Transcriptomic Analysis of Induced Pluripotent Stem Cells Derived from Patients with Bipolar Disorder from an Old Order Amish Pedigree.

Selection of fibroblasts from the NIGMH repository for generation of iPSCs.Fibroblasts from patients diagnosed with Type I BPD and their sibling controls of Old Order Amish pedigree were obtained from the NIGMS Human Genetic Cell Repository at Coriell Institute for Medical Research for reprogramming into iPSCs using the Sendai virus method. The pedigree has been updated according to DSM-IV. Circles and squares represent females and males, respectively. MDD = Major Depressive Disorder; Atyp. BP:NOS = Atypical Bipolar (not otherwise specified). Other diagnosis for the patient from which ABP12 was derived was post-partum depression.

Quantitative RT-PCR of RNA samples from iPSCs and NPs.RNA samples were collected from iPSC and NPs and gene expression of markers of developmental stages were evaluated. Changes in pluripotency and neuroprogenitor markers were measured (A-F). Each bar represents mean ± standard error, n = 8. Statistical analysis was performed using unpaired student t-test. *** p < 0.0001, ** p < 0.001.

Quantitative RT-PCR of RNA samples from NPs and neurons.RNA samples were collected from NPs, E and L neurons and markers of neuronal gene expression were evaluated. Pan-neuronal markers, (A,B), cortical neuronal marker (C), and synaptic markers (D,E) were evaluated. Each bar represents mean ± standard error (n = 8, combining 4 BPD and 4 controls). One-way ANOVA was performed and Bonferroni’s Multiple Comparison post-hoc test was done. * p < 0.01, ** p < 0.001, *** p < 0.0001 compared to NP.

Microarray data analysis.A. Principal Component Analysis (PCA) plot shows three distinct clusters: iPSCs, NPs and E plus L neurons as outlined with blue, green and red lines, respectively. The symbols represent undifferentiated iPSCs (blue x), control NP (green cross; +), control E (black circle; ○), control L (red triangle; Δ) and BPD NP (grey asterisk; *), BPD E (turquoise diamond; ♢) and BPD L (pink inverted triangle; ▽). B. Clustered heat map of differentially expressed genes in both control and BPD neurons demonstrates that the genes are clustered closely for E and L neurons regardless of disease status and separate from NPs and iPSCs. Horizontal axis shows genes used for clustering in the S2 Table and vertical axis shows sample clustering. C. Changes in expression of genes associated with axonal guidance (Ingenuity pathway analysis) shown as a clustered heat map show that the expression of genes associated with axonal guidance was higher in E and L neurons than in NPs. Horizontal axis shows genes used for clustering as listed in S3 Table.

Confirmational quantitative RT-PCR.RNA samples collected during differentiation at NP, E and L neuron stages for microarray studies were analyzed by quantitative RT-PCR. Genes previously implicated to be involved with BPD from genomic studies were evaluated: ANK3, ODZ4 and CACNA1C were analyzed (A-C) and did not show differences in expression at any stage. GSK3B did not show significant differences in BPD and control at any stage (D). SCN4B showed trends of down regulation both in E and L neurons (E), but did not meet the statistical significance. Expression of GAD1 was upregulated in L of BPD compare to control. The box on the plots represents the 25 and 75 percentiles. The horizontal bar within the box represents the median. Individual data points are represented by triangles (BPD) or squares (Controls). n = 4 except for SCN4B NP and L controls and E BPD where n = 3. Two-way ANOVA was performed and Bonferroni’s Multiple Comparison post-hoc test was done. ** p < 0.01 when compared to control.