Fig. 3

CoMo-NSCs generate astrocytes and functional neurons upon in vitro differentiation. a, b Changing morphology of differentiating CoMo-NSCs towards large flat cell type after 40 days treatment with astrocyte-inducting media. c–f Expression of human-specific GFAP (hGFAP) and CD44 in CoMo-NSCs-derived astrocytes and human fetal brain-derived astrocytes. A comparable expression pattern for both markers can be seen. g Representative flow cytometry plots from fixed/permeabilized cells at day 21 of astrocyte differentiation. CoMo-NSC-derived astrocytes were nearly all CD44⁺, with a fraction expressing GFAP. Primary fetal astrocytes (ScienCell) were used as a positive control, compared to CoMo-NSCs as a negative control. All cells lacked signal when analyzed in the absence of antibodies (data not shown). h Expression of synapsin promoter-driven GFP and appearance of neuronal morphology in CoMo-NSC-derived neurons at 6 weeks after induction using BDNF, GDNF, and cAMP. i–l Expression of neuronal markers (DCX, MAP2, human-specific axonal neurofilament HO14 and NeuN) in CoMo-NSC-derived neurons at 6 weeks after induction. m–p Patch-clamp recording in Syn-GFP neurons in vitro: voltage-clamp recording in Syn-GFP + neurons with fast inward (Na⁺) and persistent outward (K⁺) currents in depolarized membrane potentials (characteristic of neuronal cells) can be seen (o). In current-clamp recording (membrane potential − 65 mV), action potentials are triggered by depolarizing current pulses (p) (scale bars: a, b 100 μm; c, e 10 μm; h 200 μm; i–k 50 μm; l 25 μm)