HAND factors regulate cardiac lineage commitment and differentiation from human pluripotent stem cells

Figures

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  Fig. 1. Establishment of H1-KO, H2-KO and H1/H2-dKO hESC lines. A Schematic diagram of cardiomyocyte differentiation protocol. Mesoderm stage was at day 2. Cardiac mesoderm stage was at day 3. Cardiac progenitor stage was at day 5. Cardiomyocytes started beating at day 7. RB-: RPMI/B27-, RB: RPMI/B27, CHIR: CHIR99021, Vc: Vitamin C. B Temporal expression of HAND1 and HAND2 during cardiomyocyte differentiation. Relative to GAPDH expression (n = 3). C Schematic diagram of CRISPR/Cas9 mediated HAND1/2 genome editing. PAM: protospacer adjacent motif. D Expression of OCT4, SOX2 and NANOG in WT and HAND KO hESC lines. Relative to GAPDH expression (n = 3). E Immunofluorescence staining of SSEA-4 and OCT4 in WT and HAND KO hESC lines. Scale bar = 100 μm. F Flow cytometry analysis of SSEA-4+ cells in WT and HAND KO hESC lines. CTRL represented the negative control with secondary antibody incubation only. G qPCR analysis of HAND1 and HAND2 in WT and HAND KO hESC lines-derived day 10 cardiomyocytes. Relative to GAPDH expression (n = 3). One-way ANOVA. H Western blot analysis of HAND1 and HAND2 expression in WT and HAND KO hESC lines-derived differentiation day 10 cardiomyocytes. GAPDH served as…

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  Fig. 2. HAND1 deficiency promoted SHF and its derived cardiomyocyte differentiation. A The percentage of NKX2.5-eGFP+ cells in WT and H1-KO cells at different time points of cardiomyocyte differentiation (n = 3). B, C Expression of FHF (B) and SHF (C) markers in early cardiomyocyte differentiation of WT and H1-KO cells. Relative to GAPDH expression (n = 3). Unpaired t test. D, E Expression of NKX2.5 (D) and ventricular, atrial and outflow tract cardiomyocyte (VCM, ACM and OFT) markers (E) in WT and H1-KO-derived day 30 cardiomyocytes. Relative to GAPDH expression (n = 3). Unpaired t test. F Western blot analysis of CX43 and MYL2 expression in WT and H1-KO-derived day 30 cardiomyocytes. GAPDH served as loading control. Corresponding uncropped full-length gels and blots are presented in Additional file 8: Fig. S8. G The percentage of MYL2+ cardiomyocytes in WT and H1-KO cells at differentiation day 30 (n = 5). H Immunofluorescence staining of NR2F2 in WT and H1-KO-derived day 30 cardiomyocytes. Scale bar = 100 μm. I The field potential and simulated action potential recorded by MEA in WT and H1-KO-derived day 30 cardiomyocytes. Black lines represented the field potential while red lines…

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  Fig. 3. HAND2 knockout impaired SHF-derived cardiomyocyte differentiation. A The percentage of NKX2.5-eGFP+ cells in WT and H2-KO cells at different time points of cardiomyocyte differentiation (n = 3). B, C. Expression of FHF (B) and SHF (C) markers in early cardiomyocyte differentiation of WT and H2-KO cells. Relative to GAPDH expression (n = 3). Unpaired t test. D, E Expression of NKX2.5 (D) and ventricular cardiomyocyte (VCM) markers (E) in WT and H2-KO-derived differentiation day 30 cardiomyocytes. Relative to GAPDH expression (n = 3). Unpaired t test. F Western blot analysis of MYL2 expression in WT and H2-KO-derived differentiation day 30 cardiomyocytes. GAPDH served as loading control. Corresponding uncropped full-length gels and blots are presented in Additional file 8: Fig. S9. G The percentage of MYL2+ cardiomyocytes in WT and H2-KO cells at differentiation day 30 (n ≥ 3). H Expression of atrial and OFT cardiomyocyte (ACM, OFT) markers in WT and H2-KO-derived differentiation day 30 cardiomyocytes. Relative to GAPDH expression (n = 3). Unpaired t test. I Immunofluorescence staining of NR2F2 in WT and H2-KO-derived differentiation day 30 cardiomyocytes. Scale bar = 100 μm. J The…

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  Fig. 4. HAND1/2 double knockout impeded the differentiation of cardiomyocytes and impaired their electrophysiological activity. A The phase and fluorescence images of WT and H1/H2-dKO cells at differentiation day 7. B The percentage of NKX2.5-eGFP+ cells in WT and H1/H2-dKO cells at different time points of cardiomyocyte differentiation (n = 3). Unpaired t test. C The beating onset of WT and H1/H2-dKO-derived cardiomyocytes (n = 10). Unpaired t test. D Expression of FHF and SHF markers in early cardiomyocyte differentiation of WT and H1/H2-dKO cells. Relative to GAPDH expression (n = 3). Unpaired t test. E Expression of ventricular, atrial and outflow tract cardiomyocyte (VCM, ACM and OFT) markers in WT and H1/H2-dKO-derived day 30 cardiomyocytes. Relative to GAPDH expression (n = 3). Unpaired t test. F Western blot analysis of CX43 and MYL2 expression in day 30 WT and H1/H2-dKO cardiomyocytes. Corresponding uncropped full-length gels and blots are presented in Additional file 8: Fig. S10. G The percentage of MYL2.+ cardiomyocytes in WT and H1/H2-dKO cells at differentiation day 30 (n = 3). H Immunofluorescence staining of NR2F2 in WT and H1/H2-dKO-derived day 30 cardiomyocytes. Scale…

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  Fig. 5. Identifying the transcriptomic characteristics of H1-KO, H2-KO and H1/H2-dKO cardiomyocytes. A Schematic diagram of the samples for RNA-seq. WT, H1-KO, H2-KO and H1/H2-dKO cardiomyocytes were collected at differentiation day 7. B PCA analysis of WT, H1-KO, H2-KO and H1/H2-dKO cardiomyocytes. Each group had three duplicates. C The DEGs between different groups. DEGs were defined with |fold change (FC)|> 2 and adjusted P-value < 0.05. D The expression of representative FHF, SHF and cardiomyocyte (CM) genes in RNA-seq. E Gene expression cluster analysis of RNA-seq. Genes included in clustering were at least differentially expressed in one comparison in (C). F GO enrichment analysis of each gene expression cluster. G KEGG pathway enrichment analysis of each gene expression cluster

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  Fig. 6. HAND1/2 modulated cardiomyocyte differentiation through TBX5. A Venn diagram of HAND1 and HAND2 ChIP-seq and downregulated DEGs in H1/H2-dKO cells compared to WT cells in RNA-seq. B Expression of some TFs in WT and H1/H2-dKO sorted NKX2.5-eGFP+ cells at differentiation day 7. Relative to GAPDH expression (n = 3). Unpaired t test. C The ratio of Luciferase and Renilla activity in HAND1 and HAND2 overexpression 293T cells (n = 6). CTRL represented 293T transfected with pGL3-TBX5-Luciferase and Renilla only. One-way ANOVA. D TBX5 overexpression after DOX induction verified by western blot at differentiation days 5 and 10. GAPDH served as loading control. Corresponding uncropped full-length gels and blots are presented in Additional file 8: Fig. S11. E, F The percentage of NKX2.5-eGFP+ cells in WT and TBX5-OE cells (−DOX and + DOX) at differentiation day 7 (n ≥ 3). One-way ANOVA. G Expression of NKX2.5, MYH6 and TNNT2 in WT and TBX5-OE cells (−DOX and + DOX) at differentiation day 7. Relative to GAPDH expression (n = 3). One-way ANOVA. H Expression of SHF markers in WT and TBX5-OE cells (−DOX and + DOX) at differentiation days 5 and 7. Relative to GAPDH expression (n = 3). One-way…