Fig 2: Human NSCs tend to differentiation upon FGF2 withdrawal accompanied by an increase in asymmetric LAMP1 segregation during mitosis.(A) Representative brightfield images and immunostaining of rosette-type NSCs from two healthy control cell lines (Ctrl#1 and Ctrl#2) for NSC markers (Sox2 and Nestin) and neuron markers (Tubb3 and HuC/D). (B and C) Immunostaining for Sox2 and HuC/D and respective quantification of the amount of NSCs (Sox2+) and neurons (HuC/D+) after FGF2 withdrawal from Ctrl#1-NSCs over a time course of 7 days (n = 3, two-way ANOVA with Bonferroni’s multiple comparison test, means + SEM). (D and E) Representative images of symmetric and asymmetric segregation of LAMP1+ and CD63+ vesicles during mitosis of Ctrl#1-NSCs. Yellow lines indicate ROIs surrounding daughter cells, defined by DAPI and phalloidin staining (see also fig. S1E). (F and G) Quantification of LAMP1 and CD63 asymmetry based on sum intensity ratios between paired daughter cells (see fig. S1D) in conditions with and without FGF2 (N = 6 coverslips from three independent experiments with n = 42 to 58 cells per coverslip, two-way ANOVA with Bonferroni’s multiple comparison test, means + SEM). (H) Representative images of LAMP1 costained with Notch1 receptors in different mitotic phases of Ctrl#1-NSCs. (I) Correlation of asymmetry indices A of LAMP1 and Notch1 in Ctrl#1 and Ctrl#2-NSCs. Dots represent individual mitotic event, and linear regression is shown. Scale bars, 100 µm (A and B), 10 µm (D, E, and H), 5 µm [zoomed in (H)]. DNA was counterstained with DAPI. *P < 0.05 and ***P < 0.001. n.s., not significant. See also fig. S1.

Fig 3: Neurogenic cell divisions in forebrain organoids show biased localization of LAMP1 and Notch1 in the apical daughter cell.(A) Representative image of 20-day-old forebrain organoid immunostained for NSC marker Sox2 and neuronal marker Tubb3 with cortical loops indicated by white dashed line. (B) Illustration of forebrain organoids with cortical loops and NSCs dividing in a planar, intermediate, or delaminating manner at the apical membrane of the loops. (C) Immunostaining of forebrain organoids for phalloidin, LAMP1, and Notch1. Dividing cells at the apical membrane (yellow dashed line) were identified by phalloidin staining and grouped into planar, intermediate, or delaminating cell division. LAMP1 and Notch1 signal within the area of the ROIs (yellow) is shown. (D) Distribution of cell division modes as defined in (B) (n = 3 independent experiments, means + SEM). (E) Quantification of LAMP1 and Notch1 asymmetry based on sum intensity ratios (see also fig. S1) between paired daughter cells (N = 60/28/19 of planar/intermediate/delaminating cell divisions from three independent experiments, Kruskal-Wallis test with Dunn’s multiple comparison test, box plot with dots for individual mitotic events). Scale bars, 300 µm (A) and 5 µm (C). Nuclei were counterstained with DAPI in (A). **P < 0.01.

Fig 4: Egr2 overexpression reduces the myelination frequency in a mouse sciatic nerve engraftment model, analyzed at one month post engraftment. (A) Frequency of myelination, reported as percentage of graft-derived (GFP+) cells that are associated with MBP+ segments. (B) Density of graft-derived cells. (C) Representative images of anti-MBP staining. Inserts show images at a higher magnification. (D) Axon density determined by staining against ß-(III)-tubulin (Tubb3), with dashed line indicating the level found in a contralateral control nerve. (E) Tissue cross-section area after removal of the supporting silicon tube, with dashed line indicating the level found in a contralateral control nerve. (F) Representative images of anti-Tubb3 staining. Inserts show images at a higher magnification. All samples were analyzed in transverse cross-sections at the mid-graft level, with n = 4 animals per group. Scale bars = 50 µm. *p < 0.05. (G–H) Percent GFP+ cells was comparable between the two litters used for in vivo experiments one day after isolation from Plp-eGFP mice.