Article Figures & Data
Figures
- Figure 1. Patient-specific iPSCs show impaired neuronal maturation.
(A) Immunostaining for PAX6 and NESTIN in PSC-derived NPCs. (B) Immunostaining of PSC-derived cortical neurons for MAP2 and CTIP2. (C) Comparative protein analysis for SHANK3 and JIP2 as well as for JNK proteins, NPC- and pan-neuronal markers for control and patient lines. (D) Quantification of protein levels in neurons. Data are shown as mean ± SEM (n = 3). Two-tailed unpaired t test, *P < 0.05. (E) Quantification of normalized neuron content plated on MEAs. Data are shown as mean ± SEM (n = 10/11 images per condition, respectively). (F, G) MAP2 immunostaining of age/culture-matched (not density-matched) neurons used for MEAs. (H) Representative phase-contrast image showing neurons seeded on 64 electrodes/well. (I) Quantification of average spike count during in vitro maturation. Average of three wells (three independent cultures) for 5-min measurement intervals are shown as box plots ± SEM. (J) Comparative raster plots as recorded on day 71 of differentiation. Every line in an individual plot represents one electrode. Every vertical dash represents a detected field potential. Recording interval: up to 450 s. (K) Quantification of number of active electrodes at day 75 and day 85. Data are represented as mean ± SEM. Two-tailed unpaired t test, **P < 0.005. (L) Quantification of average spike rate in Hz at early and late stage differentiation. Data are represented as mean ± SEM. Two-tailed unpaired t test, *P < 0.05, **P < 0.005.
- Figure S1. Patient-specific cortical neurons show synaptic and intracellular signaling phenotype.
(A) Scheme of post-synaptic density (PSD) proteins and down-stream signaling pathways (modified from Kelleher et al [2012]). (B) Selected PSD protein analysis comparing PSC-derived control and two independent patient neuron cultures. (C) Selected down-stream pathway protein analysis comparing PSC-derived control and two independent patient neuron cultures.
- Figure 2. Patient neural progenitors show a neurodevelopmental phenotype.
(A) CRISPR/Cas9 targeting scheme for fluorescent PAX6-GFP reporter. (B) Southern blot analysis for targeted genomic locus showing 5′ external and internal probe for control and patient specific iPSC lines. (C) Representative immunostaining for GFP upon NPC differentiation. (D) FACS scheme separating three distinct cell populations. (E) Heat map of differentially expressed genes comparing GFP-positive, GFP-negative, and unsorted cell populations. (F) Scatter plot representing global gene expression levels. Blue dots represent up-regulated genes in GFP+ and GFP− fraction, respectively. Red dots highlight selected NPC marker genes in the GFP+ fraction. (G) GO term analysis of genes up-regulated in GFP+ fraction. (H) Quantitation of relative gene expression (rel. to CTRL NPCs) across patient-specific deletion. Selected NPC marker gene expression is shown as reference for PAX6-GFP sorted populations. (I) Differentially expressed genes comparing GFP+ cells form controls and patient line. (J) GO term analysis of genes down-regulated in patient NPCs (GFP+ cells).
- Figure S2. PAX6-GFP reporter lines allow specific purification of human PSC-derived neural progenitors.
(A) Sequencing of the second allele of three independent clones. (B) FACS purification of three independent clones plus negative control after NPC differentiation. (C) Comparative gene expression profiles: global versus differentially expressed genes. (D) Comparative transcript profiling of a subset of NPC markers. (E) qRT–PCR validation of a subset of NPC markers. Error bars represent SEM. (F) Gene expression upstream and across the 22q13 deletion. Data are represented as log2 RPM values.
- Figure 3. Isogenic ESC–derived neural cell types recapitulate patient-specific phenotypes.
(A) CRISPR/Cas9 targeting scheme to introduce patient-specific deletion in WIBR#3 ESC line. (B) PCR assay shows untargeted control line and three heterozygous deletion lines. (C) Genomic DNA sequence of CRISPR target sites flanking the deletion. (D) Immunostaining for control, isogenic deletion, and iPSC lines after NPC differentiation. (E) Quantification of NPC fraction. Normalized data are shown as mean ± SEM (n = 4). (F) Comparative protein analysis of control, isogenic deletion, and iPSC lines upon neuronal differentiation. Arrows indicate three isoforms of SHANK3. (G) Quantification of immunoblots shown in (F). Data are represented as mean ± SEM (n = 3).
- Figure 4. Pharmacological activation of the NRP1 recues JNK expression in maturing patient neurons.
(A) Mechanistic scheme of the semaphorin pathway in neurons. (B) Comparative transcript analysis (RNAseq) of key factors of semaphorin pathway and JNK target genes. (C) Protein analysis of mature neurons derived from control and two independent patient iPSC lines. (D) Quantification of immunoblots shown in (C). Data are represented as mean ± SEM (n = 3). (E) Transcript analysis (qRT–PCR) of key factors of the semaphorin pathway upon treatment with recombinant SEMA3A. Data are represented as mean ± SEM (two biological replicates including three technical replicates each). Two-tailed paired t test, *P < 0.05, **P < 0.005. (F) Mechanistic molecular model for 22q13DS.
Tables
CTRL (hESC) Gender Age No. of clones Induction method Diff. PAX6-GFP WIBR3 Female Blastocyst derived (Lengner et al, 2010) 1 N/A Yes Yes Rutgers ID (iPS lines) Gender Age Category Location (deletion) Ref. genome Del. start Del. end Size (bp) No. of clones Induction method Diff. PAX6-GFP MH0148130 (22q13_1) Female (patient 1) 35 Terminal deletion 22q13.3 NCBI36, HG18 49210245 49691432 481187 4 RNA Yes Yes MH0148137 Female 12 Terminal deletion 22q13.3 NCBI36, HG18 49479705 49522658 42953 4 RNA No No MH0148157 (22q13_2) Male (patient 2) 11 Terminal deletion 22q13.3 NCBI36, HG18 49369190 49463084 93894 4 RNA Yes No Isogenic ESCs Gender Age Category Location (deletion) CRIPSR Seq. Clones picked Assay Size (del.) No of clones Induction method Diff. PAX6-GFP WIBR3-1 (Δ22-1) Female N/A Terminal deletion 22q13.3 Guide I: 5′-TAATGTCACTTTTGCTGCAGTGG-3′ 30 PCR ∼10 kb 1 N/A Yes Yes WIBR3-2 (Δ22-2) Female N/A Terminal deletion 22q13.3 30 PCR ∼10 kb 1 N/A Yes Yes WIBR2-1 (Δ22-3) Male N/A Terminal deletion 22q13.3 Guide II: 5′-CTGCTTGCCTGGGCTCCAGCTGG-3′ 30 PCR ∼10 kb 1 N/A No No
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