In Vitro Modeling of the Bipolar Disorder and Schizophrenia Using Patient-Derived Induced Pluripotent Stem Cells with Copy Number Variations of PCDH15 and RELN

Generation and characterization of iPSCs derived from a BP patient. A, Schematic diagram of the strategy to explore the phenotypes of BP and SCZ in vitro. B, Subjects list containing basic information. C, CNVs in chromosome 10 were detected in blood and iPSCs derived from two BP patients by aCGH. D, The exonic deletions of PCDH15 identified in this study were validated by the TaqMan copy number assays. Bars indicate copy numbers predicted by the TaqMan copy number assays. Capped bars indicated the minimum and maximum copy number calculated for the sample replicate group (n = 4). Controls carried no aCGH-detected CNVs of PCDH15 (copy number = 2) and were used to calibrate the assays. E, The generated iPSCs expressed the pluripotent markers Oct4, Nanog, SSEA4, and Tra1-60. Scale bar, 100 μm. F, Representative images of immunocytochemical analysis for in vitro three-germ layer differentiation. βIII-Tubulin, αSMA, and AFP are pluripotent markers of the ectoderm, mesoderm, and endoderm, respectively. Blue indicates Ho, and green indicates the pluripotent marker. Scale bar, 100 μm.

Neuron differentiation via EB formation. A, Overview of the protocol for EB formation. DSi represents SB431542 and LDN193189. B, Representative images of EBs on day 7. Scale bar, 200 μm. C, Quantification of EB sizes (n = 3 independent experiments; mean ± SD; Dunnett’s test, no significant differences were observed). D, The form factor of the EBs was calculated as an indicator of roundness. E, Gene expression of iPSCs and DSi-EBs. E, Relative gene expression levels of PCDH15 and RELN in DSi-EBs derived from six control lines, two BP lines (BP1-1 and BP1-2), and two SCZ lines (SCZ1-1 and SCZ1-2; n = 3 independent experiments; mean ± SD; Dunnett’s test). Values were normalized to that of the control, which was considered to be 1.0. One sample of 1210B2-derived DSi-EBs, in which PCDH15 expression was under the detection limit, was removed from the analysis. F, Overview of the protocol for neuron differentiation via EB formation. DSi represents SB431542 and LDN193189. G, Intensity levels of three germ layer markers, namely, βIII-tubulin, αSMA, and AFP. Intensity levels were normalized to that of the control, which was considered to be 1.0 (n = 3 independent experiments; mean ± SD; Dunnett’s test among each group, no significant differences were observed). H, Schematic diagram of the analysis protocol. Fiber length and number of βIII tubulin⁺ cells were quantified from binarized image data obtained from stained images. I, Representative images of βIII-tubulin⁺ neurons. Scale bar, 300 μm. J, Time-dependent changes of βIII-tubulin⁺ mean neurite length per neurite fiber (n = 3 independent experiments; mean ± SD; *p < 0.05, **p < 0.01; Dunnett’s test among each group). Mean neurite length is shown as the mean length of βIII-tubulin⁺ neurite fiber.

Neuron differentiation by transcription factor overexpression. A, Overview of the protocol for differentiation into glutamatergic neurons. B, Immunocytochemical analysis of neuronal markers (VGluT2, MAP2, and βIII tubulin). Scale bar, 40 μm. C, Ratio of positive cells for each marker; βIII tubulin⁺ cells/all cells (βIII tubulin⁺/Ho⁺), MAP2⁺ cells/βIII tubulin⁺ cells (MAP2⁺/ βIII-tubulin⁺), and VGluT2 and βIII tubulin double⁺ cells/βIII tubulin⁺ cells (VGLUT2⁺βIII tubulin⁺/βIII tubulin⁺). (n = 3-6 independent experiments; mean ± SD; *p < 0.05; Tukey’s test, the population of βIII tubulin⁺ cells in BP2-1-derived neurons was significantly lower than those in 1210B2-, 201B7-, BP1-1-, and BP1-2-derived neurons). D, Relative gene expression levels of PCDH15 (primer set1) and RELN in NEUROG2-transduced iPSCs and induced neurons (n = 3 independent experiments; mean ± SD; Dunnett’s test among iPSCs and among neurons, no significant differences were observed). Values were normalized to that for 1210B2-derived neurons, which was considered to be 1.0. Some iPSC samples, in which PCDH15 expression was under the detection limit, were removed from the analysis. Specifically, two samples of BP1-1, and one sample of 1210B2, 201B7, BP2-1, and SCZ1-1 were excluded from the analysis. E, Overview of the protocol for differentiation into GABAergic neurons. F, Immunocytochemical analysis of neuronal markers (MAP2, GABA, and βIII tubulin). Scale bar, 40 μm. G, Ratio of positive cells for each marker; βIII tubulin⁺ cells/all cells (βIII tubulin⁺/Ho⁺), MAP2⁺ cells/βIII tubulin⁺ cells (MAP2⁺/βIII tubulin⁺), and GABA and βIII-tubulin double-positive cells/βIII tubulin⁺ cells (GABA⁺βIII tubulin⁺/βIII tubulin⁺). (n = 3-6 independent experiments; mean ± SD; Tukey’s test; no significant differences were observed). H, Relative gene expression levels of PCDH15 and RELN in ASCL1- and DLX2-transduced iPSCs and induced neurons. (n = 3 independent experiments; mean ± SD; Dunnett’s test among iPSCs and among neurons; no significant differences were observed). Values were normalized to that for 1210B2-derived neurons, which was considered to be 1.0. Some iPSC samples, in which PCDH15 expression was under the detection limit, were removed from the analysis. Specifically, each one sample of 1210B2 and BP2-1 was excluded from the analysis.

Comparison of the global gene expression profiles. A, PCA of the gene expression data of glutamatergic neurons. B, Hierarchical clustering analysis of DEGs in glutamatergic neurons. C, Venn diagram of DEGs of BP or SCZ glutamatergic neurons compared with control neurons (fold change, >2.0). A total of 498 genes were common between the two groups compared. D, Featured GO and pathway terms for the 498 common DEGs among the control vs BP and control vs SCZ. Adhesion- and neuron-associated terms were extracted. E, PCA plot of the gene expression data of GABAergic neurons. Green, control (1210B2, 201B7); blue, BP; red, SCZ. F, Hierarchical clustering analysis of DEGs in GABAergic neurons. G, Venn diagram of DEGs of BP or SCZ GABAergic neurons in comparison with control neurons (fold change, >2.0). A total of 522 genes were common among the two comparisons. H, Featured GO and pathway terms for 522 common DEGs among the control vs BP and control vs SCZ. Adhesion- and neuron-associated terms were extracted.

Neurons induced from patient-derived iPSCs exhibit abnormal phenotypes of dendrite length and synapse formation. A, Schematic diagram of the protocol for phenotypic analysis of dendrite lengths and number of synaptic markers of the neurons. B, C, Representative images of immunocytochemical analysis of a dendrite marker (MAP2; scale bar, 60 μm; B) and synaptic markers (Homer I, Synapsin I; scale bar, 10 μm; C) in glutamatergic neurons. D, Quantitative analysis of dendrite length in glutamatergic neurons (n = 3-6 independent experiments; mean ± SD; **p < 0.01; Dunnett’s test). E, Quantitative analysis of the number of synaptic marker puncta in glutamatergic neurons (n = 3-6 independent experiments; mean ± SD; *p < 0.05, **p < 0.01; Dunnett’s test). Homer I puncta, Synapsin I puncta and Homer-Synapsin I colocalized puncta on βIII-tubulin⁺ cells were counted. F, G, Representative images of immunocytochemical analysis of a dendrite marker (MAP2; scale bar, 60 μm; F) and synaptic markers (Gephyrin, Synapsin I; scale bar, 10 μm; G) in GABAergic neurons. H, Quantitative analysis of dendrite length in GABAergic neurons (n = 3-6 independent experiments; mean ± SD; **p < 0.01, Dunnett’s test). I, Quantitative analysis of the number of synaptic marker puncta in glutamatergic neurons (n = 3-6 independent experiments; mean ± SD; *p < 0.05, **p < 0.01, Dunnett’s test). Gephyrin puncta, Synapsin I puncta, and Gephyrin-Synapsin I colocalized puncta on βIII tubulin⁺ cells were counted.

Generation of isogenic PCDH15 deletion iPSCs by targeted genome editing. A, The target sites of CRISPR-sgRNAs in exon 9 of the PCDH15 gene (NM_001142763). Red bars represent PAM (protospacer adjacent motif) sequences. B, Analysis of CRISPR-sgRNA activity by the T7E1 assay using HEK293FT. NTC, No transfection. Among the constructed sgRNAs, sgRNA#3 showed the strongest cleavage activity. C, Indel pattern of the isogenic line using CRISPR-sgRNA#3. Blue letters represent stop codon. D, Representative images of immunocytochemical analysis for in vitro three-germ layer differentiation. Blue indicates Ho and green indicates the pluripotent marker (βIII-tubulin, αSMA, and AFP). Scale bar, 100 μm. E, Relative gene expression levels of PCDH15 (primer set2) in glutamatergic or GABAergic neurons on day 28 (n = 3 independent experiments; mean ± SD; *p < 0.05, Student’s t test). F, Information of isogenic RELN-deleted iPSCs generated in the previous study (Arioka et al., 2018). G, Relative gene expression levels of RELN in induced glutamatergic or GABAergic neurons on day 28 (n = 3 independent experiments; mean ± SD; **p < 0.01, Student’s t test).

Isogenic PCDH15 or RELN deleted neurons showed partial phenotypes of dendrite and synapse formation. A, Representative images of immunocytochemical analysis of neuronal markers (scale bar, 40 μm) and synaptic markers (scale bar, 10 μm) in glutamatergic neurons. B, Ratio of positive cells for each marker; βIII tubulin⁺ cells/all cells (βIII tubulin⁺), MAP2⁺ cells/βIII tubulin⁺ cells (MAP2⁺), and VGLUT2 and βIII tubulin double-positive cells/βIII tubulin⁺ cells (VGluT2⁺βIII tubulin⁺). (n = 4–6 independent experiments; mean ± SD; **p < 0.01, Student’s t test between each pair of control and isogenic lines). C, Quantitative analysis of dendrite length in glutamatergic neurons (n = 4–6 independent experiments; mean ± SD; *p < 0.05, Student’s t test). D, Quantitative analysis of the number of synaptic marker puncta in glutamatergic neurons (n = 3–6 independent experiments; mean ± SD; *p < 0.05, Student’s t test). E, Representative images of immunocytochemical analysis of neuronal markers (scale bar, 40 μm) and synaptic markers (scale bar, 10 μm) in GABAergic neurons. F, Ratio of positive cells for each marker; βIII tubulin⁺, MAP2⁺, and GABA and βIII tubulin double-positive cells/βIII tubulin⁺ neuronal cells (GABA⁺βIII tubulin⁺). (n = 4–6 independent experiments; mean ± SD; *p < 0.05, Student’s t test between Control 1 and PCDH15del or between Control 2 and RELNdel). G, Quantitative analysis of dendrite length in GABAergic neurons (n = 4–6 independent experiments; mean ± SD; **p < 0.01, Student’s t test). H, Quantitative analysis of the number of synaptic marker puncta in GABAergic neurons (n = 4–6 independent experiments; mean ± SD; *p < 0.05, Student’s t test).