Uncovering cell type-specific phenotypes using a novel human in vitro model of transthyretin amyloid cardiomyopathy

Figures

• 

  Fig. 1. Characterization of in vitro generated TTR fibrils​. (A) TTR fibrils were formed in the mild acidic buffer. (B) Amytracker 540 fluorescence of TTR solutions with (Blue) or without (Red) mild acidic buffer. Data are presented as mean ± SEM of N = 3 independent experiments. (C) ThT spectral fluorescence reading for TTR and TTR fibrils showing the fluorescence emission of unbound ThT (solid lines) and ThT bound to fibrils (dashed lines). λ excitation = 450 nm and λ emission = 482 nm. (D) Congo red stained TTR fibrils examined under brightfield and crossed polarized light (scale bar, 50 μm). (E) Dot Blot analysis of TTR, TTR fibrils and insulin fibrils using anti-TTR and anti-conformational amyloid fibril antibodies. Full blots are presented in Supplemental Fig. 6. P-values were determined using the two-way ANOVA, followed by Tukey’s test. **P < 0.01, ****P < 0.0001

• 

  Fig. 2. hiPSC-CM cell viability and immunostaining of TTR fibrils and sarcomeres. (A, B) Cytotoxicity of TTR and TTR fibrils as shown by AlamarBlue viability and Live/Dead assays. Data are presented as mean ± SEM of N = 4 independent experiments. (C) Fold change in sarcomere disruption in TTR, TTR fibrils and control groups. (D) Live/Dead staining images of hiPSC-CMs stained for Hoechst 33,342 (Blue), Calcein-AM (Green) and EthD-1 (Red) (scale bar, 500 μm). Data are presented as mean ± SEM of N = 4 independent experiments. (E) Confocal images of hiPSC-CMs stained for Hoechst 33,342 (Blue), TTR (Green) and cTnT (Red) (scale bar, 100 μm). For A and C, P-values were determined using the one-way ANOVA, followed by Tukey’s test. For B, P-values were determined using the one-way ANOVA nonparametric Kruskal-Wallis test, followed by Dunn’s test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

• 

  Fig. 3. The effect of TTR fibrils on hiPSC-CMs calcium handling. (A) Schematic of calcium transient analysis. (B) Representative normalized calcium transients over 500ms. (C-F) Quantification of the calcium transient amplitude, rise time, decay time, and CTD90. Data are presented as mean ± SEM of N = 4 independent experiments. (G) Representative calcium transients and time-lapse images of hiPSC-CMs stained with Cal520. Scale bar 500 μm. For C, P-values were determined using the one-way ANOVA nonparametric Kruskal-Wallis test, followed by Dunn’s test. For D, P-values were determined using the one-way ANOVA, followed by Tukey’s test. *P < 0.05, ***P < 0.001

• 

  Fig. 4. TTR fibrils decrease endothelial cell viability and migration capacity. (A, B) Cytotoxicity of TTR and TTR fibrils was determined by AlamarBlue and Live/Dead staining. Data are presented as mean ± SEM of N = 5 independent experiments. (C) Histogram shows the percentage of wound closure. Data are presented as mean ± SEM of N = 4 independent experiments. Areas were determined by Image J. (D) Live/Dead staining images of ECs stained for Hoechst 33,342 (Blue), Calcein-AM (Green) and EthD-1 (Red) (scale bar, 500 μm). (E) Confocal images of ECs stained for Hoechst 33,342 (Blue), CD31 (Red) and TTR (Green) (scale bar, 100 μm). (F) Brightfield microscopy images of ECs migration at 0 and 12 h (scale bar, 100 μm). For A and C, P-values were determined using the one-way ANOVA, followed by Tukey’s test. For B, P-values were determined using the one-way ANOVA nonparametric Kruskal-Wallis test, followed by Dunn’s test. *P < 0.05, **P < 0.01, ****P < 0.0001

• 

  Fig. 5. hiPSC-FBs are resilient to TTR fibrils. (A, B) Cytotoxicity of TTR and TTR fibrils as shown by AlamarBlue and Live/Dead assays. Data are presented as mean ± SEM of N = 4 independent experiments. (C) Bar graph shows the average a-SMA fluorescent intensity in hiPSC-FBs. Fluorescent intensity value was determined by Image J. Data are presented as mean ± SEM of N = 3 independent experiments. (D) Live/Dead images of hiPSC-FBs stained for Hoechst 33,342 (Blue), Calcein-AM (Green) and EthD-1 (Red) (scale bar, 500 μm). (E) Confocal images of hiPSC-FBs stained for Hoechst 33,342 (Blue), Vimentin (Red) and TTR (Green) (scale bar, 100 μm). (F) Representative confocal images of hiPSC-FBs stained for Hoechst 33,342 (Blue), a-SMA (Red) and TTR (Green) after plating (scale bar, 100 μm). For B, P-values were determined using the one-way ANOVA nonparametric Kruskal-Wallis test, followed by Dunn’s test. *P < 0.05

• 

  Fig. 6. Distinct cellular responses to TTR fibrils in hiPSC-CMs, ECs, and hiPSC-FBs. (A, B, C) The left panels show enrichment dot plots representing selected enriched gene sets from a gene set enrichment analysis (GSEA) comparing each fibril condition against the negative control in (A) hiPSC-CMs, (B) ECs and (C) hiPSC-FBs, using the Gene Ontology (GO) biological processes (GOBP), GO molecular functions (GOMF), Reactome, WikiPathways (WP) and Hallmarks databases. Transparency represents -log10(adjusted p-value), dot size the Normalized Enrichment Score (NES), and color the direction of enrichment (blue = downregulated, red = upregulated in fibrils). The right panels show bar plots representing selected gene sets from a gene over-representation analysis (ORA) for the same databases, considering the differentially expressed genes (adjusted p-value < 0.05) retrieved across the three fibril conditions versus the control condition (see Supplemental Fig. 11)