Abstract
Background: Most mesenchymal stem cells (MSCs) die shortly after transplantation into a myocardial infarcted area. Dead MSCs (dMSCs) are phagocytized by macrophages (pMΦ) in vivo and in vitro; however, the effects of pMΦ on cardiac stem cells (CSCs) remain unknown. Methods: MSCs, CSCs, and macrophages were obtained from bone marrow, hearts, and peritoneal cavity of mice, respectively. dMSCs were harvested after hypoxia for 24 h, and incubated with macrophages (2:1) for another 2 days with or without lipopolysaccharide (LPS, 50 ng/mL) and sorted by flow cytometry to obtain pMΦ. Viability and apoptosis of CSCs were respectively evaluated with the cell counting kit-8 (CCk-8) assay and Annexin V-PE/7-AAD staining at 0, 6, 12, and 24 h of culture with supernatant fluids from macrophages (MΦ), LPS-stimulated macrophages (LPS-pMΦ), pMΦ, and MSCs. GATA-4 and c-TnI expression was measured by flow cytometry on the seventh day. Expression of inflammation and growth factors was assessed by real-time polymerase chain reaction (RT-PCR) in MΦ, LPS-pMΦ, and pMΦ cells. Results: pMΦ expressed higher levels of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β)and lower levels of tumor necrosis factor-α(TNF-α)and IL-6 than LPS-pMΦ, higher levels of growth factors and of GATA-4 and c-TnI at the 7th day, which were similar to those in MSCs. CSCs cultured with supernatant fluids of pMΦ exhibited higher proliferative, anti-hypoxic, and differentiation activities. Conclusion: The supernatant fluids of macrophages that had phagocytized dead MSCs encouraged changes in phenotype and growth factor expression, enhanced proliferation, differentiation, and anti-hypoxic activity of CSCs, which is relevant to understanding the persistent therapeutic effect of MSCs after their massive demise upon transplantation in myocardial infarction. Furthermore, some miRNAs or proteins which were extracted from the supernatant fluids may give us a new insight into the treatment of myocardial infarction in the future.
Highlights
We demonstrated that mesenchymal stem cells (MSCs) transplanted into a porcine myocardial ischemia/reperfusion model were engulfed by cardiac macrophages, which had a beneficial effect on cardiac function [10]
We found that macrophages stimulated by LPS exhibited enhanced phagocytosis of Dead MSCs (dMSCs) [11]
We co-cultured with or without LPS (50 ng/mL)-stimulated macrophages labeled with PE-anti-F4/80 and dMSC marked with the FITC-green fluorescent protein (GFP) for 48 h, at a cell ratio of 1:2. dMSCs were engulfed by LPS-stimulated macrophages, as was obviously visible under fluorescence microscopy (Figure 1B)
Summary
Cardiovascular disease, especially myocardial infarction, remains the leading cause of mortality and morbidity around the world [1] and the focus of intense clinical and basic research—in particular on transplantation of stem and progenitor cells to rescue the injured myocardium [2,3].Bone marrow-derived mesenchymal stem cells (MSCs), a type of multipotent stem cell that can differentiate into various cell types, such as osteoblasts, muscle cells, and cardiomyocytes [4,5], have unique immunological characteristics that minimize immune rejection [5,6] and render them most suitable for stem cell therapy, as evidenced by basic science and clinical studies on their benefits for myocardial healing [7,8]. Our in vitro study showed that supernatant fluids obtained from macrophages that phagocytized dead MSCs (pMΦ) improved survival of hypoxic cardiomyocytes [11]. Dead MSCs (dMSCs) are phagocytized by macrophages (pMΦ) in vivo and in vitro; the effects of pMΦ on cardiac stem cells (CSCs) remain unknown. Viability and apoptosis of CSCs were respectively evaluated with the cell counting kit-8 (CCk-8) assay and Annexin V-PE/7-AAD staining at 0, 6, 12, and 24 h of culture with supernatant fluids from macrophages (MΦ), LPS-stimulated macrophages (LPS-pMΦ), pMΦ, and MSCs. GATA-4 and c-TnI expression was measured by flow cytometry on the seventh day. Conclusion: The supernatant fluids of macrophages that had phagocytized dead MSCs encouraged changes in phenotype and growth factor expression, enhanced proliferation, differentiation, and anti-hypoxic activity of CSCs, which is relevant to understanding the persistent therapeutic effect of MSCs after their massive demise upon transplantation in myocardial infarction. Some miRNAs or proteins which were extracted from the supernatant fluids may give us a new insight into the treatment of myocardial infarction in the future
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