316 Chapter 12 Figure 6. hCS morphology after AAV.I-1c treatment. (A) Schematic overview of the expression cassette containing the cytomegalovirus (CMV) promoter, the inhibitor-1 (I-1c), and Inverted terminal repeats (ITR) and the AAV vector design, combining the Rep protein of AAV2 with the Capsid protein AAV8. hCSs transfected with AAV. Ic-1 MOI.10.000 for 14 days before the Ca2+ analysis was performed. (B) Representative cardiac spheroid morphology and 6 representative 10-second Ca2+ transients of non-treated hCSs from the PLN-R14del (D4iR14del) line. Scale bar: 400 μm. (C) Representative cardiac spheroid morphology and 6 representative 10-second Ca2+ transients of AAV.I-1c treated hCSs from the PLN-R14del (D4iR14del) line. Scale bar: 400 μm. (D) Representative size of all hiPSC lines of untreated and AAV.I-1c treated hCSs. Scale bar: 400 μm. (E) Bar graphs of the size quantification from untreated vs. AAV.I-1c treated hCSs spheroids. Each dot represents one individual spheroid. (F) Bar graph of engineered heart tissue (EHT) force analysis from non-treated vs. AAV.I-1c treated EHTs. Each dot represents one individual EHT. *** P < 0.001, **** P < 0.0001 untreated vs AAV.I-1c of each group by Student’s T-Test or one-way ANOVA. (G) Heat map and hierarchical clustering of gene expression data. Log 2-fold changes of gene expression are represented, and the colour scale indicates the level of change (blue, low; red, high). The hierarchical clustering was generated using the Euclidean distance correlation metric based on the square root of the sum of the square differences (https:// software.broadinstitute.org/morpheus). (H) Representative bright field of an EHT and a 4-second force transient from all hiPSC lines of untreated and AAV.I-1c treated EHTs. Scale bar: 1 mm. (I) Bar graph of engineered heart tissue (EHT) force analysis from non-treated vs. AAV.I-1c treated EHTs. Each dot represents one individual EHT. *** P < 0.001, **** P < 0.0001 untreated vs AAV.I-1c of each group by Student’s T-Test or one-way ANOVA.
RkJQdWJsaXNoZXIy MTk4NDMw