Fig 2: Proof of concept AAVS1 targeted cell lines expressing CRISPRi and CRISPRa machinery. Strategy for targeted integration of a Cre-activatable dCas9-KRAB (CRISPRi) (A) and dCas9-SAM (CRISPRa) (B) at the AAVS1 locus. Relative mRNA expression levels of OCT4, SOX2, and NANOG 48- and 96-hours posttransfection (C) and relative proportion of median fluorescence intensity of FACS quantification for OCT4, SOX2, and NANOG protein expression (D) of the KOLF-AAVS1-KRAB iPS cells with sgRNAs designed against the OCT4, SOX2, and NANOG promoter, relative to a nontargeting sgRNA. Statistical comparisons against the theoretical mean of 1 were performed with a 1 sample t-test. (E) Expression levels of HHEX and NGN2 48-hours posttransfection of the KOLF-AAVS-SAM iPS cells with sgRNA against NGN2 and/or HHEX, relative to a nontargeting sgRNA. (F) Immunohistochemical staining of NGN2 (red), TRA-1-81 (green), and DAPI (Blue) for KOLF-AAVS-SAM cells transfected with either the NGN2 sgRNA or a nontargeting sgRNA (50-μm scale bar).

Fig 3: 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 (βIIITUBULIN, red) and NSC (PAX6, red) markers (scale bar = 100 μm). (C) Percentage of OCT4, PAX6, and βIIITUBULIN positive cells over the total number of cells at Day 10. n = 3 independent replicates (mean ± SEM); ns, not significant.

Fig 4: Expression of Dlx2 and its function in the dorsal cortex.(A) (a) Images of E17.5 Emx1-Cre/Ai9 mouse brains stained with Ngn2 and Dlx2 antibodies. Bar, 150 µm. (b–e) Magnified view of the square region in (a). Arrowheads; blue, Dlx2 + /Ngn2 + /tdTomato + ; yellow, Dlx2 + /Ngn2-/tdTomato + ; white, Dlx2 + /tdTomato-. Bar, 15 µm. (f, g) Quantification of the stained images (n = 4 brains each). Total counted cells of Dlx2 + , Ngn2 + ; 311, 282. (B) Plasmids to induce OE of p18 mixed with pCAG-H2B-TagRFP were delivered at E15.5 and then fixed after 1–3 d. Sections were stained using Dlx2 antibody. (a) Images of fixation at E18.5 are presented. Arrowheads; double-positive cells to Dlx2 and H2B-TagRFP. Bar, 30 µm. (b) Magnified view of the square region in (a). Arrowheads; double-positive cells to Dlx2 and H2B-TagRFP. Bar, 10 µm. (C) Quantification of the Dlx2-positive cells among EGFP-positive cells in (B) (n = 3 brains each). Total counted cells of Control, p18 OE; 410, 549 (E16.5); 453, 413 (E17.5); 309, 167 (E18.5). Two-tailed t test; error bars show mean ± SEM. **P = 0.00118109 (b), *P = 0.03660486 (c). (D) Plasmids to induce OE and KD of Dlx2 mixed with pCAG-NLS-EGFP were delivered at E15.5 and 16.5, respectively, and then fixed after 1 d. Sections were stained using Ngn2 antibody. (a) Images of IUE at E15.5 for Dlx2 OE are presented. Arrowheads; double-positive cells for Ngn2 and EGFP. Bar, 30 µm. (b) Magnified view of the square region in (a). Arrowheads; double-positive cells to Ngn2 and EGFP. Bar, 10 µm. (E) Quantification of the Ngn2-positive cells among EGFP-positive cells in (D) (n = 3 brains each). Total counted cells of Control, Dlx2 OE; 337, 438; Control, Dlx2 KD; 425, 322. Two-tailed t test; error bars show mean ± SEM. ***P = 0.00003530 (a), *p = 0.03660486 (b). (F) Luciferase assay using the promoter region of Ngn2 in HEK293T cells transfected with plasmids to induce p18, Dlx2, and Hes1 expression (n = 3 independent cultures). One-way ANOVA with Tukey’s test; error bars show mean ± SEM. From top to bottom, ***P = 0.00049901, ***P = 0.00069031, ***P = 0.00051001, ***P = 0.00070606. (G) Plasmids to induce OE and KD of Dlx2 mixed with pCAG-NLS-EGFP were delivered at E15.5 and then fixed at E17.5. Sections were stained using AldH1l1 antibody. (a) Arrowheads; double-positive cells for AldH1l1 and EGFP. Bar, 30 µm. (b) Magnified view of the square region in (a). Arrowheads; double-positive cells to AldH1l1 and EGFP. Bar, 10 µm. (H) Quantification of the AldH1l1-positive cells among EGFP-positive cells in (G) (n = 3 brains each). Total counted cells of Control, Dlx2 OE; 515, 468; Control, Dlx2 KD; 649, 581. Two-tailed t test; error bars show mean ± SEM. *P = 0.01812292 (a), **P = 0.00895741 (b). Source data are available online for this figure.

Fig 5: Setting of the experimental conditions for NGN2 mRNA based transcriptional programming of hiPSCs. (A) Time course of NGN2 protein expression evaluated by means of immunofluorescence after a single NGN2 mmRNA transfection in hiPSCs. (B) Time course of NGN2 protein production evaluated by means of immunofluorescence two NGN2 mmRNA transfections in hiPSCs. (C) Representative image of the microfluidic platform used in our experiments. Shown is the reconstruction of a single microfluidic channel in bright field with the magnification of an area of hiPCSs transfected with GFP mRNA (green). (D) Percentage of GFP-positive hiPSCs after transfection with GFP mmRNA combining different cell densities, mmRNA doses and the presence of Rock-inhibitor (Ri). n = 3 independent replicates (mean ± SEM). P-values: *p < 0.05. (E) Top: Experimental outline. hiPSCs were seeded at Day 0 (D0) in pluripotency medium in the microfluidic chambers and transfected daily with NGN2 mmRNA. Medium was replaced with neural medium from Day 1 until Day 3 (D1–3) and supplemented with neurotrophic factors from Day 5 till Day 10 (D5–10). The outcome analysis was performed at Day 3 and Day 10 (D3 and D10). Bottom: Representative bright field images of hiPSCs after 1, 3, and 10 days of transfection with NGN2 mmRNA (scale bar = 100 μm). (F) Representative image of a single microfluidic channel of hiPSCs at Day 10 of NGN2 mmRNA transcriptional programming. Shown are nuclei (blue) and neurons (red). The image was created merging single microscopy images next to each other (scale bar = 1 mm); ns, not significant.