CHARGE syndrome modeling using patient-iPSCs reveals defective migration of neural crest cells harboring CHD7 mutations
- Keio University School of Medicine, Japan
- Tokyo Metropolitan University, Japan
- Kanagawa Children's Medical Center, Japan
- Juntendo University School of Medicine, Japan
- Stanford University School of Medicine, United States
- Howard Hughes Medical Institute, Stanford University School of Medicine, United States
Figures
Figure 1 with 1 supplement
Characterization of CHARGE patient-derived iPSCs.
(A) Representative images of generated iPSCs from CHARGE patients CH1 and CH2 showing human ESC-like morphologies. Bar: 300 μm. (B) TRA1-60 and TRA1-81 protein, pluripotent markers, expression in …
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Figure 1—source data 1
Features and phenotypes of the enrolled CHARGE patients, and raw data and statistical data of Figure 1.
Tab1: Features and phenotypes of the enrolled CHARGE patients. Both patients showed symptoms typical of CHARGE syndrome. Tab2: Statistical data of Figure 1F. qRT-PCR analysis of CHD7 using control and CHARGE iPSC lines.
- https://doi.org/10.7554/eLife.21114.005
Figure 1—figure supplement 1
Direct sequencing analysis of the CHD7 mutations in all iPSC lines used in this study.
https://doi.org/10.7554/eLife.21114.004
Figure 2
Differentiation of patient iPSCs into NCCs.
The iPSC-NCCs in (B) and (C) were obtained by Method A, and the iPSC-NCCs in (D)-(K) were obtained by Method B. (A) Schematic presentation of the protocol for NCC differentiation from iPSCs using …
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Figure 2—source data 1
Raw data and statistical data of Figure 2.
- https://doi.org/10.7554/eLife.21114.007
Figure 3 with 1 supplement
Comparative gene expression analysis suggests migration defects in CHARGE iPSC-NCCs.
(A) Scatter plot of control vs. CHARGE iPSC-NCCs obtained by Method B. Control: 201B7 iPSC-NCCs; CHARGE: CH2#16 iPSC-NCCs. (B) Hierarchical clustering of 338 differentially expressed genes (FC >1.25)…
Figure 3—figure supplement 1
Examples of differentially expressed genes between control and CHARGE iPSC-NCCs.
(A) qRT-PCR analysis of control and CHARGE iPSC-NCCs. Ctrl: WD39, 201B7; CH: CH1#25, CH2#16. Error bars represent standard deviations from samples by four independent inductions per group …
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Figure 3—figure supplement 1—source data 1
Statistical data of Figure 3—figure supplement 1
Tab1: Statistical data of Figure 3—figure supplement 1A. Tab2: Statistical data of Figure 3—figure supplement 1B.
- https://doi.org/10.7554/eLife.21114.010
Figure 4 with 2 supplements
Defective Scattering of CHARGE iPSC-NCCs in vitro.
(A) Representative images of control and CHARGE iPSC-NCCs obtained by Method A. The control iPSC-NCCs residing at the outermost periphery began to scatter apart (Figure 4A–a). In contrast, the …
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Figure 4—source data 1
Raw data and statistical data of Figure 4.
Tab 1: Statistical data of Figure 4C. a. Statistical data of Figure 4C (left). Control (WD39): t = 0 vs t = 8 hr. b. Statistical data of Figure 4C (right). CHARGE (CH1#25): t = 0 vs t = 8 hr. c. Statistical data of Figure 4C (left and right). Tab 2: Statistical data of Figure 4D. Tab 3: Statistical data of Figure 4F. Tab 4: Raw data of Figure 4F. N: the number of neighbouring cells attached with the nine outermost cells in each of the eight 45 degree-sector of a sphere. White columns show the number of N = 0, 1, or >1 cells per sphere.
- https://doi.org/10.7554/eLife.21114.012
Figure 4—video 1
Time-lapse movies of attached control and CHARGE iPSCs-NCCs.
Representative images of control and CHARGE iPSC-NCCs scattering from an attached sphere on day16. Figure 4—video 1: control (WD39). Figure 4—video 2: CHARGE(CH1#25). Bar: 100 μm; time interval: 10 …
Figure 4—video 2
Time-lapse movies of attached control and CHARGE iPSCs-NCCs.
Representative images of control and CHARGE iPSC-NCCs scattering from an attached sphere on day16. Figure 4—video 1: control (WD39). Figure 4—video 2: CHARGE(CH1#25). Bar: 100 μm; time interval: 10 …
Figure 5 with 1 supplement
Migratory disabilities of CHARGE iPSC-NCCs.
(A) xCELLigence. The migration index of iPSC-NCCs was measured using xCELLigence, by which migrating cells through microelectrode sensors were monitored automatically. (B) Representative curve of …
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Figure 5—source data 1
Raw data of xCelligence assay of iPSC-NCCs in vitro.
Tabs 'Experiment 1' - 'Experiment 13': Raw data of migration indexes in each experiment. Each table in a sheet shows an independent experiment. Orange column shows migration index at 20 hr, and is used for quantitative analysis in Figure 5C. Tab 1: Statistical data of Figure 5C. Quantitative analysis of migration index after 20 hr of monitoring with xCELLigence, normalized to the control iPSC-NCCs migration index. Tab 2: Raw data and statistical data of Figure 5—figure supplement 1A. Control iPSC-NCCs migration index upon treatment with aphidicolin or vehicle for 36 hr. Tab 3: Raw data and statistical data of Figure 5—figure supplement 1B. BrdU assay of control and CHARGE iPSC-NCCs at 24 hr after replating. Tab 4: Raw data and statistical data of Figure 5—figure supplement 1C. Cell adhesion assay of control and CHARGE iPSC-NCCs to fibronectin.
- https://doi.org/10.7554/eLife.21114.017
Figure 5—figure supplement 1
Control and CHARGE iPSC-NCCs exhibit similar proliferation and adhesion.
(A) Representative curves of control iPSC-NCCs migration index upon treatment with aphidicolin (10 μg/ml) or vehicle (DMSO) for 36 hr. No significant difference was observed. (p=0.66; Two-way …
Figure 6 with 1 supplement
Single cell motility analysis of iPSC-NCCs in vitro.
(A) Representative images of migratory 201B7 (control; green) and CH1#25 (CHARGE; red) iPSC-NCCs at 0 hr and 16 hr, along with a combined view. Bar: 50 μm. (B) Average velocities of migratory …
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Figure 6—source data 1
Raw data and statistical data of Figure 6
Tab 1: Statistical data of Figure 6B. Average velocities of migratory iPSC-NCCs assessed by tracking control and CHARGE iPSC-NCCs for 16 hr. Tab 2: Raw data of single cell motility analysis of iPSCs-NCCs in vitro. Quantitative analysis in Figure 6B was calculated using the data below. Tab 3: Statistical data of Figure 6C. Tab 4: Raw data of single cell motility analysis of iPSCs-NCCs in vitro. Quantitative analysis in Figure 6C was calculated using the data below.
- https://doi.org/10.7554/eLife.21114.021
Figure 6—figure supplement 1
Single-cell motility analysis of iPSC-NCCs using other control and CHARGE iPSC-NCC lines.
Number of cells tracked: WD39 (control), 170 cells tracked; CH2#16 (CHARGE), 133 cells tracked. (A) Migratory velocities of control and CHARGE iPSC-NCCs. *p<0.05, **p<0.01, ***p<0.001 (Sidak's …
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Figure 6—figure supplement 1—source data 1
Raw data and statistical data of Figure 6—figure supplement 1.
Tab 1: Statistical data of Figure 6—figure supplement 1A. Average velocities of migratory iPSC-NCCs assessed by tracking control and CHARGE iPSC-NCCs over 16 hr. Tab 2: Raw data of single cell motility analysis of iPSCs-NCCs in vitro. Quantitative analysis in Figure 6—figure supplement 1A was calculated using the data below. Tab 3: Statistical data of Figure 6—figure supplement 1B. Quantitative analysis of the directionality of migratory iPSC-NCCs tracked. Tab 4: Raw data of single cell motility analysis of iPSCs-NCCs in vitro. Quantitative analysis in Figure 6—figure supplement 1B was calculated using the data below.
- https://doi.org/10.7554/eLife.21114.020
Figure 7 with 2 supplements
Defective migration of CHARGE iPSC-NCCs in chick embryos.
(A) (Left) Representative image of in vitro control and CHARGE iPSC-NCCs that were prestained with Vybrant DiI and DiO, respectively. (Right) Representative image of chick embryo at the HH 8–10 …
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Figure 7—source data 1
A list of transplanted cells and scores.
Tab 1: A list of transplanted cells and scores. Detais of the graph in Figure 7C. Transplanted control and CHARGE cells were stained with Vybrant DiI or DiO. We transplanted iPSC-NCCs into 93 embryos (HH8-10) in total, and 17 surviving embryoswere analyzed in this assay. Comparison of the maximum distance of Ctrl and CH shows that control cells migrated a greater distance from the site of transplant in some embryos evenly scored.
- https://doi.org/10.7554/eLife.21114.025
Figure 7—figure supplement 1
Time-lapse analysis of transplanted NCCs in chick embryos.
(A) Time-lapse images of transplanted cells stained with Vybrant DiO (control iPSC-NCCs) and Vybrant DiI (CHARGE iPSC-NCCs) in an HH stage 8–10 chick embryo (embryo #2). Bar: 100 μm. (B) Average …
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Figure 7—figure supplement 1—source data 1
Raw data of Figure 7—figure supplement 1
Tab 1: Raw data of Figure 7—figure supplement 1. In all, 4–14 cells per cell line were tracked for 6–7 hr as migrating cells in each experiment. Velocity was calculated at each time point and analyzed statistically by two-way ANOVA.Velocity of migrating cells: Control > CHARGE (p=0.03; Wilcoxon signed-rank test). Tabs, 'embryo #1' - 'embryo #9': Raw data of control and CHARGE cell velocities tranplanted in each embryo. The cell velocities of individual cells were calculated by manual tracking using the Manual Tracking plugin of the Fiji software.
- https://doi.org/10.7554/eLife.21114.024
Figure 7—video 1
A time-lapse movie of transplanted NCCs in chick embryos.
A time-lapse image of transplanted cells stained with Vybrant DiO (control iPSC-NCCs, green) and Vybrant DiI (CHARGE iPSC-NCCs, red) in an HH stage 8–10 chick embryo (embryo #2). Bar: 100 μm; time …
Figure 8
Model summarizing defective migration of CHARGE NCCs using patient-derived iPSCs.
Defects in cell delamination, migration, and motility in our model reflect phenotypes in CHARGE syndrome that develop in utero. Various aspects of NCC migration were not well coordinated in CHARGE …
Author response image 1
Author response image 2
The results of flow cytometric analysis of control and CHARGE iPSC-NCCs from method-B (control; red line, CHARGE; blue line).
Isotype controls were used as negative controls (black line).
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.21114.028
