Autism Patient-Derived Neural Stem Cells Display Neurite Extension and Migration Defects
Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by impaired social interaction and communication and the presence of repetitive restrictive behaviors. Invaluable studies in ASD mouse models have shown changes in neuronal connectivity and migration that replicate findings of human neuropathology and brain-MRI. However, mouse models cannot capture the complexity of human neurodevelopment. Thus, in order to understand a uniquely human disorder like ASD, we need techniques to model human neurodevelopment. Recently, induced pluripotent stem cell (iPSC) technology has allowed the generation of neural stem cells (NSCs) and neurons from individuals with neuropsychiatric disorders thereby allowing study of human neuropathology. Using iPSCs we have generated NSCs from 8 boys with severe idiopathic autism and their unaffected-brothers (Sib) as controls.
Our goals are to study neurite outgrowth and neurosphere migration in our patient-derived NSCs using developmentally relevant extracellular (EC) factors such as PACAP, serotonin (5HT) and nerve-growth-factor (NGF).
Neurite Assay: Confluent NSCs were dissociated and plated at low density on fibronectin+ poly-d-lysine coated plates in control media, or media containing EC factors. At 48h, the proportion of cells with neurites >2 cell diameters were counted blind in 1 cm rows. 3 dishes were quantified per condition per experiment. At least 200 cells were counted per dish
Neurosphere Migration: Neurospheres were formed by plating NSCs in absence of substrate. After 48-96h, spheres were plated on Matrigel with control or EC factor media and fixed at 48h. Using phase-contrast images, areas of the inner-cell-mass and total sphere outgrowth were measured. 15-21 spheres were measured blind per condition per experiment. Migration: total sphere area-inner cell mass area.
We have extensively studied neurite outgrowth and migration in NSCs from one family. To ensure that the phenotypes observed were not a consequence of the viral-reprogramming involved in iPSC generation, we have assessed multiple NSCs derived from 2 iPSC clones for Sib and multiple NSCs derived from 3 iPSC clones for ASD.
In control media, Sib NSCs had a significantly higher percent of cells with neurites (15%) than ASD NSCs (8%, p<0.0001). Under stimulation of PACAP, there was a 51% increase in cells bearing neurites in the Sib NSCs (p<0.0004) but no change in ASD NSCs (p>0.5). Similarly, under stimulation of NGF, there was a 58% increase in neurites in Sib NSCs (P<0.005) but no change in ASD NSCs (p>0.7). Finally, Sib NSCs displayed a dose-dependent increase in neurites with 5HT stimulation while ASD NSCs only responded at the maximal tested dose of 5HT indicating a difference in sensitivity.
In control media, Sib neurospheres migrated further than ASD NSCs (p<0.05). Under the stimulation of PACAP there was a 50% increase in migration of Sib neurospheres (P<0.001) but no change in migration in ASD neurospheres (p>0.7).
Conclusions: Our studies of patient-derived NSCs reveal neurobiological abnormalities that may provide insight into impaired brain development and function in ASD. Our studies also illustrate the value of using developmentally-relevant factors to uncover patient-specific abnormalities that may facilitate the development of personalized ASD therapies