Introduction: A major limitation of cardiac stem-cell transplantation following myocardial infarction (MI) is poor retention of cells in the anoxic infarct microenvironement. Pathways related to paracrine signaling are being recognized as major contributors to improve cardiac function after stem-cell transplantation into the infarct area. Our study aims to: (1) characterize human cardiac outgrowth cells (hCOC) derived from patient biopsies and (2) to study the regulation of cytokines in hCOC subjected to anoxia.
Methods: hCOC were obtained by plating patient cardiac biopsy tissue in cardiac explant media (CEM). hCOC were collected and passaged 3 to 4 times and analyzed by fluorescence-activated cell sorting (FACS). Cells were placed in anoxia for 8, 16, 20, and 24 hours and cell viability assay was performed at each timepoint with calcein/ethidium homodimer uptake. Cytokine array analysis was performed on cells incubated in anoxia for 20 hours as this timepoint demonstrated the greatest difference in viability between anoxia and normoxia groups.
Results: FACS analysis demonstrated Sca-1 (90%), CD105 (71%), and ckit (27.6%) positivity, as well as CD90, CD34, CD45, and CD31 in lesser proportions. 20 hours of anoxia resulted in 1.7 fold increased cell viability (more living and less dead cells) compared to normoxia (P≤0.05). Cytokine array analysis of cells incubated in 20 hours of anoxia demonstrated increased expression of proliferative, angiogenic, and pro-differentiation cytokines, including VEGF, TGF-alpha, and angiogenin. Proinflammatory cytokines such as MCP1, CXCL-5, IL-17, and PTX3 were downregulated.
Conclusions: These findings demonstrate a new, previously unknown response of hCOC to anoxia, resulting in both increased viability and expression of cytokines associated with proliferation, differentiation, and angiogenesis and a reduction of inflammatory cytokines. The results suggest a possible reparative role of this population of cells within the human heart and the role anoxic preconditioning can play in priming cells prior to transplantation. Future studies will also explore the role of the secreted cytokines as exogenous agents that may potentially provide benefits in the setting of MI.