Fig 2: Transient activation of lentiviral mediated Ngn2 expression does not allow PAX6 positive cell appearance in culture. (A) Experimental design. Note that dox was provided only for 6 h in culture (B) Representative images of hiPSCs with a transient activation of Ngn2 stained for pluripotency (OCT4, green) neuronal (ßIIITUBULIN, red) and NSC (PAX6, red) markers (scale bar = 100 µm). (C) Percentage of OCT4, PAX6, and ßIIITUBULIN positive cells over the total number of cells at Day 10. n = 3 independent replicates (mean ± SEM); ns, not significant.

Fig 3: Lentiviral mediated transcriptional programming does not lead to the generation of PAX6 positive cells. (A) Experimental design. hiPSCs are seeded at Day-2 (D-2) in the microfluidic chamber and infected with dox inducible lentiviral vector coding for Ngn2. From Day 0 to Day 10 Ngn2 expression was activated by dox treatment. Infected cells were selected with puro treatment from Day 1 to Day 4. (B,C) Representative images of induced neural cells at Day 10, treated (top) or not (bottom) with puro, in the absence (B, CONTROL) or presence (C, FGF2 + DAPT) of FGF2 and DAPT. Shown are staining for ßIIITUBULIN (red), PAX6 (red) and OCT4 (green) markers counterstained with Hoechst (H, blue) (scale bar = 200 µm) (D–F) Percentage of cells expressing ßIIITUBULIN (D), PAX6 (E), and OCT4 (F) in all the conditions at Day 10. n = 3 independent replicates (mean ± SEM). P-values: *p < 0.05; **p < 0.01; ns, not significant.

Fig 4: Characterization of the NSC and neuronal population at D10 and D20. (A) Representative images of NSC (PAX6, NESTIN, SOX2) and neuronal (ßIIITUBULIN and NEUN) markers at Day 10 of differentiation (scale bar = 50 µm). (B) Representative images of NSC (PAX6), neuronal (ßIIITUBULIN, MAP2, and NEUN) and synaptic (vGLUT1 and PSD95) markers at Day 20 of differentiation. The presence of synapses is highlighted by arrow heads (scale bar = 50 or 20 µm). (C) Representative images of neurons derived from Synapsin I-GFP iPSC line transfected 10 days with NGN2 mmRNA and kept in culture for additional 10 days (D20). Shown are MAP2 positive neurons expressing SYNI-GFP (scale bar = 50 µm).