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. 2015 Sep 15;78(6):e29-34.
doi: 10.1016/j.biopsych.2014.12.028. Epub 2015 Jan 30.

Altered WNT Signaling in Human Induced Pluripotent Stem Cell Neural Progenitor Cells Derived from Four Schizophrenia Patients

Aaron Topol  1 , Shijia Zhu  2 , Ngoc Tran  1 , Anthony Simone  3 , Gang Fang  2 , Kristen J Brennand  4
Affiliations

Altered WNT Signaling in Human Induced Pluripotent Stem Cell Neural Progenitor Cells Derived from Four Schizophrenia Patients

Aaron Topol et al. Biol Psychiatry. .
No abstract available

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig. 1
Fig. 1. RNAseq comparisons of control and SZ hiPSC NPCs
A. Multidimensional scaling (MDS) of RNAseq gene expression of hiPSC NPCs from each of six control and four SZ patients segregates samples along the two leading fold change dimensions. Gene expression analysis was performed on passage-matched hiPSC forebrain NPCs cultured on matrigel. Cells were lysed in RNA BEE (Tel-test, Inc). RNA was chloroform extracted and treated with RQ1 RNAse-free DNAse (Promega). RNAseq samples were prepared using the Illumina HiSeq 2500 RNA kit for 100nt/single end reads, four samples were run per lane. Raw cDNA reads were aligned to the hg19 reference with the spliced gap aligner Spliced Transcripts Alignment to a Reference (STAR) software, with count-based quantitation carried out via the Subread package featureCounts (http://bioconductor.org/packages/release/bioc/html/Rsubread.html) at both the geneic and exonic levels for UCSC and ensemble annotation builds. B. Pathway enrichment analysis based on DAVID. X-axis represents fold enrichment; Y-axis denotes pathways. The FDR are labeled on the right of the bar plot. C. Heat map of control and SZ hiPSC NPCs of 848 unique genes (FDR<0.05). The count data were normalized and modeled as over-dispersed Poisson data using a negative binomial model in the Bioconductor package edgeR (15). Fold changes, p-values and false discovery rates (FDRs) are obtained from the same package for integrative analysis. D. Volcano plots of −log10 p-value versus log2 fold-change mRNA levels for control and SZ hiPSC NPCs. Key canonical WNT signaling genes, including Lymphoid Enhancer-Binding Factor 1 (LEF1), Dickkopf-1 (DKK1), DKK2, Secreted frizzled-related protein-2 (SFRP2), SFRP4, growth differentiation factor 5 (GDF5) and GDF10, are indicated. E. Wnt signaling pathway. The differentially expressed genes by RNAseq are marked by red stars. F. qPCR validation of perturbed WNT gene expression, normalized to the expression of the housekeeping genes GAPDH and ACTIN: LEF1, DKK1, DKK2, SFRP2, SFRP4, and GDF10. Error bars are s.e. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 2
Fig. 2. Perturbed WNT signaling in SZ hiPSC forebrain NPCs
A. Comparison of canonical WNT activity (assayed as LV-TOPFLASH reporter levels relative to LV-renilla florescence) between control and SZ hiPSC forebrain NPCs, averaged by diagnosis. Luciferase levels were determined using the Dual-Glo Luciferase Assay System (Promega), measured on a FlexStation 3 (Molecular Devices) and then normalized to LV-renilla florescence. B. Increased β-CATENIN protein levels in SZ hiPSC forebrain NPCs. Western blot comparison of β-CATENIN and β-ACTIN levels in control and SZ hiPSC NPCs. Western blots were repeated twice using independent protein lysates; Student’s T tests were used to test statistical differences between control and SZ western blot β-CATENIN levels. β-ACTIN was used as a loading control because we have found no evidence, by microarray or Nanostring nCounter gene expression assays or SILAC quantitative protein mass spectrometry, that it is differentially expressed in SZ hiPSC NPCs or neurons (13, 14). C. No effect of WNT on aberrant migration in SZ hiPSC forebrain NPCs. Control and SZ neurosphere outgrowth when cultured with PBS, canonical WNT3A (20 ng/ml) and noncanonical WNT7B (5 ng/ml). Error bars are s.e. *P < 0.05, **P < 0.01.
Fig. 3
Fig. 3. Experimental variability in assaying WNT signaling in SZ hiPSC forebrain NPCs
A. Six experimental replicates comparing canonical WNT activity (assayed as LV-TOPFLASH reporter levels relative to LV-renilla florescence) between control and SZ hiPSC forebrain NPCs, averaged by diagnosis. With increasing passage, NPC lines can show reduced ability to differentiate to neurons or undergo spontaneous transformation to a highly proliferative cell with rounded morphology that cannot undergo neural differentiation at all; when either event occurred, that NPC line was dropped from subsequent experiments, for this reason, not all NPC lines were analysed in independent experiments. B. Six experimental replicates comparing canonical WNT activity (assayed as LV-TOPFLASH reporter levels relative to LV-renilla florescence) between control and SZ hiPSC forebrain NPCs, averaged by individual. (Top row: Mean +/− s.e. Bottom row: Variability chart showing individual data points). For phenotypic analysis, statistical analysis was performed using JMP (Carey, NC). Box-Cox transformation of raw data was performed to correct non-normal distribution of the data and means were compared within diagnosis by Oneway analysis using both Student’s T test and Tukey Kramer HSD. A nested analysis of values for individual patients was performed using standard least squares analysis comparing means for all pairs using Student’s T test for specific pairs and Tukey Kramer HSD for multiple comparisons. Error bars are s.e. *P < 0.05, **P < 0.01, ***P < 0.001.
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