Reprogramming human iPSC into sensory neurons to study Rett syndrome

Sharmin S1,2, Lesperance LS1, Wei W1, Piekna A1, Prescott SA1 and Ellis J1

  1. The Hospital for Sick Children, Toronto, ON, Canada.
  2. The University of Queensland, Brisbane, QLD, Australia.

There is a large and growing population of individuals diagnosed with neurodevelopmental disorders. Rett syndrome is one of the major neurodevelopmental disorders. Symptoms include difficulties in social interaction, communication and repetitive behaviours as well as altered touch and pain sensitivity. Sensory neurons in the peripheral nervous system respond to somatosensory input including touch and pain. Our studies are focused on evaluating sensory neurons from control subjects for comparison neurons from subjects with Rett syndrome. To this end we adapted a previously published protocol (Chambers et al., Nature Biotechnology, 2012) to produce populations of sensory neurons from induced Pluripotent Stem Cell (iPSC) lines derived from healthy control and Rett syndrome individuals (Cheung et al., Human Molecular Genetics, 2011). Our modification of the growth factor and media composition in the Chambers et al. protocol resulted in 85% of the cells exhibiting a repetitive spiking pattern at 5 weeks consistent with nociceptor-like neurons. In contrast, preliminary lentivirus-mediated NGN2 infections and addition of neurotrophin-3 (NT-3) may accelerate the formation of single spiking mechanoreceptor / proprioceptor cell populations with repetitive spiking nociceptor population in sensory neuron culture. Western blots for TRK family proteins and immunostaining for sensory neuron markers support these conclusions and FACS analysis is underway. Preliminary screening of Rett syndrome patient lines using the adapted protocol suggests TRKA and glutamate receptor 1(GLUR1) are down regulated whereas gamma amino-butyric acid (GABA) receptor seems unchanged in western blot which indicates potential synaptic / morphological abnormalities. In future, electrophysiology and morphological analysis will be performed for more depth analysis.