2C treatment induced dedifferentiation of hESC-derived CMs toward ISL1-expressing cells.

Cells induced from hESC-derived CMs by treatment with DMSO (NC) or 2C for 60 hours. (A) Phase contrast images showing cell morphology. (B) Immunofluorescence staining of the ISL1 (ISL1, green), and the CM marker cardiac troponin T (TNNT2, red) in the cells. Nuclei were stained by DAPI (4′,6-diamidino-2-phenylindole) and presented in DNA blue. (C), Cytosolic and nuclear areas of the cells. Data are shown as mean ± SD. (n=3 independent experiments, represented as dots). Two-way ANOVA with Dunnett’s multiple comparisons test. ****P < 0.0001. (D) Changes in cell number after 2C treatment. Total cell number, TNNT2+ cell number and ISL1+ cell number were normalized to the negative control DMSO (NC). Data are shown as mean ± SD (n=3 independent experiments, represented as dots). Two-way ANOVA with Šidák’s multiple comparisons test. ns, not significant (P > 0.05), ****P < 0.0001. (E) Fraction of ISL1+ cells in TNNT2+ cells. Data are shown as mean ± SD (n=3 independent experiments, represented as dots). Two-way ANOVA with Šidák’s multiple comparisons test. **P < 0.01. (F) Western blot and quantitative analysis of ISL1 expression in DMSO (NC) or 2C-treated CMs for 60 hours. Data are shown as mean ± SD. Unpaired t test. *P < 0.05. (G) Relative gene expression of embryonic cardiogenesis marker genes (MESP1, ISL1, NR2F2, FUT4, and LEF1), pan-cardiac genes (GATA4, TBX5, and NXK2-5), and CM marker genes (MEF2C, TNNT2, MYL2, and MYL7) in the cells treated by DMSO (NC) or 2C for 60 hours (60h). Data are shown as mean ± SD (n=3 independent experiments, represented as dots). Multiple unpaired t tests. ns, not significant (P > 0.05), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Regenerative ability of 2C-induced RCCs.

(A-C) Immunofluorescence staining (A) and statistical analysis (B and C) of the ISL1 (green) and BrdU (red) double positive RCCs induced from CMs by treatment with DMSO (NC) or 2C for 60 hours. The ISL1+ cell number and ISL1+/BrdU+ cell number were normalized to the negative control DMSO (NC). DAPI (4′,6-diamidino-2-phenylindole) staining labeled nuclei as blue. Data are shown as mean ± SD (n=3 independent experiments, represented as dots). Multiple unpaired t tests in (B), ****P < 0.0001. Two-way ANOVA with Šidák’s multiple comparisons test in (C), **P < 0.01, ****P < 0.0001. (D) Phase contrast images of hESC-derived CMs treated by DMSO (NC) or 2C for 60 hours (60h) and subsequently cultured in the absence of 2C for another 3 days (60h+3d). (E) Immunostaining showed the expression of ISL1 (green) and TNNT2 (red) in the cells under the same condition in (D). (F) Statistical analysis of TNNT2+ cell numbers under the same condition in (D). Data are shown as mean ± SD (n=3 independent experiments, represented as dots). Two-way ANOVA with Šidák’s multiple comparisons test. ns, not significant (P > 0.05), ****P < 0.0001. (G) Schematic diagram of directed differentiation of 2C-induced RCCs towards cardiomyocytes (CMs), smooth muscle cells (SMCs) and endothelial cells (ECs). (H) Immunostaining showed the expression of EC markers (CD31, green and CD144, red), SMC marker (SMA, red), and CM marker (TNNT2, green). DAPI (4′,6-diamidino-2-phenylindole) staining labeled nuclei as blue.

Lineage tracing demonstrated 2C induced dedifferentiation of TNNT2+ CMs to ISL1-expressing RCCs.

(A) Immunofluorescence images showing expression of endogenous ISL1 (green) and ISL1-mCherry (red) reporter in the cells differentiated from K9 hESC KI reporter line at day 6 (D6). DAPI (4′,6-diamidino-2-phenylindole) staining labeled nuclei as blue. (B-C) Flow cytometry analysis of the percentage of mCherry+/ISL1+ cells in the cells differentiated from K9 at D6. (D-E) Flow cytometry analysis of the percentage of mCherry-negative cells at selection day 4 (SD4) in lactate purification medium. (F) Cells induced from mCherry-negative CMs by treatment with or without 2C for 60 hours. Images showing the expression of mCHERRY (red) and TNNT2 (green) in the cells. (G) Relative gene expression of ISL1, mCHERRY, LEF1, TNNT2 and MYL2 in K9-derived mCherry-negative CMs treated with DMSO (NC) or 2C for 60 hours. Data are shown as mean ± SD (n=2 independent experiments with 4 replicates each). Two-way ANOVA with Šidák’s multiple comparisons test. ***P < 0.001, ****P < 0.0001. (H-I) Flow cytometry analysis of the percentage of mCherry-positive cells induced from mCherry-negative CMs by treatment with or without 2C for 60 hours. Data are shown as mean ± SD (n=3 independent experiments, represented as dots). (J-K) Flow cytometric plots showing EGFP-labeled CMs by lineage-tracing of K9-derived mCherry-negative CMs (J), and bar graph showing the percentage of mCherry-negative CMs expressing EGFP (K). Data are shown as mean ± SD (n=3 independent experiments). (L) Images showing the expression of ISL1 (red) and EGFP (green) in the cells induced from EGFP-positive/mCherry-negative CMs by treatment with or without 2C for 60 hours. DAPI (4′,6-diamidino-2-phenylindole) staining labeled nuclei as blue.

Heart regeneration via 2C-induced dedifferentiation of CMs.

(A) Immunofluorescence staining of ISL1 (green) and TNNT2 (red) in cross-sectioned hearts from 2C or vehicle (DMSO)-treated (NC) adult 129SvJ mice. Ao, aorta. PA, pulmonary artery. LA, left atrial. RA, right atrial. IAS, interatrial septum. DAPI (4′,6-diamidino-2-phenylindole) staining labeled nuclei as blue. (B) Schematic illustration of the method used to examine the prophylactic effect of 2C in 129SvJ mice post MI. (C) Body weight of mice pre-treated with vehicle (DMSO) or 2C as shown in (B) at Day 12, Day 16, Day 20, and Day 35 after MI (dpi). Error bars represent SD. ns, not significant (P > 0.05). (D) Survival curve of sham-operated mice and mice pre-treated with vehicle (DMSO) or 2C as shown in (B), at indicated time points before or after MI. (E) Ejection fraction (EF) of sham-operated mice and mice pre-treated with vehicle (DMSO) or 2C as shown in (B), before MI (baseline) or at Day 1, Day 8, Day 25, and Day 35 after MI (dpi). Data are shown as mean ± SD. Two-way ANOVA with Tukey’s multiple comparisons test. ns, not significant (P > 0.05), **P < 0.01, ***P < 0.001. (F) Schematic illustration of the method used to examine therapeutic effect of 2C in the 129SvJ mice post MI. (G) Serial fMRI measurements showing the cardiac function from sham-operated mice and mice treated with vehicle (DMSO) or 2C at as shown in (F). Data are shown as mean ± SD. Two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05. (H) Echocardiography of sham-operated mice and mice treated with vehicle (DMSO) or 2C as shown in (F) at Day 35 post MI. (I) Masson staining of serial transverse sections of hearts from sham-operated mice and mice treated with vehicle (DMSO) or 2C as shown in (F) at Day 35 post MI.

Bulk RNA-seq of analysis of 2C-treated ISL1/mCherry-negative CMs.

(A) Scheme of bulk RNA-seq analysis of K9-derived mCherry-negative CMs with DMSO (NC) or 2C treatment for 60 hours. (B) Heatmap of differentially expressed genes (DEGs) in ISL1/mCherry-negative CMs treated with DMSO (NC) or 2C for 60 hours. (C) Volcano plot showing genes significantly changed by 60 hours of 2C treatment. (D-E) Gene Ontology (GO) analysis of downregulated (D) and upregulated (E) genes in ISL1/mCherry-negative CMs by 2C treatment for 60 hours, compared to DMSO (NC) treated cells. (F) Plotting GO terms of upregulated genes by 2C treatment with cnetlpot. (G) Relative expression fold-changes of indicated genes in K9-derived ISL1/mCherry-negative CMs by 60 hours of DMSO (NC) or 2C treatment. Data are shown as mean ± SD. Multiple unpaired t tests. **P < 0.01, ***P < 0.001.

Single-cell RNA-seq of 2C-treated mCherry-negative CMs.

(A) UMAP analysis showing 7 clusters in cells induced from K9-derived mCherry-negative CMs by treatment with DMSO (NC) and 2C for 60 hours. (B) The percentage of cells in the 7 indicated clusters, following DMSO (NC) or 2C treatment. (C) Heatmap showing the differentially expressed genes in the cells from 7 indicated clusters. The representative marker genes of 7 indicated clusters were listed on the right. (D) Violin plots showing the expression levels of marker genes of CMs (MYL2, MYH6), ICs (COL1A1, ACTA2), and RCCs (ISL1, BMP4, FGF20) among cells from 7 indicated clusters. (E) UMAP analysis showing the second-level clustering of cluster 2 into 4 subclusters (left), which exhibited dramatic distinction under condition of 2C or NC (right). (F) The percentage of cells in the 4 indicated subclusters within cluster 2, following DMSO (NC) or 2C treatment. (G) Heatmap showing the differentially expressed genes among cells from 4 subclusters of cluster 2. Genes related to RCCs are highlighted in the green blocks on the right. (H-I) Pseudotime trajectory showing changes across various cell states upon 2C treatment, which were presented with different developmental pseudotime points (H) and cell states (I). (J) Curves showing the dynamic expression of representative genes of RCCs (ISL1, FGF20), ICs (COL1A1), and CMs (TNNT2) along indicated pseudotime points.

Chromatin immunoprecipitation-sequencing (ChIP-seq) analyses of chemical-treated H9 hESC-derived CMs.

(A) Schematic illustration of ChIP-seq analysis of H9-derived CMs subjected to DMSO, A-485, CHIR99021, or 2C treatment for 60 hours. (B) Average ChIP-seq signal profiles showing the indicated histone modifications around the TSS in the input and ChIP samples prepared from DMSO and 2C-treated cells. (C) Heatmap showing the whole-genome wide distribution of H3K9Ac, H3K27Ac, and H3K4me3 peaks within a range of ±4kb from TSSs in the cells treated with DMSO or 2C for 60 hours. (D) H3K9Ac and H3K27Ac peaks surrounding CM genes (TNNT2 and TNNI1) and RCC genes (LEF1 and ISL1) in the cells treated with DMSO, A-485, CHIR99021 or 2C for 60 hours, and H3K4me3 peaks surrounding the same genes in the cells treated by DMSO or 2C. The y-axis represents the number of counts. (E-F) Veen diagram showing the number of annotated genes with H3K9Ac or H3K27Ac enrichment in the cells treated with DMSO, A-485, CHIR99021 or 2C for 60 hours. Red circles indicate the number of genes with unique H3K9Ac or H3K27Ac enrichment induced by 2C treatment; black circles indicate the number of genes with H3K9Ac or H3K27Ac enrichment unaffected by any chemical treatment. (G-J) The annotated genes with the most significant changes in H3K9Ac enrichment following treatment with DMSO (G) or 2C (I) were ranked by Log10LR and analyzed by GOs (H and J), respectively. (K) ISL1 binding motifs identified from the cells treated with A-485 or 2C.