Epicardial transplantation of antioxidant polyurethane scaffold based human amniotic epithelial stem cell patch for myocardial infarction treatment

Figures

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  Fig. 1. Schematic illustration of PUR-hAESC cell patch for myocardial infarction treatment. RGD modified PUR scaffold was used as a carrier for hAESCs to construct a PUR-hAESC cardiac patch. The patch provides a beneficial microenvironment for cardiac repair by facilitating a desirable immune response, paracrine modulation and limited oxidative milieu, consequently improving cardiac remodeling and function. PUR porous antioxidant polyurethane, hAESCs human amniotic epithelial cells, RGD arginine-glycine-aspartic adhesion peptide.

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  Fig. 2. Biocompatibility of PUR scaffold with hAESCs in vitro. a Representative fluorescent image of phalloidin (red) showing the morphology of hAESCs on PUR, n = 3 independent experiments. b Representative SEM image of the PUR-hAESC patch. c Higher-magnification image of the white frame in b, white arrow indicating the migration of hAESCs cultured on PUR. d 3D-reconstructed confocal images of hAESCs after culture for 1, 3 and 5 days on PUR porous scaffolds. e Migration depth of hAESCs into the porous scaffold, 1 d vs 3 d (p = 0.0011), 3 d vs 5 d (p = 0.0104), n = 3 independent experiments. f Viability of hAESCs in PUR porous scaffold for 1, 3 and 5 days assayed via CCK-8 assay, 1 d vs 3 d (p < 0.0001), 3 d vs 5 d (p < 0.0001), n = 3 independent experiments. g Calcein-AM (live)/EthD-1 (dead) staining to determine the viability of hAESC with or without PUR scaffold after oxygen glucose deprivation (OGD) injury; quantification summary in h, p < 0.0001, n = 5 independent experiments. i TUNEL staining (green) and phalloidin staining (red) revealed the viability and morphology of hAESC on PUR scaffold after OGD injury and quantification summary in j, p < 0.0001, n = 5 independent experiments.…

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  Fig. 3. PUR-hAESC patch ameliorated ventricular dysfunction and reduced pathological cardiac remodeling after acute MI. a Schematic showing the study design. Cardiac function was assessed 7 and 28 days after MI. b Representative echocardiography images and quantitative analysis of c left ventricular ejection fraction (LVEF), d left ventricular fractional shortening (LVFS), e end-diastolic volume (EDV) and f end-systolic volume (ESV) by echocardiography post implantation for 28 days, sham vs MI (p < 0.0001 in c, d, f, p = 0.0013 in e), MI vs hAESC (p = 0.3688 in c, p = 0.7070 in d, p = 0.9639 in e, p = 0.0048 in f), MI vs PUR-hAESC (p < 0.0001 in c and f, p = 0.0002 in d, p = 0.0047 in e), hAESC vs PUR-hAESC (p = 0.0010 in c, p = 0.0014 in d, p = 0.0123 in e, p = 0.0049 in f), n = 5 rats per group. g Masson’s trichrome staining of the whole heart; scale bars: 2 mm. Representative images in the infarct zones; scale bars: 100 μm. Quantitative analysis of infarction size (h) in the whole heart and LV wall thickness (i) in MI region. Normal hearts of the sham group were stained for comparison, sham vs MI (p < 0.0001 in h and i), MI vs hAESC (p < 0.0001 in h and i), MI vs PUR-hAESC (p < 0.0001…

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  Fig. 4. PUR-hAESC patch promoted angiomyogenesis in the post-MI heart. Representative immunofluorescent images and quantification of α-SMA+ blood vessels (a, b) and CD31+ area (c, d) in the infarction region post implantation for 28 days, White dashed lines indicate the border locations of the patches, and white asterisks point out the porous patch locations, Sham vs MI (p = 0.0005 in b, p = 0.0107 in d), MI vs hAESC (p = 0.0026 in b, p = 0.0214 in d), MI vs PUR-hAESC (p < 0.0001 in b, d), hAESC vs PUR-hAESC (p < 0.0001 in b, p = 0.0002 in d), n = 5 rats per group. e Phase-contrast microscope images of HUVECs to evaluate the migration capability; quantitative results are shown in g, 12 h (P = 0.0033), 18 h (p < 0.0001), 24 h (p < 0.0001), n = 5 independent experiments. f Representative images of tubes formed on Matrigel; quantification summary in h, P = 0.0008, n = 5 independent experiments. Scale bars: 100 μm in a, c, e and f. All data are presented as means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs control, one-way ANOVA in b, d, followed by Tukey’s multiple comparisons test; two-way ANOVA in g, followed by Bonferroni’s multiple comparisons test and two-tailed…

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  Fig. 5. Single-cell RNA sequencing of cardiac CD45+ cells reveals the presence of 27 cell clusters. a Schematic overview of the experimental setup. CD45+ immune cells isolated from hearts of MI and PUR-hAESC group 7 days after surgery. b Heat map showing the top 50 most differentially expressed genes in each cluster identified through unsupervised clustering of cardiac immune cells. Purple indicates lower expression; yellow indicates higher expression. Known cell type markers strongly and specifically associated with major cell types are shown on the right. c Heat map representing Pearson correlations between the 27 cell populations identified through unsupervised clustering of cardiac immune cells. Pearson’s correlation coefficients are shown on the right, colored according to the degree of correlation (from light dark blue to dark red). d t-SNE image showed the identified cell types are shown on the right. e Bar plot showing the proportions of cells in the 18 identified clusters.

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  Fig. 6. PUR-hAESC patch reduced neutrophil and inflammatory macrophage subsets. a t-SNE projection of the three neutrophil clusters identified in the heart of MI and PUR-hAESC rats. b Expression distribution (violin plot) showing normalized expression levels of genes encoding S100a9, S100a8, Il1b and Slpi in neutrophil clusters. c Bar plot showing the proportions of cells in each condition (MI and PUR-hAESC) for each neutrophil cell cluster (0, 5 and 17) identified. d, e Representative immunohistochemical and immunofluorescent images for the neutrophil cell markers MPO and S100a9 (green), n = 5 independent experiments. f t-SNE projection of the two macrophage clusters identified in the hearts of MI and PUR-hAESC rats. g Expression distribution (violin plot) showing normalized expression levels of genes encoding CD68, CD14, Hif1α and Spp1 in macrophage clusters. h Bar plot showing the proportions of cells in each condition (MI and PUR-hAESC) for macrophage cell cluster (7, 9) identified. i Representative images of immunofluorescent staining for the macrophage cell marker CD68 (red) and cluster 7 marker Hif1α (green), n = 5 independent experiments. j Representative images of…