Fig. 1

Generation and characterization of isogenic TSC1-iPSCs. a Bright field images of a TSC1-iPSC colony (right) generated from skin fibroblasts of a TSC1 patient (left). b Representative normal karyotype of heterozygous TSC1-iPSCs. A total of 20 cells were counted to confirm normal diploidy of 46 and a total of 8 cells were analyzed in which the chromosomes were compared band by band to their homologues and a total of 4 were karyotyped. c Upon differentiation, real-time PCR in embryoid bodies (EB) from TSC1-iPS cells show decreased pluripotency markers (OCT4, NANOG, and REX1) at 8 days post-differentiation (8dEB) versus undifferentiated iPSCs and increased expression of the 3 germ layers markers (GFAP for ectoderm, AFP for endoderm, and Brachyury for mesoderm). Error bars represent standard deviation on 3 biological replicates. Data were normalized to the undifferentiated control. Mean values are shown, **p < 0.001 calculated with Student’s t test d CRISPR/Cas9-mediated correction of TSC mutation. Sequencing of original and corrected control iPSC lines derived from a TSC1 patient. e Quantitative RT-PCR shows increased TSC1 (left) expression in CRISPR-corrected iPSCs (Corr-WT) compared to heterozygous (Het) original iPSCs. Data were normalized to the Corr-WT. Mean values are shown, error bars represent standard deviation of 3 biological replicates, **p < 0.001 calculated with Student’s t test. No change in expression of TSC2 was noted. f Isogenic iPSCs heterozygous (Het) and corrected (Corr-WT), immunostained for pluripotency markers OCT4 (green) and NANOG (NAN, red). Bright field and nuclear DAPI (blue) are also shown. Scale bar = 200 μm. g Schematic representation of the two TSC1-Null clones of iPSCs generated using CRISPR/Cas9. h Immunoblot of TSC1 iPSC lines compared to an unrelated TSC iPSCs control (CT). Expression of TSC1 is reduced in TSC1-Het clones and completely lost after introducing a second somatic mutation, and mTORC1 is activated in both the Het and Null clones as shown by elevated expression of pS6. a–c Independent iPSC clones of TSC1-Het (TSC1+/−) or TSC1-Null (TSC1−/−). Expression of TSC2, TSC1, phosphorylated S6 (pS6), and total amount of S6 (S6) were quantified from at least 3 independent experiments. Data were normalized to the Corr-WT. Mean values ± S.D. of three separate experiments are shown, *p < 0.01, **p < 0.001 calculated with Student’s t test