Enteric Nervous System Dysfunction in Autism Spectrum Disorder: Development of an in Vitro Ips-Derived Model System Using Patient Cells

Background:  Autism Spectrum Disorders (ASDs) are characterized by central nervous system (CNS) dysfunction that manifests in behavioral and cognitive deficits. A number of genes, especially those that code for synaptic proteins, have been shown to harbor mutations and/or deletions that result in functional consequences in the CNS of ASD individuals. Many of these genes, for example Shank3 and Neuroligin3, are also expressed in the enteric nervous system (ENS), making it likely that mutations causing CNS dysfunction are also operating at the level of the ENS. In order to explore this relationship we have undertaken the development of a patient-specific induced pluripotent stem cell (iPSC)-derived in vitro model system that can be used to generate and functionally characterize enteric neurons.

Objectives: The overall goal of this project is to develop a model system to study ENS dysregulation in ASD children. We propose to do this by selecting patients from the Autism Genetics Resource Exchange (AGRE) who meet the criteria for suspected CNS and ENS involvement and who have a functionally-relevant single nucleotide polymorphism (SNP) and/or deletion in a key synaptic protein-coding gene. Cells from these individuals (and controls) will be used to differentiate enteric neurons which can then be assessed for function using established in vitro assays.  

Methods:  The steps involved in our model development are fourfold: (1) identify ASD individuals who have ENS involvement (as measured by gastrointestinal dysfunction; e.g. GERD or hypomotility) and CNS involvement, and who have a known mutation/deletion in at least one of the genes known to negatively  impact the function of a key synaptic protein, (2) use lymphoblastoid cell lines (LCLs) from these individuals to generate induced pluripotent stem cells, (3) direct the patient-specific iPSCs down neuronal lineages to make enteric neurons, and (4) characterize the function of these neurons, compared to those derived from individuals with unaffected synaptic proteins, in a smooth muscle co-culture system.    

Results:  Epstein Barr Virus immortalized-LCLs were obtained from two male sibling probands from the AGRE: one with a SNP in the gene coding for Shank3 and another SNP in the gene coding for Homer1; the second with only the Homer1 SNP. IPSCs, generated from EBV-LCLs transfected with Epi5 Episomal iPSC reprogramming plasmids, were apparent at Day16 post-transfection. Clonal iSPC lines are being evaluated for patient-specificity, normal karyotype, expression of pluripotency markers, and loss of OriP/EBNA-1 expression vectors. In parallel experiments, neural progenitor cells and neural crest cells, differentiated from human embryonic stem cells, showed proper gene expression and cell morphology. Neural lineage differentiation methods have now been optimized and will be applied to the ASD-specific LCL-derived iPSCs for the generation of enteric neurons. 

Conclusions:  Using a state-of-the-art reprogramming system, LCLs from ASD individuals in the AGRE repository have been used to generate iPSCs. We have also demonstrated that stem cells can be differentiated to neural progenitor and neural crest cells. Our next step will be to differentiate the ASD-specific iPSCs into enteric neurons and then compare their function to enteric neurons derived from non-ASD cells.