Derivation of human post-mitotic cardiomyocytes from tetraploid iPSCs

Figures

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  Fig. 1. Generation of 4N-iPSCs by fusion of 2N-iPSCs. a Venus or mRFP1-positive Japanese male iPSC line, WTC11, was established by lentiviral gene transduction. These cells were fused using Hemagglutinating virus of Japan Envelope (HVJ-E). Fused cells were detected and isolated as Venus and mRFP1 double-positive colonies, followed by single-cell cloning. b Fluorescent images of 4N-iPSC colonies. Scale bar, 200 µm. c Karyotype of the isolated 4N-iPSCs. Autosomes and sex chromosomes were duplicated without obvious chromosomal abnormality. d The comparative genome hybridization (CGH) analysis of the isolated 4N-iPSCs using the original diploid WTC11 as a reference. No chromosomes showed insertions or deletions.

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  Fig. 2. Enhanced sensitivity of 4N-iPSCs to oxidative stress. a Diameter of 2N- and 4N-iPSCs (n = 6). b Doubling time of 2N- and 4N-iPSCs (n = 3). Statistical significance was determined by Student’s t-test (**P < 0.01, ***P < 0.001). c A volcano plot to visualize differentially expressed genes (DEGs) between 2N- and 4N-iPSCs. Red and blue dots represent genes significantly upregulated or downregulated in 4N-iPSCs, respectively (P < 0.05, |fold-change| > 2). Differentially expressed Metallothionein (MT) genes are highlighted in purple. d Heatmap representation of the expression of MT genes in 2N- and 4N-iPSCs. e The gene ontology (GO) biological process analysis of 2N- and 4N-iPSCs. The top 10 predicted differentially regulated processes between 2N- and 4N-iPSCs are shown. f Crystal violet staining showing cell survival of 2N- and 4N-iPSCs in the presence of different concentrations of H2O2, CuSO4, and CdCl2.

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  Fig. 3. The decreased expression of mitotic genes and improved mitochondrial functions in 4N-iPS-CMs. a Immunofluorescent staining of cardiac troponin T (cTnT) in 2N- and 4N-iPS-CMs. Scale bars, 50 µm (high magnification) and 200 µm (low magnification). b Electron microscopy of the sarcomeric structure. Scale bar, 5 µm. c The flow cytometric analysis of the DNA content of 2N- and 4N-iPS-CMs. d A heatmap representation of the mitotic gene expression in 2N- and 4N-iPS-CMs. e The GO biological process analysis of 2N- and 4N-iPS-CMs. The top 10 predicted differentially regulated processes between 2N- and 4N-iPS-CMs. f The copy number ratio of mitochondrial DNA to genomic DNA in 2N- and 4N-iPS-CMs (iPS-CMs) and pluripotent 2N- and 4N-iPSCs (Pluripotent iPSCs) quantified by digital PCR (n = 15). g Representative images of mitochondrial staining (green) in 2N- and 4N-iPS-CMs. h Quantification of mitochondrial pixel numbers per nucleus in 2N- and 4N-iPS-CMs, as determined by an image analysis (n = 25). Statistical significance was determined by Student’s t-test (NS not significant, P > 0.05, ***P < 0.001, *****P < 0.00001). i Evaluation of mitochondrial functions by a flux analyzer. The arrows…

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  Fig. 4. Increased impedance and resistance to a potassium channel blocker in 4N-iPS-CMs. a Representative cardiac impedance waveforms of 2N- and 4N-iPS-CMs. Base impedance (b), impedance amplitude (c), and upstroke velocity (d) of 2N- and 4N-iPS-CMs over two weeks after plating on a sensor plate (n = 15 for 2N-iPS-CMs, n = 11 for 4N-iPS-CMs). e Overlayed waveforms of 2N- and 4N-iPS-CMs to highlight the stronger amplitude and faster upstroke velocity of 4N-iPS-CMs. f Representative fold-changes of impedance amplitude of 2N- and 4N-iPS-CMs after the addition of different concentrations of Terfenadine. g Amplitude reduction rate of 2N- and 4N-iPS-CMs at 30 minutes after the addition of different concentrations of Terfenadine (n = 6 for DMSO and 500 nM, n = 3 for 900 nM Terfenadine). Statistical significance was determined by Student’s t-test (NS not significant, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, *****P < 0.00001).

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  Fig. 5. Generation of 4N-iPSCs and 4N-iPS-CMs from two different individuals. a The Japanese male iPSC line, WTC11, and the Caucasian female iPSC line, HPS0076, were fused. We established WTC11 and HPS0076 iPSC lines with Venus and puromycin-resistance genes, and mRFP1 and blasticidin-resistance genes, respectively. These cells were fused using HVJ-E. Fused cells were detected and isolated as Venus and mRFP1 double-positive, and puromycin and balsticidin double-resistant colonies, followed by single-cell cloning. b The karyotype of the isolated hybrid 4N-iPSCs. Autosomes and sex chromosomes were duplicated without obvious chromosomal abnormalities in this case. c The CGH analysis of the isolated hybrid 4N-iPSCs using standard male diploid iPSCs as a reference. No chromosomes showed insertions or deletions. d The principal component analysis (PCA) of WTC11 2N-iPSCs, HPS0076 2N-iPSCs, and hybrid 4N-iPSCs. Hybrid 4N-iPSCs were positioned between the two 2N-iPSCs. e Immunostaining of cardiac troponin T (cTnT, magenta) to confirm successful cardiomyocyte differentiation of hybrid 4N-iPSCs. f PCA of WTC11 2N-iPS-CMs, HPS0076 2N-iPS-CMs and hybrid 4N-iPS-CMs. Hybrid 4N-iPS-CMs were positioned…