Intramyocardial injection of adult bone marrow–derived stem cells has recently been proposed as a potential therapy to repair damaged myocardium after acute infarction1–4. The mechanisms underlying their therapeutic effect have not been clearly defined, with an intense debate over differentiation versus fusion2, 5. In this report, we provide evidence for a novel mechanism that involves the secretion of paracrine cytoprotective factor (s) from the stem cells. We have previously reported that rat bone marrow–derived mesenchymal stem cells (MSCs) overexpressing the survival gene Akt1 (Akt-MSCs) are superior to control MSCs transduced with green fluorescent protein (GFP-MSCs) for cell therapy of acute myocardial infarction. Specifically, intramyocardial injection of Akt-MSCs prevents ventricular remodeling and restores cardiac function when measured 2 weeks after infarction6. We have recent evidence7 that these effects occur in less than 72 h. Because this early recovery cannot be explained by the occurrence of meaningful regeneration resulting from donor cell cardiomyogenic differentiation, we postulated that it is achieved through protection of the ischemic myocardium by paracrine mediator (s) released in situ by the MSCs.

To test our hypothesis, we first assessed the effects of conditioned medium from cultured MSCs in vitro on adult rat ventricular cardiomyocytes (ARVCs) subjected to hypoxia. Serum-free conditioned medium (α-minimum essential medium, α-MEM) was collected from MSCs after 12 h of exposure either to normoxia or hypoxia. Hypoxic conditions were created by incubating the cells at 37 C in an airtight Plexiglas chamber with an atmosphere of 5% CO2 and 95% N2 and a controlled oxygen level of 0.5%. Initially, the ARVC standard growth medium was replaced with α-MEM control medium, normoxicconditioned medium or hypoxic-conditioned