenosine monophosphate (+BGAA), or in basal medium (−BGAA). The analysis shows that transcriptomic profiles of neuronal cultures differentiated for 35 days more closely approximate that of human cortex than of cerebellum, suggesting cortical-like properties. Inclusion of BGAA increases the kinetics and/or extent of neuronal maturation

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