Ansgene re-expression. (C-D) The GG3.1 cell line displays similar expression levels of the Dlk1-Dio3 locus genes Gtl2 and Rian, which is an indirect measure of complete reprogramming. Equivalent expression of these genes was validated using 2 different primer sets; one novel and one published by Stadtfeld et al., 2010. Values are mean ?SD for 2-3 independent samples. Supplementary Figure 3 - Expression of neural lineage and subtype specific genes throughout Ni of early-passage GG3.1 iPSCs. (A-C) Representative micrographs showing the presence and abundance of HuC/D, Map2, neurofilament (NF) and Calretinin (Calr) positive cells at Ni day 7. Scale bars represent 150 m. (D) The pluripotency marker Rex1 is downregulated during differentiation. (E) The anterior neurodevelopmental gene Otx2 is expressed by day 5 of EB. The neurotrophin receptor TrkB is expressed during the EB stage, but expression is elevated by day 7 of Ni. Calretinin is expressed by Ni day 3. (F) Markers of glutamatergic neurons, vesicular glutamate transporter 2 (VGLUT2) and the AMPA receptor subunit GluR2 are highly expressed by days 7 and 15 of Ni. Likewise, the GABAergic neuronal marker, glutamic acid decarboxylase 1 (GAD1) is unregulated by days 7 and 15. Values are mean ?SD for 2-3 independent samples. Supplementary Table 1 Pluripotent stem cell lInes. Supplementary Table 2 - Primers. Supplementary Table 3 - Antibodies.Prior to differentiation and at days 3 and 7 of neural differentiation, cultures were fixed with 4 paraformaldehyde for 30 min. Chamber slides were incubated in blocking solution and then with a primary polyclonal and a monoclonal antibody together. Primary antibodies used in this study are listed in Supplementary Table 3 (Additional File 1). Immunoreactivity with monoclonal and polyclonal antibodies was visualized by using an Alexa Fluor 488 conjugated anti-mouse IgG and Alexa Fluor 568 conjugated anti-rabbit IgG, respectively. For visualizing cellular nuclei, the specimens were counterstained with DAPI (Vector, VectaShield). Expression of certain proteins was quantified using the imageJ (NIH) cell counting plug-in. Regions with moderate cellular densities were chosen at random for 3 biological samples unless stated otherwise.ElectrophysiologyWhole cell patch-clamp recordings were conducted as described previously [29]. Briefly, experiments were performed using an EPC-10 amplifier, and data was acquired using the Pulse program (HEKA Electronics). Putative bipolar neurons were selected for recording based on morphology. The pipette solution contained: 140 mM KCl, 5 mM MgCl2, 5 mM EGTA, 2.5 mM CaCl2, 4 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/16989806 mM ATP, 0.3 mM GTP, and 10 mM Hepes, pH 7.3 (adjusted with KOH). The bathing solution contained: 140 mM NaCl, 1 mM MgCl2, 5 mM KCl, 2 mM CaCl2, 10 mM Hepes, and 10 mM glucose, pH 7.3 (adjusted with NaOH). Voltage-clamp and currentclamp data was analyzed using the Pulsefit (HEKAAcknowledgements The authors would like to thank Elizabeth Tobin, Emily Beans and Riddhi Trivedi for their helpful comments and technical support during the preparation of this manuscript. This work was supported by the National Institutes of Health grants RC1DC010706 (to EH) and R01NS053422 (to TRC), as well Capecitabine as by funds provided from the French Centre National de la Recherche Scientifique (CNRS), Institut National de la Sant?et de la Recherche M icale (INSERM), the Minist e de l'Education Nationale, theKoehler et al. BMC Neuroscience 2011, 12:82 http://www.biomedcentral.com/1471-22.