Homozygous MESP1 knock-in reporter hESCs facilitated cardiovascular cell differentiation and myocardial infarction repair

Figures

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  Figure 1. Generation of MESP1 knock-in H9 hESC. (A) Schematic view of MESP1 targeting strategy. Donor plasmid: T2A-mTomato-pA is a T2A self-cleaving peptide sequence joint with mTomato and a polyadenylation sequence (pA); PGK, phosphoglycerate kinase promoter; puroRFP, puromycin resistance gene fused with red fluorescence protein (RFP) gene; loxP, loxP sites for Cre recombinase-mediated PGK-puroRFP removal; MC1-TK, MC1 promoter driving TK gene for negative selection. White arrow indicates the cutting site of the MESP1 TALEN pair. Below, MESP1 TALENs and their recognition sequence. The DNA binding specificity of the repeat variable di-residue (RVD) is: NI = A (red), HD = C (yellow), NN = G (green), NG = T (blue). (B) Schematic view of MESP1-mTomato knock-in locus and positions of genomic DNA PCR primers (red arrows). The wild type (WT) fragment is 2.3 kb, and the knock-in locus (MT) fragment is 4.1 kb. EcoR I (E) digestion of the MT fragment generates two fragments of 2.9 kb and 1.2 kb. Hind III (E) digestion generates two fragments of 1.1 kb and a 3 kb. P (loxP). (C) Genomic DNA PCR confirming correct targeting. Lane 1: WT fragment (2.3 kb), lane 2: MT fragment (4.1 kb), lane 3-5: EcoR I…

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  Figure 2. Canonical Wnt signaling controls the induction of MESP1-mTomato+ cells. (A) Cartoon depicting the CM differentiation protocol. (B) Flow cytometry analysis of mTomato fluorescence in cells during 168 h (7 days) of CM differentiation (n = 3). (C) Western blot showing endogenous MESP1 protein levels during CM differentiation (n = 3). (D) Quantification of MESP1 protein level based on the western blot. (E) Experiment timeline for signaling pathway test: 4 days after replating and culturing in E8 medium, MESP1-mTomato reporter cells were treated with small molecules activating or inhibiting various signaling pathways for 72 h, then harvested for flow cytometry analysis. (F) Bar graph quantification of MESP1-mTomato+ cell percentage after growth factor or small molecule treatment (as listed at the bottom) (n = 3). “-” no small molecule added. Data is shown as mean ± SEM, *p < 0.05, **p < 0.005, ***p < 0.001, Student's t-test.

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  Figure 3. Transcriptome analysis of MESP1-mTomato+ cardiovascular progenitor cells. (A) Flow chart of MESP1-mTomato cell gene expression analysis. (B) PCA graph showing that hESCs, MESP1-mTomato+ cells at day 3 and 5 (MT_day3, MT_day5), and MESP1-mTomato- cells (MT_neg) were separated from each other. (C) Representative gene expression patterns in day 3 and day 5 MESP1+ cells. The number of genes in each category, representative GO terms and enrichment P values were listed on the right side. (D) Heatmaps showing that many key regulators of cardiovascular development were differentially expressed in day 3 or day 5 MESP1+ cells, including Wnt pathway members, heart development genes, endothelial genes, hematopoietic cell genes and skeletal development genes. (E) GSEA plot of selective biological process enriched in MESP1+ or MESPI- cells, day 3 or day 5 MESP1 cells.

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  Figure 4. MESP1 represses canonical Wnt/β-CATENIN signaling during hESC cardiac differentiation. (A) Dox inducible MESP1 expression system. Left, dosage effect of Dox on MESP1-Flag protein expression (Dox concentration is indicated at the top). Middle and right, MESP1-Flag protein was induced 3 h after Dox addition and disappeared 6 h after Dox withdraw. ACTIN was used as the loading control in all experiments. (B) Schematic view of the differentiation protocol. Cells were treated with 25 ng/mL BMP4 and 2 µM CHIR99021 for 24 h. 48 h after BMP4 and CHIR99021 removal, 5 µM IWP2 or 400 ng/mL Dox was added for 48 h. After IWP2 or Dox treatment, cells were cultured in RPMI/B27 minus insulin medium until day 7, then changed to RPMI/B27 medium until day 12. (C) Bar graph showing cTnT+ cell percentage in untreated, Dox, or/and IWP2 treated cells based on flow cytometry analysis (n = 3). Data represent mean ± SEM. (D) Q-PCR analysis of genes induced by MESP1. Dox or IWP2 were added during day 3-5. The expression levels of cardiac and Wnt pathway genes were normalized against GAPDH. The expression levels in untreated cells were set as “1”. (n = 3). (E) Western blot showing MESP1, phosphorylated…

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  Figure 5. Efficient tri-lineage differentiation based on optimizing MESP1-mTomato expression. (A) Schematic view of the tri-lineage differentiation system. (B) Immunostaining of α-actinin (red) and cTnT (red) in CMs, DNA in blue, scale bars, 20 µm. (C) Flow cytometry analysis of cTnT+ cells on day 12. (D) Di-4-ANEPPS fluorescence intensity measurement to show the action potential change in MESP1-mTomato+ cells derived CMs. Most beating cells showed ventricular-like electrophysiology characteristics (left), and some displayed nodal-like potential change (right). (E) Time-lapse images of Ca2+ transients in CMs. The time was indicated on the top-right corner; the white line circled the contracting area. (F) Drug response of CMs differentiated from MESP1-mTomato+ cells. Beating cells stained with Fluo-4 AM were first filmed. Then they were treated with 5 µmol/L Isoproterenol and filmed again. Images were recorded at 10 frames per second. Fluorescence intensity of the circled area in (E) was quantified before and after Isoproterenol addition. (G) Immunostaining of CD31 (red), αSMA (green) and SM22a (red). Scale bars, 100 µm. (H) Flow cytometry analysis of CD31-CD144 for ECs and SM22a for SMCs.…

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  Figure 6. Transplantation of MESP1+ cardiac progenitor cells improved the function of rat MI hearts. (A) Representative M-mode echocardiographic images obtained in Sham, Control, and MESP1 groups. (B-C) Quantitative cardiac function analysis of LVFS and LVEF 28 days after permanent LAD occlusion of the three groups. Data expressed as mean ± SD. Sham (n = 6), Control (n = 6), and MESP1 (n = 5). *p < 0.05, **p < 0.005, ***p < 0.001, t-test. (D) Table summarizing the echocardiography result. (E) Distribution of left ventricular remodelling index (LVRI) in Sham, Control, and MESP1 groups. (F) The survival curve of rats in the Control (n = 10) and MESP1 (n = 7) groups. (G) Representative images of rat hearts (Masson's trichrome staining) for Sham, Control, and MESP1 groups. (H) Infarct size as percent area of myocardial scarring in MI hearts. Data are mean ± SD measured from Masson's trichrome staining, **p < 0.005, t-test. (I and J) Immunostaining of hCD31, human nuclei (hNA) and human cTnT (hcTnT) 28 days after transplantation; scale bar: 50 µm. The color of the antigen is as indicated on the top, DNA, blue. (K) Quantification of vessel density in the myocardial scar and border zone 28 days…