Abstract 350: Early Cell-Cell Coupling Contributes to Loss of Transplanted Stem Cell Retention and Efficacy

Abstract

Bone marrow-derived mesenchymal stem cell (BM-MSC) replacement therapy is beneficial to the heart following ischemia but a significant loss of these cells within hours of administration could diminish their effect. We hypothesized that early coupling between BM-MSC and ischemic cardiomyocytes through gap junctions (GJ) may play a detrimental role in stem cell survival and retention in the acute phase of cell therapy. We seeded HL-1 cardiomyocytes in either normoxic (Nx) or ischemic (Isc) conditions for four hours. Subsequently, BM-MSC were seeded on the HL-1 monolayer and the co-cultures were returned to incubation either in their previous conditions (Nx, Isc) or switched from Isc to Nx condition (ischemia-reperfusion; Isc/Rep) for an additional two hours. Co-cultures were labeled with Annexin V, Sytox Red, and Sca-1 (BM-MSC), and subjected to flow cytometry. Ischemia induced a greater proportion of dead BM-MSC over the two-hour co-culture compared to the Nx group. Isc/Rep resulted in significantly higher early apoptotic but fewer dead BM-MSC. The presence of the GJ inhibitor carbenoxolone (CBX; 100 µM) in the co-culture reduced the number of dead and apoptotic cells in Isc and Isc/Rep groups by 3-5 fold (p<0.05). To determine the effect of GJ inhibition in vivo, we induced ischemia in mice by 90-minute LAD ligation followed by reperfusion for 24 hours. BM-MSC, CBX-treated BM-MSC, or CBX alone were injected at the end of the 90 min Isc period. Twenty-four hours after cell injection, left ventricular diastolic and systolic function was assessed by pressure-volume loop analysis. Isc/Rep caused impaired cardiac function which was attenuated by BM-MSC injection. CBX-treated BM-MSC further enhanced the cardiac function (MSC vs. MSC+CBX: Ees 7.3 ± 1.66 vs. 15.0 ± 5.81; Emax 18.8 ± 6.50 vs. 27.5 ± 9.33; PRSW 49.5 ± 9.89 vs. 99.4 ± 17.4; mean ± SD; p≤0.05; n = 6) while CBX alone did not. While long term integration of stem cells within the myocardium relies on functional GJ, early GJ communication may represent a novel paradigm whereby ischemic cardiomyocytes cause a “bystander effect” in newly transplanted stem cells and thus impair retention and functional benefits.