Analysis of induced pluripotent stem cells carrying 22q11.2 deletion.

Reduction in size of patient-derived neurospheres. (a) Bright-field and immunofluorescent images of neurospheres stained for SOX2 (green), derived from control and patient-derived human-induced pluripotent stem cells (hiPSCs). Dotted white circles in the right panel show the outline of neurospheres. Scale bars, 100 μm. (b) Quantitative analysis of the mean size of neurospheres derived from control or patient hiPSCs. The mean size of neurospheres derived from patient hiPSCs was significantly smaller than that from control hiPSCs (n=180–210 neurospheres per cell line). (c and d) Quantitative analysis of the number of neurospheres derived from control or patient-derived hiPSCs. The number of neurospheres with a diameter of less than 100 μm or more than 200 μm were significantly different in patient-derived neurospheres, but the total number of neurospheres was not significantly different (n=4 for each group). Error bars show mean±s.e.m. (*P<0.05, **P<0.01; two-tailed t-test).

Patient-derived neurospheres undergo abnormal neural differentiation. (a) Representative images of neurite outgrowth from neurospheres. The neurites were visualized by immunocytochemical staining of βIII-tubulin. The average distance between the sphere and the neurite tip (white dashed line) was calculated. Scale bars, 100 μm. (b) Quantitative analysis of the neurite length. The neurite lengths were significantly decreased in patient-derived neurospheres (n=300 neurites per cell line). (c) Representative images of cellular migration from neurospheres. Nuclei were visualized by immunocytochemical staining with NeuN. The average distance between the sphere and the most distant cell (white dashed line) was calculated. Scale bars, 100 μm. (d) Quantitative analysis of cellular migration. The cellular migrations were significantly decreased in patient-derived neurospheres (n=300 cells per cell line). (e) Representative images of neural differentiation from neurospheres. Neural cells derived from neurospheres expressed βIII-tubulin and glial fibrillary acidic protein (GFAP) in the patient and control-derived samples, but not OLIG2. The magnified pictures of GFAP-positive cells are shown in the insets. Scale bars, 100 μm. (f) The analysis of neural differentiation efficiencies between control and patient-derived neurospheres (n=10 per cell line). The error bars show mean±s.e.m. (*P<0.05, **P<0.01; two-tailed t-test).

Downregulation of the miRNAs of miR-17 family and miR-17/92 cluster in patient-derived neurospheres. (a) Quantitative real-time PCR (RT-PCR) analysis of DGCR8 in fibroblasts, human-induced pluripotent stem cells (hiPSCs) and neurospheres (n=3–4 for each group). (b) Heat map showing differential expression of 19 miRNAs between patient and control-derived neurospheres. (c) Sequences of the members of the miR-17, 18, 19 and 92 family. The sequences are divided into four families according to the miRNA seed sequences (marked in blue). Members of the miR-17/92 cluster are shown in right panel with information on their chromosomal location. Red: members of the miR-17 family; blue: members of the miR-18 family; green: members of the miR-19 family; yellow: members of the miR-92 family. (d) Quantitative RT-PCR analysis of eight miRNAs in neurospheres. U6 snRNA was used as an internal control (n=4 for each group). Error bars show mean±s.e.m. (*P<0.05, **P<0.01; two-tailed t-test). miRNA, microRNA; snRNA, small nuclear RNA.

Effects of p38 protein on controlling neurogenic competence in patient-derived neurospheres. (a) Expression levels of p38α in human-induced pluripotent stem cell (hiPSC)-derived neurospheres examined by western blotting using an anti-p38α antibody. (b) Quantitative analysis of p38α protein levels in neurospheres derived from control or patient hiPSCs. The p38α protein levels were significantly increased in patient-derived neurospheres (n=4 for each group). (c) Representative images of neural differentiation from neurospheres treated with SB203580 (1.0 μm). Neurons and astrocytes were visualized by immunocytochemical staining of βIII-tubulin and glial fibrillary acidic protein (GFAP), respectively. Scale bars, 100 μm. (d and e) Analysis of the effects of p38 on neural differentiation efficiencies between control and patient-derived neurospheres. In the total differentiated cells derived from patient neurospheres treated with SB203580 (1.0 μm), neuronal population was significantly increased and astrocyte population was significantly reduced (n=10 per cell line). The cells used here were prepared separately from those in Figure 2f. Error bars show mean±s.e.m. (*P<0.05; two-tailed t-test).

mRNA expression analyses of MAPK14, GFAP, IL1B and IL6 and MAP2 in postmortem brains. Expression levels of MAPK14 (a), GFAP (b), IL1B (c), IL6 (d), MAP2 (e) and GFAP/MAP2 (f) in postmortem brain tissues (Brodmann's area 8; BA8) of schizophrenia patients and controls were analyzed using real-time quantitative RT-PCR. The P-values were calculated using two-tailed Mann–Whitney U-test. Horizontal bars show mean±s.d. GFAP, glial fibrillary acidic protein; IL, interleukin; MAP, microtubule-associated protein; MAPK, mitogen-activated protein kinase; mRNA, messenger RNA.