Dual Stem Cell Therapy Improves the Myocardial Recovery Post-Infarction through Reciprocal Modulation of Cell Functions

Abstract

Mesenchymal stromal cells (MSC) are promising candidates for regenerative therapy of the infarcted heart. However, poor cell retention within the transplantation site limits their potential. We hypothesized that MSC benefits could be enhanced through a dual-cell approach using jointly endothelial colony forming cells (ECFC) and MSC. To assess this, we comparatively evaluated the effects of the therapy with MSC and ECFC versus MSC-only in a mouse model of myocardial infarction. Heart function was assessed by echocardiography, and the molecular crosstalk between MSC and ECFC was evaluated in vitro through direct or indirect co-culture systems. We found that dual-cell therapy improved cardiac function in terms of ejection fraction and stroke volume. In vitro experiments showed that ECFC augmented MSC effector properties by increasing Connexin 43 and Integrin alpha-5 and the secretion of healing-associated molecules. Moreover, MSC prompted the organization of ECFC into vascular networks. This indicated a reciprocal modulation in the functionality of MSC and ECFC. In conclusion, the crosstalk between MSC and ECFC augments the therapeutic properties of MSC and enhances the angiogenic properties of ECFC. Our data consolidate the dual-cell therapy as a step forward for the development of effective treatments for patients affected by myocardial infarction.

Keywords: dual stem cell therapy; endothelial colony forming cells; mesenchymal stromal cells; myocardial infarction; proteomic profiling.

Figure 1

The effect of dual-cell therapy on infarcted myocardium. (A) Schematic representation of the experimental timeline; (B) ex vivo fluorescent imaging of a murine heart at 7 days after transplantation with CMFDA-labeled MSC and CMTPX -labeled ECFC at a cell ratio of 9:1 (MSC: ECFC). The white star indicates the ligature and the arrow shows the injection site and the direction of the infusion (towards the apex). Note the local retention of both MSC (green) and ECFC (red) at this time-point. (C) Representative echocardiography images obtained at 7 and 14 days after surgery in B-mode parasternal long axis; (D) ejection fraction and stroke volume determined at days 7 and 14 post-transplant. Data are mean ± SEM; (E) Western blot images showing the levels of CX43 and ITGA5 in the ventricular samples of infarcted mice with or without cell therapy at 14 days after surgery. The densitometry quantification relative to the no cells group is illustrated on the right side for both proteins. (Statistics: two-way and one-way ANOVA, followed by Tukey’s multiple comparison test; * p value < 0.05; ** p value < 0.01, ***p < 0.001).

Figure 2

Angiogenic effects of MSC and ECFC. (A) Phase-contrast microscopy images showing vascular networks of endothelial cells assembled on Matrigel in the presence of MSC- and ECFC-CM. The diagrams illustrate comparable effects of the MSC- and ECFC-CM in terms of the number of junctions, total tube length, and closed structures formed by endothelial cells in one representative experiment performed in duplicates; (B) proteomic screening of cytokines secreted by MSC and ECFC in vitro, as assessed by Human Angiogenesis Proteome Profiler array; (C) ELISA quantification of VEGF in MSC and ECFC secretomes; (D) tube-like structures formed by fluorescent ECFC when co-cultured with unlabeled MSC in different cell ratios.

Figure 3

Gene and protein expression modulation following MSC + ECFC co-culture. (A) Schematics of the experimental settings used for MSC + ECFC direct contact (upper image) and indirect co-culture (lower image). (B) Gene expression analysis of ITGA5 in individual cultures and MSC + ECFC direct co-culture, indicating the induction after co-culture. (C) Western blot showing protein levels of Integrin alpha-5 subunit after direct MSC–ECFC interaction. (D) Gene expression analysis of ITGA5 in MSC (left side) and ECFC (right side), in individual cultures (control) and after indirect-contact co-culture. (E) Western blot showing ITGA5 protein level in MSC and ECFC following indirect contact co-culture. Data are mean ± SD. (Statistics: one-way ANOVA followed by Tukey’s multiple comparison test and two-tailed, paired t test; * p value < 0.05; ** p value < 0.01).

Figure 4

Proteome array analysis of the secretome produced by MSC and ECFC co-cultured in direct contact. (A) Secretory profiles of MSC, ECFC, and MSC + ECFC co-cultures were identified by antibody-based Human XL Cytokine Array. Shown in red are proteins whose cumulative levels were induced following co-culture as compared to their expected levels; (B) heatmap representation of the highly secreted cytokines, illustrating the major secretion source and biological processes associated to the cytokines; (C) the relative levels of proteins in the co-culture supernatant, which were found to be induced by more than 35% after MSC-ECFC co-culture.