Supplementary Materials

The PDF file includes:

  • Materials and Methods
  • Fig. S1. Family 1 to family 5 pathogenic mutations in NHE6 cDNA.
  • Fig. S2. Sanger sequencing chromatograms of paired control and CS iPSCs.
  • Fig. S3. Morphological characterization of paired control and CS iPSCs.
  • Fig. S4. Karyotype analysis of paired control and CS iPSC lines.
  • Fig. S5. Staining for alkaline phosphatase activity in paired control and CS iPSC lines.
  • Fig. S6. Immunostaining of paired control and CS iPSC colonies for pluripotency markers.
  • Fig. S7. Fluorescence-activated cell sorting analysis of paired control and CS iPSCs for stem cell–specific markers.
  • Fig. S8. Quantitative reverse transcription polymerase chain reaction analyses of pluripotency markers in undifferentiated iPSCs as compared with hESC-derived embryoid bodies.
  • Fig. S9. In vitro differentiation potential of paired control and CS iPSCs.
  • Fig. S10. In vivo differentiation potential of paired control and CS iPSCs.
  • Fig. S11. Northern blot analysis of family 4 and family 5 paired control and CS iPSCs.
  • Fig. S12. Conservation of the G383 residue across NHE homologs.
  • Fig. S13. Protocol for neuronal differentiation from iPSCs.
  • Fig. S14. Neuronal and cortical marker expression in iPSC-derived neurons.
  • Fig. S15. Gene expression of cell fate markers in iPSC-derived neurons.
  • Fig. S16. Unsupervised hierarchical clustering of iPSCs and iPSC-derived neurons by gene expression.
  • Fig. S17. Volcano plot of gene expression changes in iPSCs as compared with iPSC-derived neurons.
  • Fig. S18. Volcano plots of gene expression changes in CS versus control iPSCs and iPSC-derived neurons.
  • Fig. S19. NHE family member gene expression changes in CS iPSCs and iPSC-derived neurons.
  • Fig. S20. V-ATPase gene expression changes in CS iPSCs and iPSC-derived neurons.
  • Fig. S21. Weighted Gene Co-expression Network Analysis (WGCNA)-based network construction and module detection in CS iPSCs.
  • Fig. S22. Analysis of iPSC-derived neuronal cultures for proliferation and apoptosis.
  • Fig. S23. Rescue of deficits in neuronal arborization complexity by reexpression of NHE6 in CS lines with frameshift or nonsense mutations but not in the CS missense mutation line.
  • Fig. S24. Validation of control and CRISPR-Cas9–induced NHE6-null HEK293T cell lines and characterization of endosomal pH.
  • Fig. S25. Establishment of isogenic, genome-corrected iPSCs for family 1 using genome editing.
  • Fig. S26. Calibration curve and flow cytometer profiles for ratiometric analysis of endosomal pH using pH-sensitive and pH-insensitive fluorescent conjugates of transferrin.
  • Fig. S27. Analysis of endosomal pH in paired control and CS iPSCs from family 1 to family 5 using the transferrin method.
  • Fig. S28. Analyses of endosomal pH in paired control and CS iPSCs from family 1 to family 5 using the VAMP3-pHluorin2 method.
  • Fig. S29. Analysis of endosomal pH in paired control and CS iPSC–derived neurons.
  • Fig. S30. Establishment of iPSCs with NHE6 mutations and isogenic controls using genome editing of a normal founder line.
  • Fig. S31. Analysis of endosomal pH in paired isogenic control iPSCs and iPSCs with induced NHE6 mutations using the transferrin method.
  • Table S1. Characterization of peripheral blood mononuclear cell–derived iPSC lines.
  • Table S2. Statistical analysis of genes included in the blue module using a hypergeometric test.
  • Legends for data files S1 to S7
  • References (3251)

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Other Supplementary Material for this manuscript includes the following:

  • Data file S1 (Microsoft Excel format). NanoString customized probe set.
  • Data file S2 (Microsoft Excel format). Average normalized NanoString count data of gene regulation in iPSCs and iPSC-derived neurons from patients with CS and controls.
  • Data file S3 (Microsoft Excel format). By-sample normalized NanoString count data of gene regulation in iPSCs and iPSC-derived neurons from patients with CS and controls.
  • Data file S4 (Microsoft Excel format). By-sample raw NanoString count data of gene regulation in iPSCs and iPSC-derived neurons from patients with CS and controls.
  • Data file S5 (Microsoft Excel format). Genes in each gene module from the WGCNA-based analyses.
  • Data file S6 (Microsoft Excel format). Primary data for main text figures.
  • Data file S7 (Microsoft Excel format). Primary data for supplementary figures.