Figure 2: Generation of UiNPCs in simplified medium.

HUCs were transfected with lentivirus vectors encoding reprogramming factors and cultured in defined medium E8, E7 or E8 adding A8301 on Matrigel, the morphology of colonies formed in different mediums were different between E8 and E7 or E8 adding A8301 (A). The colonies were randomly picked and directly analyzed for OCT4 and SOX2 gene expression by qRT-PCR (B). The same two genes were detected during the process of reprogramming (C). (D) Micrographs show immunostains of colonies for the indicated markers (D18). Representative morphology of UiNPCs cultured on Matrigel (E) or in suspension as neuospheres (F) is shown. (G) qRT-PCR analysis of markers expressed by expanded UiNPCs (P5). (H) Differentiation of UiNPCs in vitro. Immunostains of spontaneously differentiated UiNPCs with antibodies against the indicated markers: GFAP, astrocyte marker; TUJ1, pan-neuronal marker; MAP2, mature neuronal markers; glutamate (Glu), GABA and TH, subtype-specific neuronal markers. (I) The electrophysiological properties of UiNPCs derived neurons. (a) Representative voltage-gated ion currents recorded from UiNPC-derived neuron, Whole-cell Na⁺ (inward) and K⁺ (outward) currents (upper panel) were elicited by test pulses to potentials between −50 mV and +60 mV in steps of 10 mV from a holding potential of −70 mV (lower panel). (b) Current-clamp recording showing a representative train of action potentials in a UiNPC-derived neuron (upper panel), intracellular injected currents were step currents incremented by 2 pA from 14 to 22 pA with 800 ms duration (middle panel). Action potentials evoked by 20 pA injected current (lower panel). (c) and (d) showed the current response to AMPA (1 mM) and GABA (1 mM). (e) Representative traces of spontaneous postsynaptic currents (PSCs) recorded from a UiNPC-derived neuron at a holding potential of −70 mV, arrowhead pointed to the magnified view of a typical postsynaptic current. Scale bars: 50 μm; zoom insert in D, 20 μm. Error bars, s.d., n = 3 experiments. *p < 0.05, **p < 0.01, ***p < 0.001.