ISDN2012_0223: Rett syndrome iPS cell derived neurons reveal novel electrophysiology alterations

N. Farra 1,2, W.B. Zhang 1, P. Pasceri 1, J.H. Eubanks 2,3, M.W. Salter 1,2, J. Ellis 1,2,∗ 1 Hospital for Sick Children, Canada 2 University of Toronto, Canada 3 Toronto Western Hospital, Canada E-mail address: jellis@sickkids.ca (J. Ellis). Introduction: Rett (RTT) syndrome is a neurodevelopmental autism spectrum disorder primarily caused by mutations in the methyl CpG-binding protein 2 (MECP2) gene on the X chromosome. Here we describe the first characterization and neuronal differentiation of iPS cells derived from Mecp2-deficient mice. Methods: iPS cells were derived by 3 factor retrovirus delivery of Oct4, Sox2 and Klf4 into fibroblasts from wild-type and Mecp2308 mice including heterozygous females and hemizygous males. The iPS cell lines were directed to differentiate into functional glutamatergic neurons by generating embryoid bodies that were treated with Retinoic Acid. These neurons were stained for MAP2 and other markers, and patch clamp recordings were made from several hundred neurons to examine electrophysiology alterations in Rett syndrome. Results: The iPS cell lines are fully reprogrammed. They express endogenous pluripotency markers, silence the retrovirus transgenes, reactivate the X-chromosome in female cells, and differentiate into cells of the three germ layers in vitro and form teratomas in vivo. iPS cell-derived neurons are electrically excitable, generate action potentials, and form functional excitatory synapses. Neurons from heterozygous female and hemizygous male iPS cells show defects in generating evoked action potentials and glutamatergic synaptic transmission, as previously reported in Mecp2-null mouse brain. Further, we identify novel previously untested deficits in Mecp2-deficient iPS cell-derived neurons, including diminished peak inward voltage-activated currents and higher input resistance relative to neurons differentiated from wild-type iPS cells. Discussion: Taken together, these results indicate that neuronally differentiated MeCP2-deficient iPS cells recapitulate deficits observed previously in MeCP2-null mouse neurons, and newly identified phenotypes further illustrate the requirement for MeCP2 in development or maintenance of normal neuronal function. Thus, our results provide validation for the use of iPS cells to delineate mechanisms underlying RTT pathogenesis, and also identify deficiencies that can be readily targeted for in vitro translational screens.