Modeling Enteric Nervous System Function in Children with Phelan Mcdermid Syndrome

Background: Shank3, a critically important scaffolding protein controlling CNS post-synaptic density, is one of the most well characterized genes known to have a functional role in Autism Spectrum Disorders (ASD). Phelan McDermid Syndrome (PMDS), a genetic disorder caused by the deletion of a portion of chromosome 22 containing the Shank3 gene, provides an excellent model in which to study the effects of Shank 3 haploinsufficiency in ASD since PMDS patients exhibit many relevant similarities, including an ASD diagnosis (in >84%) and chronic GI symptoms (reported in >50%).  Shank3 haploinsufficiency is thought to be the main contributor to neurodevelopmental abnormalities in PMDS and Shank3 is also expressed in the enteric nervous system (ENS). Therefore, it is likely that deletions/truncations causing neurological dysfunction in the CNS also have a role in the ENS. To explore this relationship we have developed a patient-specific induced pluripotent stem cell (iPSC)-derived in vitro model system that can be used to generate and functionally characterize enteric neurons. This model will lead to greater understanding of molecular mechanisms involved in PMDS and ASD, specifically those with GI symptoms.

Objectives: The overall goal of this project is to develop a model system to study ENS dysregulation in ASD. This will be accomplished by selecting lymphoblastoid cell lines (LCL) from PMDS children who have an ASD diagnosis and chronic GI symptoms. PMDS patients who meet these criteria provide a compelling model for the investigation of cross nervous system synaptic dysfunction to increase our understanding of the relationship between of ASD and GI symptoms.

Methods: The steps involved in our model development are fourfold: (1) identify PMDS individuals who have chronic GI disturbances (e.g. GERD or hypomotility) and an ASD diagnosis, (2) use lymphoblastoid cell lines (LCLs) from these individuals to generate iPSCs, (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 patients (proband and parent) from the NIMH RGR. IPSCs, generated from EBV-LCLs transfected with Epi5™ Episomal iPSC reprogramming plasmids, were apparent at Day 8 post-transfection and are currently at passage 10. 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 crest cells (NCCs), differentiated from WT-iPSCs, showed proper gene expression and cell morphology. Neural lineage differentiation methods have now been optimized and will be applied to the PMDS-specific LCL-derived iPSCs for the generation of enteric neurons.

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