Fig. 2

Characterization of TSC1 iPSC-derived NPCs. a All TSC1 NPC lines (Het, Null, Corr-WT) express expected neural progenitor markers SOX2 (upper panel) and NESTIN (panel below). DAPI in blue, SOX2 in green, and NESTIN in green. Scale bar = 100 μm. Immunostaining was performed at least 3 times. b TSC1-Het and Null NPCs display increased cell size compared to TSC1-Corr-WT as shown in bright field images (a) and by forward scatter FACS analysis; n = 3. c As expected, TSC1-Null and -Het NPCs show dose-dependent increased mTORC1 signaling (pS6 readout) compared to Corr-WT. Protein expression was quantified and normalized to the Corr-WT NPCs, n = 6, mean values ± s.e.m. are shown, *p < 0.01, **p < 0.001 calculated with Student’s t test. d Proliferation rate of NPC lines was quantified at day 0 (D0, equal cell seeding), and live cell numbers were assessed at D2, D3, and D5. Mutant TSC1 NPCs (Het and Null) revealed genotype-dependent increased proliferation compared to Corr-WT. Data was normalized to Corr-WT at D0, mean values ± S.D. of three separate experiments are shown,*p < 0.01, **p < 0.001 calculated with Student’s t test. e, f MAP2 immunostaining showed genotype-dependent increased neurite outgrowth (number and length) in TSC1 mutant NPCs, which were quantified using a custom image analysis pipeline and HCA Vision imaging software creating neurite segmentation (representative panel shown for the DMSO-treated NPCs). Analysis on n = 6 field images per treatment with approximately 50 cells per field. Data normalized to DMSO treated Corr-WT NPCs. Mean values + s.e.m. are shown. *p < 0.05, **p < 0.001, ****p < 0.0001, n.s. = not significant, calculated with Welch’s t test (GraphPad Prism 7.05). g TSC1 NPCs (Corr-WT, Het, and Null-clone B) were treated with 100 nM of rapamycin for 24 h or with DMSO and analyzed by flow cytometry using the forward scatter height (FSC-H) gating. For each cell line the blue curve represents the rapamycin-treated cells and the red curve represents the DMSO control cells. A shift in the curves shows a cell size difference. N = 3. h Proliferation rate of NPC lines after treatment with a vehicle control (DMSO) or rapamycin (100nM) was quantified at day 0 (D0, equal cell seeding), and live cell numbers were assessed at D2, D3, D4, and D5. No significant differences were observed between DMSO or rapamycin-treated NPCs in all cell types at all time point; n = 3. Mean values ± S.D. of three separate experiments are shown, data was normalized to the Corr-WT treated with DMSO at D0