Cardiovascular regenerative therapies are pursued on both basic and translational levels. Although efficacy and value of cell therapy for myocardial regeneration can be debated, there is a consensus that profound deficits in mechanistic understanding limit advances, optimization, and implementation. In collaboration with the TACTICS (Transnational Alliance for Regenerative Therapies in Cardiovascular Syndromes), this review overviews several pivotal aspects of biological processes impinging on cardiac maintenance, repair, and regeneration. The goal of summarizing current mechanistic understanding is to prompt innovative directions for fundamental studies delineating cellular reparative and regenerative processes. Empowering myocardial regenerative interventions, whether dependent on endogenous processes or exogenously delivered repair agents, ultimately depends on mastering mechanisms and novel strategies that take advantage of rather than being limited by inherent myocardial biology. (Circ Res.

Rationale The relative actions and synergism between distinct myocardial-derived stem cell populations remains obscure. Ongoing debates regarding optimal cell population(s) for treatment of heart failure prompted implementation of a protocol for isolation of multiple stem cell populations from a single myocardial tissue sample to develop new insights for achieving myocardial regeneration. Objective Establish a robust cardiac stem cell isolation and culture protocol to consistently generate three distinct stem cell populations from a single human heart biopsy. Methods and Results Isolation of three endogenous cardiac stem cell populations was performed from human heart samples routinely discarded during implantation of a left ventricular assist device (LVAD). Tissue explants were mechanically minced into 1 mm3 pieces to minimize time exposure to collagenase digestion and preserve cell viability. Centrifugation removes large cardiomyocytes (CMs) and tissue debris producing a single cell suspension that is sorted using magnetic-activated cell sorting (MACS) technology. Initial sorting is based upon c-Kit expression that enriches for two c-kit+ cell populations yielding a mixture of cardiac progenitor cells (CPCs) and endothelial progenitor cells (EPCs). Flow through c-Kit− mesenchymal stem cells (MSCs) are positively selected by surface expression of markers CD90 and CD105. After one week of culture the c-Kit+ population is further enriched by selection for a CD133+ EPC population. Persistence of respective cell surface markers in vitro is confirmed both by flow cytometry and immunocytochemistry. Conclusions Three distinct cardiac cell populations with individualized phenotypic properties consistent with CPCs, EPCs and MSCs can be successfully concurrently isolated and expanded from a single tissue sample derived from human heart failure patients.