Cell therapy with embryonic stem cell-derived cardiomyocytes encapsulated in injectable nanomatrix gel enhances cell engraftment and promotes cardiac repair

Abstract

A significant barrier to the therapeutic use of stem cells is poor cell retention in vivo. Here, we evaluate the therapeutic potential and long-term engraftment of cardiomyocytes (CMs) derived from mouse embryonic stem cells (mESCs) encapsulated in an injectable nanomatrix gel consisting of peptide amphiphiles incorporating cell adhesive ligand Arg-Gly-Asp-Ser (PA-RGDS) in experimental myocardial infarction (MI). We cultured rat neonatal CMs in PA-RGDS for 7 days and found that more than 90% of the CMs survived. Next, we intramyocardially injected mouse CM cell line HL-1 CMs with or without PA-RGDS into uninjured hearts. Histologic examination and flow cytometry analysis of digested heart tissues showed approximately 3-fold higher engraftment in the mice that received CMs with PA-RGDS compared to those without PA-RGDS. We further investigated the therapeutic effects and long-term engraftment of mESC-CMs with PA-RGDS on MI in comparison with PBS control, CM-only, and PA-RGDS only. Echocardiography demonstrated that the CM-only and CM+PA-RGDS groups showed higher cardiac function at week 2 compared to other groups. However, from 3 weeks, higher cardiac function was maintained only in the CM+PA-RGDS group; this was sustained for 12 weeks. Confocal microscopic examination of the cardiac tissues harvested at 14 weeks demonstrated sustained engraftment and integration of mESC-CMs into host myocardium in the CM+PA-RGDS group only. This study for the first time demonstrated that PA-RGDS encapsulation can enhance survival of mESC-derived CMs and improve cardiac function post-MI. This nanomatrix gel-mediated stem cell therapy can be a promising option for treating MI.

Keywords: PA-RGDS; cardiac regeneration; cardiomyocyte; myocardial infarction; pluripotent stem cell.

Figure 1

Structure of PA-RGDS.

Figure 2

Evaluation of cellular behaviors of cardiomyocytes encapsulated in PA-RGDS. Representative Live/Dead assay images of NRCMs after 7 days of culture in normoxic conditions. Bar graph summarizes the results of the Live/Dead assay. *p < 0.0001; N = 3. Scale bars, 20 μm.

Figure 3

Cytoprotective effects of PA-RGDS encapsulation against H₂O₂. (A) Encapsulation of NRCMs with PA-RGDS increased cell survival after H₂O₂ (200 μM) treatment as determined by the Live/Dead assay. *p < 0.001 compared with CM only group; N = 3. Scale bars, 20 μm. (B) Cell viability was also measured by extracellular release of LDH. *p < 0.001 compared with H₂O₂-untreated controls.

Figure 4

Survival and engraftment of HL-1 CMs after injection into uninjured mouse hearts. Seven days after injection of dilabeled (red) HL-1 CMs encapsulated with or without PA-RGDS into intact mouse hearts, mice were sacrificed and hearts were collected. (A) Confocal microscopic images of sectioned heart tissue after DAPI staining. DiI: red fluorescence. DAPI: blue fluorescence. Scale bars, 200 μm. (B) Quantification of engrafted HL-1 CMs by flow cytometry following cardiac tissue digestion into cell suspension; N = 3. (C) Confocal microscopic images of sectioned heart tissues after staining with ACTN2 in the mice receiving HL-1 CMs encapsulated with PA-RGDS. Green: ACTN2 staining. Scale bars, 20 μm.

Figure 5

Favorable effects of mESC-CMs with PA-RGDS on mouse experimental MI. (A) Improvement of cardiac function in mice receiving mESC-derived CMs with PA-RGDS. Fractional shortening (FS: left) and ejection fraction (EF: right) were significantly higher in the mESC-CM+PA-RGDS group compared to the three other groups measured by echocardiography. Repeated-measures ANOVA was used for statistical analyses. *p < 0.05; N = 6–10 per group. (B) Representative images from the four treated groups showing cardiac fibrosis after staining with Masson’s trichrome in the hearts harvested 4 weeks after MI and their quantification results. *p < 0.05; N = 6. (C) Confocal microscopic images of heart sections collected 4 weeks after MI and cell injection showed that the engraftment of DiI-labeled mESC-CMs was substantially higher when cells were encapsulated. *p < 0.05; N = 3. (D) The Dil-mESC-CMs (red fluorescence) expressed MYH6/7 (green) and were well integrated into host myocardium. Scale bars, 20 μm.

Figure 6

Sustained therapeutic effects of mESC-CMs with PA-RGDS on a mouse model of MI. (A) EF and FS measured by echocardiography were significantly greater in the mESC-CM+PA-RGDS group compared to the mESC-CM-only injected group. Repeated-measures ANOVA was used for statistical analyses. *p < 0.05. N = 6 per group. (B) Representative confocal microscopic images showing engraftment of DiI-labeled mESC-CMs in hearts harvested at 14 weeks compared to the CM-only injected group and the CMs with PA-RGDS group and their quantification (lower panel) *p < 0.05. N = 5. (C, D) Confocal microscopic images show integration of implanted mESC-CMs (DiI, red fluorescence) into ischemic myocardium, as evidenced by expression of Myh6/7 (C, green), Actn2 (D, green), and Gja1 (D, white). Hearts harvested from the mESC-CM+PA-RGDS group at 14 weeks. Scale bars, 20 μm.