Figure 1. Engineering a human cardiomyocyte model to study polyploidization.
(A) In vivo RNA-seq analysis of adult cardiomyocyte nuclei relative to fetal (Gilsbach et al., 2018) demonstrates downregulation of G2/M cyclins CCNB1 and CCNB2 with persistence of G1/S cyclins CCND1 and CCND2.
(B) Overview of CRISPR methods to generate CCNB1-eGFP, TNNT2-T2A-NeoR, and TNNT2-mCherry iPSC lines to study polyploidization in differentiated cardiomyocytes (CMs).
(C) Representative flow cytometry plots demonstrating that iPSCs relative to CMs express more uniform CCNB1 levels that progressively increase in S-phase and peak in G2/M.
(D) Flow cytometry quantification of CCNB1-eGFP levels and Hoechst stain in CMs at differentiation day 20 to 40 shows progressive increase in DNA content paralleling inhibition of CCNB1.
(E) Representative time-lapse confocal images of live CCNB1-eGFP and cTnT-mCherry CM cell-cycle progression and cytokinesis. White arrow denotes a CM that undergoes cytokinesis into two daughter CMs. Scale bar, 10 μm.
(F) Replicative outcomes of 83 CCNB1+ CMs from time-lapse confocal imaging (green denotes CCNB1-eGFP expression and localization).
(G) CM mitosis relates to peak CCNB1-eGFP levels from time-lapse confocal imaging (n = 27 CCNB1+ CMs).
Data are n ≥ 3 and mean ± SEM; significance assessed by ANOVA with Holm-Sidak correction (D and G) and defined by *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. See also Figure S1 and Video S1.