Embryonic stem cells attenuate myocardial dysfunction and inflammation after surgical global ischemia via paracrine actions

Abstract

Stem cell treatment may positively influence recovery and inflammation after shock by multiple mechanisms, including the paracrine release of protective growth factors. Embryonic stem cells (ESCs) are understudied and may have greater protective power than adult bone marrow stem cells (BMSCs). We hypothesized that ESC paracrine protective mechanisms in the heart (decreased injury by enhanced growth factor-mediated reduction of proinflammatory cytokines) would be superior to the paracrine protective mechanisms of the adult stem cell population in a model of surgically induced global ischemia. Adult Sprague-Dawley rat hearts were isolated and perfused via Langendorff model. Hearts were subjected to 25 min of warm global ischemia and 40 min of reperfusion and were randomly assigned into one of four groups: 1) vehicle treated; 2) BMSC or ESC preischemic treatment; 3) BMSC or ESC postischemic treatment; and 4) BMSC- or ESC-conditioned media treatment. Myocardial function was recorded, and hearts were analyzed for expression of tissue cytokines and growth factors (ELISA). Additionally, ESCs and BMSCs in culture were assessed for growth factor production (ELISA). ESC-treated hearts demonstrated significantly greater postischemic recovery of function (left ventricular developed pressure, end-diastolic pressure, and maximal positive and negative values of the first derivative of pressure) than BMSC-treated hearts or controls at end reperfusion. ESC-conditioned media (without cells) also conferred cardioprotection at end reperfusion. ESC-infused hearts demonstrated increased VEGF and IL-10 production compared with BMSC hearts. ESC hearts also exhibited decreased proinflammatory cytokine expression compared with MSC hearts. Moreover, ESCs in cell culture demonstrated greater pluripotency than MSCs. ESC paracrine protective mechanisms in surgical ischemia are superior to those of adult stem cells.

Fig. 1.

Comparison of myocardial function [left ventricular developed pressure (LVDP), end-diastolic pressure (EDP), and maximal positive (+dP/dt) and negative (−dP/dt) values of the first derivative of pressure] at end reperfusion in hearts with and without intervention. A–D: LVDP, EDP, +dP/dt, and −dP/dt in hearts infused with stem cells before ischemia. Results are means ± SE; *P < 0.05 vs. controls, †P < 0.05 vs. corresponding mesenchymal stem cell (MSC) group. ESC, embryonic stem cell group. E–H: LVDP, EDP, +dP/dt, and −dP/dt in hearts infused with stem cells after ischemia. Results are means ± SE; *P < 0.05 vs. controls, †P < 0.05 vs. MSC. I–L: LVDP, EDP, +dP/dt, and −dP/dt in hearts infused with stem cells or conditioned media derived from stem cells. Results are means ± SE; *P < 0.05 vs. controls, †P < 0.05 vs. corresponding stem cell media group. Statistics: ordinary ANOVA with post hoc Bonferroni test.

Fig. 2.

Changes in myocardial function following ischemia-reperfusion (I/R) in hearts without intervention (controls, n = 8), hearts infused with MSCs before ischemia (n = 10), and hearts infused with ESCs before ischemia (n = 12). A: LVDP (% of equilibration), 2 × 10⁶ cells. B: EDP (mmHg), 2 × 10⁶ cells. C: +dP/dt maximum (% of equilibration), 2 × 10⁶ cells. D: −dP/dt maximum (% of equilibration), 2 × 10⁶ cells. E: LVDP (% of equilibration), 3 × 10⁶ cells. F: EDP (mmHg), 3 × 10⁶ cells. G: +dP/dt maximum (% of equilibration), 3 × 10⁶ cells. H: −dP/dt maximum (% of equilibration), 3 × 10⁶ cells. Results are means ± SE; *P < 0.05 vs. ESC (A–D) or MSC (E–H). Statistics: repeated-measures ANOVA with post hoc Bonferroni test.

Fig. 3.

Changes in myocardial function following I/R in hearts without intervention (controls), hearts infused with MSCs (2 × 10⁶; n = 3) after ischemia, and hearts infused with ESCs (3 × 10⁶ cells; n = 9) after ischemia. A: LVDP (% of equilibration). B: EDP (mmHg). C: +dP/dt maximum (% of equilibration). D: −dP/dt maximum (% of equilibration). Results are means ± SE. Statistics: repeated-measures ANOVA with post hoc Bonferroni test.

Fig. 4.

Changes in myocardial function following I/R in hearts without intervention (controls), hearts infused with ESCs before ischemia, hearts infused with ESC-conditioned media before ischemia (n = 3), hearts infused with MSCs before ischemia, and hearts infused with MSC media before ischemia (n = 3). A: ESC LVDP (% of equilibration). B: EDP (mmHg). C: ESC +dP/dt maximum (% of equilibration). D: ESC −dP/ dt maximum (% of equilibration). E: MSC LVDP (% of equilibration). F: MSC EDP (mmHg). G: MSC +dP/dt maximum (% of equilibration). H: MSC −dP/dt maximum (% of equilibration). Results are means ± SE; *P < 0.05 vs. ESC media. Statistics: repeated-measures ANOVA with post hoc Bonferroni test.

Fig. 5.

Representative stem cells viewed under Nikon TE2000U microscope at ×200 magnification. MSCs exhibit greater cell diameter and differing morphology than ESCs. A: unstimulated MSCs. B: unstimulated ESCs.

Fig. 6.

Stem cell activation after 24-h TNF-α (TNF; n = 4), endotoxin (LPS; n = 4), or hypoxic (1.0% O₂) (n = 4) exposure. A: expression of VEGF in ESCs. B: expression of VEGF in adult MSCs. C: expression of IL-10 in ESCs. D: expression of IL-10 in adult MSCs. Results are expressed as pg/ml, means ± SE; *P < 0.05 vs. control (Ctrl).

Fig. 7.

Myocardial expression of inflammatory cytokines in vehicle-treated control hearts, hearts infused with MSCs before ischemia, hearts infused with ESCs before ischemia, and hearts infused with ESC media before ischemia. A: TNF-α. B: IL-1β. C: IL-6. D: VEGF. E: IL-10. F: IGF-I. Results are expressed as pg/mg, means ± SE; *P < 0.05 vs. control, †P < 0.05 vs. ESC.