Introduction:
Bone marrow-derived mesenchymal stem cells (MSCs) and cardiac progenitor cells (CPCs) have been used successfully as a cell-based approach for cardiac regeneration after myocardial infarction. While MSCs and CPCs are effective, in part due to differentiation, paracrine effect, and integration into the heart, the mechanism underlying these effects remains controversial.
Objective:
We hypothesized that functional connexin 43 (Cx43) gap junctions are crucial for MSC and CPCs interaction and integration into cardiac tissue and cardiac stem cell niche formation.
Methods and Results:
Human MSCs were co-cultured with neonatal rat ventricular cardiomyocytes (NRVMs). The ability of MSCs to form gap junctions was modulated using lentiviral constructs to either knockdown (Cx43KD) or overexpress (Cx43OE) Cx43. Co-culture of Cx43OE or control MSCs with NRVMs led to the formation of beating, three-dimensional tubes whereas Cx43KD MSCs failed to form tubes (n=5, p<0.05). Furthermore, we replicated the cardiac stem cell niche by combining human CPCs and MSCs in an in vitro model (the same lentiviral constructs were used). As a result, Cx43 expressing MSCs and CPCs formed organotypic three-dimensional structures similar to human MSCs and NRVMs, whereas the lack of Cx43 significantly affected the structure formation (n=6, p<0.05). Cx43KD significantly increased proliferation (n=5, p<0.05) and changed culture phenotype (n=4, p<0.05) of MSCs or CPCs cultured separately. The angiogenic tube length, assessed by a Matrigel assay in vitro, was significantly reduced in MSCs and HUVEC Cx43 KD groups compared to control (n=5, p<0.05). The change/onset of endothelial (KDR, PECAM-1, VE-cadherin), and cardiomyocyte (Nkx2.5, Gata4, Troponin I, Na+ channel, K+ channel, Ca2+ channel, Na+-Ca2+ exchanger) gene expression was investigated in co-culture of MSCs and CPCs. Only voltage ion channel gene expression was upregulated in control and Cx43OE groups by co-culture (n=5, p<0.05), but not affected in Cx43KD group.
Conclusion:
These findings reveal that cell−cell contact mediated by Cx43 gap junctions enables MSCs to interact with CPCs, integrate and reconstitute cardiac stem cell niches.
Cardiac Stem Cell Niche, MMP9, and Culture and Differentiation of Embryonic Stem Cells