iPSC-derived neurons as a higher-throughput readout for autism: promises and pitfalls

Abstract

The elucidation of disease etiologies and establishment of robust, scalable, high-throughput screening assays for autism spectrum disorders (ASDs) have been impeded by both inaccessibility of disease-relevant neuronal tissue and the genetic heterogeneity of the disorder. Neuronal cells derived from induced pluripotent stem cells (iPSCs) from autism patients may circumvent these obstacles and serve as relevant cell models. To date, derived cells are characterized and screened by assessing their neuronal phenotypes. These characterizations are often etiology-specific or lack reproducibility and stability. In this review, we present an overview of efforts to study iPSC-derived neurons as a model for autism, and we explore the plausibility of gene expression profiling as a reproducible and stable disease marker.

Keywords: autism; gene expression; high-throughput assay; iPSC.

Figure I. Example of comprehensive transcriptomic map for reprogramming and neuronal differentiation

Principal component analysis of whole-transcriptome profiles for blood, lymphoblast cell lines, brain tissue, fibroblasts, induced pluripotent stem cells, embryonic stem cells, primary neural progenitors and derived neurons showing clustering of cell types based on the first two principal components (PC1 and PC2). This database is comprised of 899 gene expression samples belonging to 25 series performed on the Illumina HumanRef-8 v3.0 expression beadchips that were obtained from NCBI’s Gene Expression Omnibus (GEO)[, , –127]. Interestingly, the gene expression signature of primary neuronal cultures (neural progenitor cells (NPCs) at 0, 2, 4 and 8 weeks) consistently shifts towards brain tissue over the course of days in culture and neural differentiation.

Figure II. Reprogramming and neuronal differentiation distribution over stemness index or pluripotency score

Each curve represents the distribution of pluripotency score values for a particular differentiation stage. The differentiation process is represented from the extreme right curve to the extreme left, with decreasing pluripotency score, clearly separating between early and late stages of differentiation. The spectrum of pluripotency in fibroblasts, induced pluripotent stem cells (iPSCs), iPS-derived neural progenitor cells (NPCs) and iPSC-derived neurons (neurons), which were derived from individuals with Timothy syndrome (A) and from control individuals (B). iPSCs were generated from dermal fibroblasts through retroviral reprogramming and then differentiated into NPCs and neurons using conditions favoring generation of cortical neurons [22]; (C) Spectrum of pluripotency in differentiating primary normal neural progenitor cells at 0, 2, 4 and 8 weeks of differentiation in vitro [121].

Figure II. Reprogramming and neuronal differentiation distribution over stemness index or pluripotency score

Each curve represents the distribution of pluripotency score values for a particular differentiation stage. The differentiation process is represented from the extreme right curve to the extreme left, with decreasing pluripotency score, clearly separating between early and late stages of differentiation. The spectrum of pluripotency in fibroblasts, induced pluripotent stem cells (iPSCs), iPS-derived neural progenitor cells (NPCs) and iPSC-derived neurons (neurons), which were derived from individuals with Timothy syndrome (A) and from control individuals (B). iPSCs were generated from dermal fibroblasts through retroviral reprogramming and then differentiated into NPCs and neurons using conditions favoring generation of cortical neurons [22]; (C) Spectrum of pluripotency in differentiating primary normal neural progenitor cells at 0, 2, 4 and 8 weeks of differentiation in vitro [121].

Figure II. Reprogramming and neuronal differentiation distribution over stemness index or pluripotency score

Each curve represents the distribution of pluripotency score values for a particular differentiation stage. The differentiation process is represented from the extreme right curve to the extreme left, with decreasing pluripotency score, clearly separating between early and late stages of differentiation. The spectrum of pluripotency in fibroblasts, induced pluripotent stem cells (iPSCs), iPS-derived neural progenitor cells (NPCs) and iPSC-derived neurons (neurons), which were derived from individuals with Timothy syndrome (A) and from control individuals (B). iPSCs were generated from dermal fibroblasts through retroviral reprogramming and then differentiated into NPCs and neurons using conditions favoring generation of cortical neurons [22]; (C) Spectrum of pluripotency in differentiating primary normal neural progenitor cells at 0, 2, 4 and 8 weeks of differentiation in vitro [121].

Figure 1. Overview of a pipeline to generate iPSCs and cortical neurons for modeling ASDs

Somatic tissue such as skin is obtained from the patient and expanded into fibroblasts. These fibroblasts are reprogrammed into iPSCs, which are later differentiated to a neural forebrain fate. Cortical neural fate may be achieved using several examples of protocols. Forebrain neural progenitors may be directed into a cortical excitatory projection fate or an inhibitory interneuron fate [52]. The resulting neurons are validated for their structural and functional properties in culture. Next, these cells may be transplanted into mouse cortex to evaluate their ability to generate networks, fire action potentials and form synapses. Sources of variation throughout this pipeline are summarized [45]. Abbreviations: BMP, bone morphogenetic protein; FGF, fibroblast growth factor; GABA, γ-aminobutyric acid; IGF, insulin growth factor; iPSCs, induced pluripotent stem cells; RA, retinoic acid; SHH, sonic hedgehog; SMAD, Sma and Mothers Against Decapentaplegic signaling; TGFβtransforming growth factor βWnt, Wingless/Int proteins.