Characterization of bipolar disorder patient-specific induced pluripotent stem cells from a family reveals neurodevelopmental and mRNA expression abnormalities.

Genomic characterization of Family-811 quartet(A) The pedigree for Family-811 is shown. Males are squares and females are circles. Affected individuals are shaded while unaffected individuals are open. Individuals from the Family-811 quartet studied herein are shown with vermillion squares and circles. The associated Coriell identification numbers are shown below the individuals. GM05224 and GM05225 were both diagnosed with BD type I. (B) The familial relationship of these individuals was confirmed using identity-by-descent (IBD) analysis. The IBD1 and IBD2 components were plotted for the parent-parent, parent-offspring and offspring-offspring comparisons. Each of these comparisons agreed with the theoretic prediction of allele sharing for the given familial relationship [(IBD0, IBD1): parent-parent = (1,0), siblings = (1/4,1/2), parent-sibling = (0,1)].34 (C) The multidimensional scaling analysis confirmed that the Family-811 quartet was ethnically related to the CEU Hap Map population and the STEP-BP cohort of individuals used to identify genetically associated bipolar disorder loci. Plotted for comparison are HapMap individuals from Northern and Western Europe (CEU), Yoruba (YRB), and Han Chinese (HCB). (D) The complement of CNVs identified in the Family-811 quartet and their segregation, the size, and encompassed genes. Dark grey squares with 2’s indicate a homozygous locus while light grey squares indicate a hemizygous deletion locus, and white squares with a 3 indicate a copy number of 3. The pedigree at the top indicates the familial relationship. (E) The orange bars indicate the CNVs as determined by SNP analysis, purple bars indicate CNVs identified by droplet digital PCR and the green bars are genes identified as RefSeq genes.

Stepwise neural differentiation of Family-811 quartet iPSCs(A) Schematic of the two-dimensional iPSC neural induction protocol used. Each of the iPSC lines generated from the Family-811 quartet was capable of generating neural rosettes with a characteristic morphology that were manually dissected, passaged and expanded in culture. Each of the iPSC lines was capable of generating SOX1+ cells that could be expanded in vitro. (B) To derive comparable populations of NPCs, a cell surface signature described by Yuan et al.36 was used with FACS to isolate CD184+/CD271−/CD24+/CD15+ cells. A representative gating scheme and plot of the events for an experiment is shown. Representative images of CD184+/CD271−/CD24+/CD15+ cells from GM08330, GM08329, GM05225 and GM05224 stained for NESTIN (red) and DAPI (blue) for DNA. (C) FACS-purified NPCs from the iPSC lines indicated were differentiated into neurons for six weeks yielding post-mitotic neurons staining for the neuronal markers TuJ1 and MAP2. (D) A cell lineage diagram summarizes the cells and cell lines generated from fibroblasts and iPSCs from of each of the individuals in the Family-811 quartet. X’s indicate NPC lines that were able to be initally isolated but were unable to be continually expand beyond four or five passages in vitro despite mutliple attempts. The scale bar is 100 µm.

Phenotypic differences of NPCs derived from both BD-patient iPSCs compared to their unaffected parents(A) BrdU incorporation and staining was used to quantify proliferation of the two BD-patient (GM05225, GM05224) and their unaffected parental (GM08330, GM08329) NPC counterparts. (B) Bar graph summarizing the percent of cells of the total NESTIN+ cells that incorporated BrdU. The BD-patient NPCs exhibited a significant decrease in BrdU incorporation compared to the mean of the unaffected parents (*** indicates significant, t-test, p=0.001). (C) Upon differentiation, BD-patient NPCs (GM05224-3 NPC and GM05225-5 NPC) were found to extend neuritic processes, similar to the unaffected counterparts, but exhibited reduced viability after two weeks of differentiation. In the case of GM05225-5 almost no neurons remained in culture after six weeks. (D) Higher magnification of post-mitotic neurons from the parental control NPC line 8329-1 shown in (c). The scale bar is 100 µm. Error bars depict standard deviation of the measurement.

Differential gene expression signature of BD-patient derived NPCs and neurons(A) Principle components analysis (PCA) of a custom PsychGene NanoString profiles from the Family-811 quartet using all 352 of the genes reveals similar grouping of cells by cell type. (B) Fibroblasts, iPSCs and NPCs were compared using probes against OCT4, NANOG, SOX2, ZFP42, DNMT3B, hTERT, MYC, GFP with total RNA from the human ES cell line H9 shown for comparison. (C) Volcano plots of [−Log10 (p-value) versus the Log2 (fold change expression)] of all genes (grey dots). Significant, differentially expressed genes (> 1.5-fold comparing BD cells to unaffected cells, moderated t-test p-value < 0.05, Benjamini-Hochberg corrected) were plotted with orange dots with a subset annotated by gene name. Those genes that were also significant when members of the Family-811 quartet were compared based on sex (i.e. GM08329 versus GM8330, GM05225 and GM05225) were colored green. Gene expression differences were found to be enriched in the BD-patient NPCs and neuronal populations. Each dot represents a gene expression assay (n=3) for the cell type indicated. Error bars depict standard deviation of the measurement.

Gene expression differences in the BD-patient iPSC neural derivativesDifferential gene expression (>1.5 fold at a significance of p<0.05 calculated using a moderated t-test and corrected using the Benjamini-Hochberg test) in (A) BD-patient CXCR4+ NPCs and (B) in BD-patient neurons relative to the unaffected parental cells. Genes with increased expression are shown in red; genes with deceased expression are shown in blue. (C) Agglomerative hierarchical clustering of the 53 differentially expressed genes between BD-patient CXCR4+ NPCs and differentiated neurons using a weighted pair-group average method and a Pearson correlation coefficient as the similarity metric. (D) DAPPLE protein-protein interaction network amongst differentially expressed genes form the PsychGene NanoString profiles between BD-patient CXCR4+ NPCs and differentiated neurons demonstrating a statistically significant high degree of interconnectivity for specific nodes (marked with *) and globally. (E) WNT module within the DAPPLE protein-protein interaction network amongst differentially expressed genes from the global RNA-seq analysis composed of WNT7B and four additional input seeds amongst the direct interactions all of which were downregulated (levels indicated with arrow) in the BD-patient CXCR4+ NPCs compared to the parental control CXCR4+ NPCs.(F) Rescue of DNA synthesis deficits in the BD-patient NPCs with WNT pathway activation. BrdU incorporation data with pairs of DMSO and CHIR-99021 treated cell lines represented with a symbol connected by a bar. Cell were treated for 16 hours with either DMSO (vehicle) or CHIR-99021 (5 µM), labeled with BrdU, fixed, and stained to detect BrdU and NESTIN. Proliferation was measured by counting BrdU labeled nuclei and quantifying percent of NESTIN+ cells labeled with BrdU. The orange symbol and bars represent averages of the BrdU incorporation experiments in the presence of either DMSO or CHIR-99021. The proliferation defect observed in the BD-patient CXCR4+ NPCs was significantly (pair-wise t-test, p=0.02) decreased with CHIR-99021 treatment while the proliferation of the unaffected parental CXCR4+ NPCs was not changed. (G) CHIR-99021 treatment (16 hours, 5 µM) alters the expression of a subset of WNT pathway responsive genes in the BD-patient CXCR4+ NPCs measured using the PsychGene NanoString probe set. Several genes were upregulated (> 1.5-fold; p-value <0.05 Benjamini-Hochberg corrected) in the BD-patient NPC cell line, including CD44, LEF1, AXIN2 and down regulated (GBX2), while others were only responsive in the parental control lines (ATOH, GSK3B) unaffected. Error bars depict standard deviation of the measurement.