Thus, this system will be generally applicable to analysis of mutations in genes with a general neuronal action, for example FMRP, neuroligins, and MECP2. However, even for analysis of diseases that manifest in specific types of neurons, such as Parkinson’s disease, Ngn2 iN cells may be useful because in many neurological diseases the pathological processes are not restricted to the specfiic types of neurons in which the disease becomes manifest. Specifically, even if disease

such Ponatinib concentration as Parkinson’s or Huntington’s disease manifest in a dysfunction of dopaminergic or striatal neurons, respectively, this manifestation probably represents a particular vulnerability of specific types of neurons to a general disease process, and not a disease

process that is restricted to these types of neurons. Thus, even for such diseases it may not only be feasible, but actually be productive to examine Ngn2-generated iN cells as a homogeneous population of glutamatergic neurons, especially in coculture with mouse neurons or after transplantation into the mouse brain. H1 ESCs were obtained from WiCell Research Resources (Wicell, WI); the iPS#1 line was derived SCR7 in vitro from dermal fibroblasts of a Dystrophic epidermolysis bullosa patient carrying homozygous mutations in COL7A1, while the iPS#2 line was derived from dermal fibroblast of a sickle cell anemia patient and genetically corrected by homologous recombination ( Sebastiano et al., 2011). The type VII collagen gene is not expressed in neurons, patients with mutations have no brain phenotypes, and our study demonstrates that the mutation of this gene does not affect the molecular and functional properties of Ngn2-mediated iN cells. Both iPSC lines were generated from by infecting with a floxed polycistronic lentiviral reprogramming vector followed by Cre-mediated loop-out of the reprogramming factors ( Sommer et al., 2010). ESCs and iPSCs were maintained as feeder-free cells in mTeSR1 medium

(Stem Cell Technologies; Xu et al., 2010). Mouse glial cells were cultured from the forebrain of newborn wild-type CD1 mice ( Franke et al., 1998). Briefly, newborn mouse forebrain homogenates were digested with papain and EDTA for 30 min, cells were dissociated by harsh trituration to avoid growing of neurons, and plated onto T75 flasks in DMEM supplemented with 10% FBS. Upon reaching confluence, glial cells were trypsinized and replated at lower density a total of three times to remove potential trace amounts of mouse neurons before the glia cell cultures were used for coculture experiment with iN cells. Mouse cortical neurons were cultured as described ( Pang et al., 2011), added to iN cells 4–5 days after infection, and cocultured for an additional 2 weeks. Lentiviruses were produced as described (Pang et al.