Induction of human neuronal cells by defined transcription factors.

Rapid generation of functional neurons from human ES cellsa, Four days after induction, ES-iN cells exhibited bipolar neuronal morphologies. b–c, Eight days after induction, ES-iN cells expressed Tuj1 (b) and MAP2 (c). d, Spontaneous action potentials presumably caused by membrane potential fluctuations recorded from an ES-iN cell 6 days after induction. Arrow: pronounced AHP. e, Representative traces of action potentials in response to step current injections 15 days after induction. Membrane potential was maintained at ~ –63mV. f, Quantification of intrinsic membrane properties in control ES cells (0 day) before and after viral transduction. membrane input resistance (Rin), resting membrane potential (RMP), capacitance (Cm), after hyperpolarization potentials (AHP). Scale bars: 10µm (a,b,c). Numbers of cells recorded are labeled in the bars. Note the heterogeneity of the parameters (see also Suppl. Fig. 1). Data are presented with mean±SEM. * p<0.05.

NeuroD1 increases reprogramming efficiency in primary human fetal fibroblastsa, Quantification of Tuj1-positive BAM-iN cells with indicated factors, 3 weeks after dox. b–c, Three weeks after dox BAM+NeuroD1 iN cells exhibited neuronal morphologies (b) and expressed Tuj1 (c) . d–f, iN cells expressed NeuN (d,) PSA-NCAM (e), and MAP2 (f) 2 weeks after dox. g–h, An iN cell expressing MAP2 (g) and synapsin (h) 4 weeks after dox and co-cultured with primary astrocytes. i, Single cell gene expression profiling using Fluidigm dynamic arrays. Rows represent the evaluated genes and columns represent individual cells. Heatmap (blue to red) represents the threshold Ct values as indicated. Data in (a) are presented as mean±SD. Scale bars: 100 µm (b, c), 10 µm (d–h).

Membrane properties of fibroblast iN cellsa, Quantification of Tuj1-positive neuronal cells from HFFs (line HFF-A) 3 weeks after dox or HPFs (line HPF-B) 4 weeks after dox. N=3 independent experiments. b, Patch clamp recording was conducted on HFF-iN cells identified by EGFP fluorescence and DIC microcopy. c, Representative traces of membrane potentials in response to step current injections (lower panel) from an HFF-iN cell 19 days after dox. Membrane potential was maintained at ~ −63 mV. d, Representative traces of membrane currents recorded with a ramp protocol (lower panel). Fast activating and inactivating Na+ currents were prominent. Three traces are shown superimposed. e–g, HPF-iN cells express Tuj1 (red) and NeuN (green) (e), Neurofilament (green) (f) and MAP2 (green) (g). h, Representative traces of membrane potentials in response to step current injections in HPF-iN cells. Action potentials were generated in cultures without glia. i, Representative traces of membrane currents recorded following a ramp protocol (lower panel) in HPF-iN cells. The Na+ currents could be blocked by TTX. Data in (a) are presented as mean±SD. Scale bars: 10 µm (a, e–g).

Synaptic responses of HFF-iN cellsa, An HFF-iN cell expressing EGFP co-cultured with mouse cortical neurons at day 35 after dox. b, Synapsin positive puncta co-localize with neurites extending from HFF-iN cells (arrow heads). c, Thirty-five days after dox, spontaneous PSCs were recorded in HFF-iN cells. d, The slow responses could be blocked by PTX. Bursting events of EPSCs were recorded in the presence of PTX. The insert shows the fast kinetics of the responses. e, In the presence of PTX and CNQX (50 µM), no spontaneous activities were observed. f, Evoked postsynaptic responses. Four traces were super imposed. Sti. = stimulation. g, In the presence of PTX, electric stimulation evoked fast-kinetic excitatory PSCs (EPSCs). h, No evoked synaptic responses were observed in the presence of PTX and CNQX. Scale bars: 100 µm (a); 10µm (b).