hESC‐Derived Epicardial Cells Promote Repair of Infarcted Hearts in Mouse and Swine

Figure 1. Cardiac reparative effects of human embryonic stem cell‐derived epicardial cells (hEPs) delivered to acutely infarcted mouse hearts. A) Flow cytometry analysis of WT1‐positive hEPs at differentiation day 12. n = 4. B) Representative immunocytochemical staining of hEPs at differentiation day 20. n = 3. Scale bar: 50 µm. C) Schematic of hEP or vehicle treatment and analysis in the myocardial infarction (MI) hearts. D) Left ventricular ejection fraction (LVEF), LV fractional shortening (LVFS), and LV end‐systolic diameter (LVESD) measured by echocardiography in M‐mode. n = 7 each. *** p < 0.001 versus the Sham group; ### p < 0.001 versus the MI group. E) Representative cross‐sectional images and quantitative analysis of scar size stained with Masson's trichrome at day 28 post‐MI. n = 6 to 7. Scale bar, 1 mm. F) Representative images and quantitative analysis of the collagen content in the border zone of infarcted hearts stained with picrosirius red at day 28 post‐MI. n = 6 to 7. Scale bar: 50 µm. Data are means ± S.E.M. Two‐way ANOVA followed by Bonferroni's multiple analysis in D. Unpaired student's t‐test in E and F. * p < 0.05.

Figure 2. Cardiomyocyte survival, angiogenesis, and lymphangiogenesis regulated by implanted hEPs and the engraftment of hEPs in the infarcted mouse hearts. A) Representative images and quantitative data of immunofluorescent staining for TUNEL+ cardiomyocytes (CMs) in the border zone of infarcted hearts at day 3 post‐MI. n = 5. Scale bar: 20 µm. B) Lactate dehydrogenase (LDH) activity in mouse serum at day 3 post‐MI. n = 5. C,D) Representative images and quantitative data of immunofluorescent staining for CD31+ endothelial cells (C) and LYVE1+ lymphatic vessels (D) at day 28 post‐MI. n = 5 to 6. Scale bar: 50 µm. E) Representative images of co‐immunofluorescent staining for human mitochondria (hMito) with an epicardial marker WT1 or mesenchymal markers (vimentin or calponin) in the infarcted hearts at day 3 post‐MI. Scale bar: 20 µm. 15 sections from 6 hearts were analyzed. F) qRT‐PCR analysis of the transcript level of human GAPDH (hGAPDH) versus mouse Gapdh (mGapdh) in the infarcted LV tissues. The percentage was calculated by normalizing to the average value detected immediately after the hEP injection (day 0). n = 3 for day 0, n = 7 for day 3. Data are means ± S.E.M. Unpaired student's…

Figure 3. hEPs reduce inflammatory responses and promote reparative macrophage polarization concomitantly with the inhibition of type I interferon (IFN‐I) responses in the mouse LV tissues at day 3 post‐MI. A) RNA sequencing analysis of differentially expressed genes (DEGs, p < 0.05 and fold changes ≥ 1.5) in the Sham and infarcted LV tissues with or without hEP treatment. n = 3 each. B) Gene oncology (GO) annotation of the DEGs downregulated in the hEP‐treated MI hearts (p value < 0.05 and fold change ≥ 1.2). C) qRT‐PCR analysis of the transcript level of cytokine and chemokines. n = 4 to 5. D) ELISA analysis of the relative IL‐6 protein levels. n = 4. E) Representative immunofluorescent images and quantification of CD11b+ cells. n = 6. Scale bar, 50 µm. F) Representative images and quantitative data of immunofluorescent staining for CD68+ cells and CD68+CD206+ macrophages in the border zones. n = 6. Scale bar: 50 µm. G) The expression of IFN‐stimulated genes (ISGs) in hEP‐downregulated DEGs. H) qRT‐PCR analysis of the transcriptional level of ISGs. n = 3 to 5. Data are means ± S.E.M. One‐way ANOVA followed by Tukey's multiple comparison test in (C,D,H). Unpaired student's t‐test in…

Figure 4. The IFN‐I receptor agonist RO8191 attenuates the anti‐inflammatory and cardiac reparative effects of hEPs. A) Cardiac performance measured by echocardiography. All mice received intragastric administration of 10 mg kg−1 RO8191 or vehicle control (corn oil) 30 min before MI surgery. n = 6, 8, 6, and 8 in the MI, MI + hEPs, MI + RO8191, and MI + hEPs + RO8191 groups. ** p < 0.01, *** p < 0.001, and n.s.(no significant difference) versus the MI group. # p < 0.05, ## p < 0.01, and n.s. as indicated. B) Representative images and quantitative analysis of scar sizes stained with Masson's trichrome at day 28 post‐MI. n = 6 to 7. Scale bar: 1 mm. C,D) Representative images and quantitative data of immunofluorescent staining for CD31+ endothelial cells (C, n = 5 to 6) and LYVE1+ lymphatic vessels (D, n = 4 to 7) in the border zones at day 28 post‐MI. Scale bar: 50 µm. E) Representative images and quantitative data of immunofluorescent staining for TUNEL+ CMs in the border zones at day 3 post‐MI. n = 4 to 5. Scale bar: 20 µm. F) Representative images and quantitative data of immunofluorescent staining for CD68+ cells and CD68+CD206+ macrophages in the border zones at day 3 post‐MI. n = 5 to…

Figure 5. hEPs abundantly secrete ITLN1 and inhibit IFN‐I responses in macrophages. A) Western blotting analysis of IFN‐I signaling‐related proteins in the LV tissues at day 1 post‐MI. n = 4. B) Mass spectrometry (MS) analysis revealed top eight enriched proteins in the co‐immunoprecipitated (co‐IP) precipitate of IFN‐β and the proteins in the hEP‐conditioned medium (hEP‐CdM). C) qRT‐PCR analysis of the transcriptional levels of candidates screened by MS in the hESC‐derived cardiomyocytes (hCMs) and hEPs. n = 3. D) ELISA analysis of the hITLN1 level in the CdM of hEPs and hCMs. n = 3 to 4. E) ELISA analysis of the hITLN1 protein level in the LV tissues at day 1 post‐MI. n = 3 to 4. F) Representative image of co‐immunofluorescent staining for hMito and ITLN1 at day 3 post‐MI in the infarcted hearts treated with hEPs. Scale bar: 50 µm. G) Co‐IP analysis showed the interaction between ITLN1 and hFc‐tagged IFNβ (hFc‐IFNβ) in the hEP‐CdM. n = 3. H) Representative images showing the morphology of mouse peritoneal macrophages after 24 h with various treatments. n = 3. Serum‐free DMEM was used as a vehicle control. The cells were treated with vehicle supplemented with 300 ng mL−1 human recombinant…

Figure 6. Knockdown of ITLN1 in hEPs weakens their cardioreparative and anti‐inflammatory effects. A) Measurements of cardiac function by echocardiography. n = 5, 6, and 7 for the MI, MI + hEPvec, and MI + hEPshITLN1 groups. * p < 0.05, n.s.: no significant difference versus the MI group; # p < 0.05 versus the MI + hEPshITLN1 group. B) Representative images and quantitative analysis of scar sizes stained with Masson's trichrome at day 28 post‐MI. n = 5 to 7. Scale bar: 1 mm. C,D) Representative images and quantitative data of immunofluorescent staining for vWF+ endothelial cells (C) and LYVE1+ lymphatic vessels (D) in the border zones at day 28 post‐MI. n = 6. E,F) Representative images and quantitative data of immunofluorescent staining for TUNEL+ CMs (E) and CD11b+ cells (F) in the border zones at day 3 post‐MI. n = 3 to 6. G) Representative images and quantitative data of immunofluorescent staining for CD68+ cells and CD68+CD206+ macrophages in the border zones at day 3 post‐MI. n = 3 to 6. Scale bar: 50 µm for all images. Two‐way ANOVA followed by Bonferroni's multiple analysis in (A). One‐way ANOVA followed by Tukey's multiple comparison test in (B–G). * p < 0.05, ** p < 0.01, and ***…

Figure 7. Intramyocardial injection of hEPs improves heart function and reverses cardiac remodeling in the infarcted porcine hearts at day 28 post‐MI. A) Schematic of hEP or vehicle treatment in porcine perfused MI model and analysis. Myocardial injury was induced by LAD occlusion for ischemia (60 min) and reperfusion, followed by intramyocardial injection of 5 × 107 hEPs (MI + hEPs) or with cell suspension vehicle (MI). Heart function was measured by cardiac multi‐detector computed tomography (MDCT). B) Representative photograph of the position for LAD occlusion after thoracotomy (left panel) and for hEP injection into the border zones (white arrowheads) after reperfusion (right panel). Representative normal electrocardiogram (ECG) before the ligation (left bottom) and the ST elevation following reperfusion (right bottom). C) Representative MDCT images of end‐systole (top rows) and end‐diastole (rows below) in each group. D,F) Analyzed data of MDCT for LVEF (D), LV end‐diastolic volume (LVEDV, E), and LV end‐systolic volume (LVESV, F). n = 4, 6, and 6 for the Sham, MI, and MI + hEPs groups. G,H) Hemodynamic measurements of maximal LV pressure rising rate (+dp/dtmax) and minimal LV…

Figure 8. Intramyocardial injection of hEPs promotes angiogenesis, reduces cardiomyocyte apoptosis, and inhibits monocyte/macrophage infiltration in the infarcted swine hearts at day 28 post‐MI. A) Representative images and quantitative data of immunofluorescent staining for α‐SMA+ arteries in the border zones. n = 6. B) Representative images and quantitative data of immunofluorescent staining for CD31+ vessels in the border zones. n = 6. C) Representative images and quantitative data of immunofluorescent staining for TUNEL+ CMs in the border zones. n = 6. D) Representative images and quantitative data of immunofluorescent staining for CD11b+ cells in the border zones. n = 6. E) Representative images and quantitative data of immunofluorescent staining for CD68+ cells and CD206+ CD68+ macrophages in the border zones. n = 5. Scale bar: 50 µm for all images. Unpaired student's t‐test in (A–E). * p < 0.05 and *** p < 0.001.