Dual stem cell therapy synergistically improves cardiac function and vascular regeneration following myocardial infarction

Abstract

Since both myocardium and vasculature in the heart are excessively damaged following myocardial infarction (MI), therapeutic strategies for treating MI hearts should concurrently target both so as to achieve true cardiac repair. Here we demonstrate a concomitant method that exploits the advantages of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) and human mesenchymal stem cell-loaded patch (hMSC-PA) to amplify cardiac repair in a rat MI model. Epicardially implanted hMSC-PA provide a complimentary microenvironment which enhances vascular regeneration through prolonged secretion of paracrine factors, but more importantly it significantly improves the retention and engraftment of intramyocardially injected hiPSC-CMs which ultimately restore the cardiac function. Notably, the majority of injected hiPSC-CMs display adult CMs like morphology suggesting that the secretomic milieu of hMSC-PA constitutes pleiotropic effects in vivo. We provide compelling evidence that this dual approach can be a promising means to enhance cardiac repair on MI hearts.

Fig. 1

Implantation of hMSC-PA into MI hearts enhances the expression of multiple factors. a Preparation of heart-derived decellularized extracellular matrix (hdECM) bioink. b Macroscopic view and illustration of 3D printing system used to produce PCL platform. c Schematic illustration of human mesenchymal stem cell patch (hMSC-PA). Scale bar: 4 cm. d Epicardially implanted hMSC-PA in MI heart at 1 week. e-g Quantitative real-time polymerase chain reaction analysis of relative mRNA expression of multiple factors in the myocardium at 1 week after hMSC-PA implantation in MI induced hearts. e angiogenesis (f) inflammation, (g) fibrosis. The y-axis represents relative mRNA expression of target genes to GAPDH. A.U. indicates arbitrary units. The data are represented as mean ± SEM. ⁺p < 0.05 compared with MI group, ^#p < 0.05 compared with PA-only group; n = 3 biologically independent samples per group. One-way ANOVA was used for statistical analyses. Control: MI control, PA-only: cell-free hdECM patch, and hMSC-PA: hMSC-loaded patch

Fig. 2

hMSC-PA implantation only improved vascular regeneration. a hMSC-PA implanted to MI hearts were perfused with GFP-conjugated IsB4 to visualize the vessels, IsB4 (green). Representative images of capillaries on the infarct zone, border zone, and the remote zone at 8 weeks after MI and their quantification summary. For quantification, the number of capillaries on five randomly selected fields in each heart was counted. The number of capillaries was counted per mm² within the infarct zone. Scale bars: 50 µm. The data are represented as mean ± SEM. *p < 0.05 compared with control group; n = 5 biologically independent samples per group. T test was used for statistical analyses. b Rats undergoing MI were implanted with a hMSC-PA or control, followed by echocardiography analysis. Both ejection fraction (EF) and fractional shortening (FS) were not significantly higher than control group. The data are represented as mean ± SEM. *p < 0.05 compared with Sham group; n = 5 animals per group. One-way ANOVA was used for statistical analyses. Sham Sham operation, MI CON MI control, PA hMSC-loaded patch

Fig. 3

Dual approach improved cardiac function, capillary density, and reduced scar formation following MI. a Improvement of cardiac function in rats receiving hiPSC-CMs with hMSC-PA. Fractional shortening (FS) and ejection fraction (EF) were significantly higher in the hiPSC-CM with hMSC-PA group compared with other groups measured by echocardiography. The data are represented as mean ± SEM. *p < 0.05 compared with Sham group, ^†p < 0.05 compared with MI CON group, ^‡p < 0.05 compared with CM group, ^§p < 0.05 compared with PA group; n = 5 animals per group. One-way ANOVA was used for statistical analyses. b Representative images of capillaries stained with FITC-IsB4 (green) on the infarct zone, border zone, and the remote zone at 8 weeks after MI and their quantification summary. For quantification, the number of capillaries on five randomly selected fields in each heart was counted. Scale bars: 100 µm. The data are represented as mean ± SEM. *p < 0.05 compared with control, ^#p < 0.05 compared with the CM group; n = 5 independent samples per group. One-way ANOVA was used for statistical analyses. c Representative images from the four experimental groups showing cardiac fibrosis after staining with Masson’s trichrome in the hearts harvested 8 weeks after MI and their quantification results. The data are represented as mean ± SEM. *p < 0.05 compared with control, ^#p < 0.05 compared with the PA group, and ⁺p < 0.05 compared with the CM group; n = 5 biologically independent samples per group. One-way ANOVA was used for statistical analyses. Sham Sham operation, MI CON MI control, CM hiPSC-CMs, PA hMSC-loaded patch, CM + PA hiPSC-CMs + hMSC-loaded patch

Fig. 4

Implantation of hMSC-PA improves the retention and maturation of hiPSC-CMs on the infarcted myocardium. a Representative immunostaining images with hiPSC-CM-GFP (green), human specific MYH7 (gray), and MYH6/7 (red). Expression of hMYH7 in the hiPSC-CM-GFP verify the identity of hiPSC-CM-GFP as human CMs. Scale bars: 50 μm. b Representative immunostaining images with hiPSC-CM-GFP (green), human-specific mitochondria (gray), and MYH6/7 (red). c Quantification of hiPSC-CM-GFP remained in the hearts receiving CM or CM + PA. The heart tissue sections at three different locations were prepared and then immunostained with the MYH6/7 antibody. Subsequently, we imaged entire left ventricle (LV) area at three different locations of heart sections and manually counted the hiPSC-CMs positive for both GFP signal (green) and MYH6/7 antibody (red). Representative immunostaining images with hiPSC-CM-GFP (green) and MYH6/7 (red) and their quantification results. The data are represented as mean ± SEM. *p < 0.05 compared with CM only group. T test was used for statistical analyses. n = 3 biologically independent samples per group. d Results of TUNEL assay performed to assess the alive hiPSC-CM-GFP in the hearts receiving CM or CM + PA. The data are represented as mean ± SEM. ***p < 0.05 compared with CM group; n = 3 biologically independent samples per group. T test was used for statistical analyses. e Representative images of hiPSC-CM-GFP (green) expressing MYH6/7 (red) when they were injected in the absence or presence of hMSC-PA in MI hearts. Scale bars: 50 μm. f Representative images of hiPSC-CM-GFP (green) expressing MYH6/7 (red) and GJA1 (gray) when they were injected in the absence or presence of hMSC-PA in MI hearts. Expression of GJA1 indicates integration of implanted hiPSC-CMs with host myocardium. Scale bars: 50 μm. The heart sections were also stained with DAPI (blue) for visualization of nuclei. CM: hiPSC-CMs. CM + PA: hiPSC-CMs + hMSC-loaded patch

Fig. 5

Secreted factors from hMSCs promote endothelial cell migration and vasculogenic potential. a Endothelial cell-migration assay. The HUVEC were incubated with 10% conditioned media harvested from hMSC cultures (hMSC-CM) or control media to evaluate the migration capability. Representative Images under an inverted microscope and quantification summary. The data are represented as mean ± SEM. *p < 0.05 compared with control group; n = 5 biologically independent samples per group. T test was used for statistical analyses. b Matrigel plug assay. The 10% hMSC-CM was treated to HUVEC on matrigel to examine the vasculogenic potential. Representative images of tubes formed on Matrigel and quantification summary. The data are represented as mean ± SEM. *p < 0.05 compared with hMSC basal media, ^#p < 0.05 compared with EGM2 group; n = 3 biologically independent samples per group. One-way ANOVA was used for statistical analyses. c Increased proliferation of HUVEC when they were co-cultured with hMSCs. Representative images of co-cultures of hMSCs and HUVEC and quantification summary. The data are represented as mean ± SEM. *p < 0.05 and **p < 0.01 compared with HUVEC only group; n = 3 biologically independent samples per group. T test was used for statistical analyses

Fig. 6

Direct cytoprotective effects of the hMSC-conditioned medium on hiPSC-CMs undergoing simulated ischemic injury. a, b Treatment with hMSC-conditioned media (hMSC-CM) increased cell survival after H₂O₂ (200 µM) treatment as determined by the (a) Annexin V and (b) lactate dehydrogenase (LDH) assay. The data are represented as mean ± SEM. *p < 0.05 compared with untreated control group, ^#p < 0.05 compared with H₂O₂-only treated control group; n = 3 biologically independent samples per group. One-way ANOVA was used for statistical analyses

Fig. 7

Characterization of conditioned media harvested from hMSC cultures by antibody arrays. Multiple array membrane incubated with hMSC-CM collected at day 7 and day 14 reveal the presence of several paracrine factors. hMSC-CM conditioned media harvested from hMSC cultures

Fig. 8

Schematic diagram of the underlying mechanism of dual treatment approach of hPSC-CMs and hMSC-patch