Darren R. Lebl

Embryonic Stem Cell Immunogenicity Increases Upon Differentiation After Transplantation Into Ischemic Myocardium

Background—We investigated whether differentiation of embryonic stem cells (ESCs) in ischemic myocardium enhances their immunogenicity, thereby increasing their chance for rejection. Methods and Results—In one series, 129/SvJ-derived mouse ESCs (ES-D3 line) were transplanted by direct myocardial injection (1×106 cells) into murine hearts of both allogeneic (BALB/c, n=20) and syngeneic (129/SvJ, n=12) recipients after left anterior artery ligation. Hearts were procured at 1, 2, 4, and 8 weeks after ESC transplantation and analyzed by immunohistochemistry to assess immune cell infiltration (CD3, CD4, CD8, B220, CD11c, Mac-1, and Gr-1) and ESC differentiation (hematoxylin and eosin). In a second series (allogeneic n=5, sham n=3), ESC transplantation was performed similarly; however after 2 weeks, left anterior descending artery-ligated and ESC-injected hearts were heterotopically transplanted into naive BALB/c recipients. After an additional 2 weeks, donor hearts were procured and analyzed by immunohistochemistry. In the first series, the size of all ESC grafts remained stable and there was no evidence of ESC differentiation 2 weeks after transplantation; however, after 4 weeks, both allogeneic and syngeneic ESC grafts showed the presence of teratoma. By 8 weeks, surviving ESCs could be detected in the syngeneic but not in the allogeneic group. Mild inflammatory cellular infiltrates were found in allogeneic recipients at 1 and 2 weeks after transplantation, progressing into vigorous infiltration at 4 and 8 weeks. The second series demonstrated similar vigorous infiltration of immune cells as early as 2 weeks after heterotopic transplantation. Conclusion—In vivo differentiated ESCs elicit an accelerated immune response as compared with undifferentiated ESCs. These data imply that clinical transplantation of allogeneic ESCs or ESC derivatives for treatment of cardiac failure might require immunosuppressive therapy.

Novel Injectable Bioartificial Tissue Facilitates Targeted, Less Invasive, Large-Scale Tissue Restoration on the Beating Heart After Myocardial Injury

Background—Implantation of bioartificial patches distorts myocardial geometry, and functional improvement of the recipient heart is usually attributed to reactive angiogenesis around the graft. With the liquid bioartificial tissue compound used in this study, we achieved targeted large-scale support of the infarcted left ventricular wall and improvement of heart function. Methods and Results—A liquid compound consisting of growth factor-free Matrigel and 106 green fluorescent protein (GFP)-positive mouse (129sv) embryonic stem cells (ESCs) was generated and injected into the area of ischemia after ligation of the left anterior descending artery in BALB/c mice (group I). Left anterior descending artery-ligated mice (group II) and mice with Matrigel (group III) or ESC treatment alone (group IV) were used as the control groups (n=5 in all groups). The hearts were harvested for histology 2 weeks later after echocardiographic assessment with a 15-MHz probe. The liquid injectable tissue solidified at body temperature and retained the geometry of the infarcted lateral wall. Immunofluorescence stains revealed voluminous GFP grafts. The quality of restoration (graft/infarct area ratio) was 45.5±10.8% in group I and 29.1±6.7% in group IV (P=0.034). ESCs expressed connexin 43 at intercellular contact sites. The mice treated with the compound had a superior heart function compared with the controls (P<0.0001 by ANOVA/Bonferroni test; group I: 27.1±5.4, group II:11.9±2.4, group III:16.2±2.8, group IV: 19.1±2.7). Conclusions—Injectable bioartificial tissue restores the heart’s geometry and function in a targeted and nondistorting fashion. This new method paves the way for novel interventional approaches to myocardial repair, using both stem cells and matrices.

Insulin‐Like Growth Factor Promotes Engraftment, Differentiation, and Functional Improvement after Transfer of Embryonic Stem Cells for Myocardial Restoration

Insulin‐like growth factor‐1 (IGF‐1) promotes myocyte proliferation and can reverse cardiac abnormalities when it is administered in the early fetal stage. Supplementation of a mouse embryonic stem cell (ESC) suspension with IGF‐1 might enhance cellular engraftment and host organ‐specific differentiation after injection in the area of acute myocardial injury. In the study reported here, we sought to enhance the restorative effect of ESCs in the injured heart by adding IGF‐1 to the injected cell population. Green fluorescent protein (GFP)–labeled sv129 ESCs (2.5 × 105) were injected into the ischemic area after left anterior descending (LAD) artery ligation in BalbC mice. Recombinant mouse IGF‐1 (25 ng) was added to the cell suspension prior to the injection (n = 5). Echocardiography was performed before organ harvest 2 weeks later. The degree of restoration (ratio of GFP+ to infarct area), expression of cardiac markers by GFP+ cells, inflammatory response, and tumorigenicity were evaluated. Mice with LAD ligation only (n = 5) and ESC transfer without IGF‐1 (n = 5) served as controls. ESCs formed viable grafts and improved cardiac function. Left ventricular wall thickness was higher in the IGF‐1 group (p = .025). There was a trend toward higher fractional shortening in the IGF‐treated group. Histological analysis demonstrated that IGF‐1 promoted expression of α‐sarcomeric actin (p = .015) and major histocompatibility complex class I (p = .01). IGF did not affect the cellular response to the donor cells or tumorigenicity. IGF‐1 promotes expression of cardiomyocyte phenotype in ESCs in vivo. It should be considered as an adjuvant to cell transfer for myocardial restoration.

Stimulation of Paracrine Pathways With Growth Factors Enhances Embryonic Stem Cell Engraftment and Host-Specific Differentiation in the Heart After Ischemic Myocardial Injury

Background—Growth factors play an essential role in organogenesis. We examine the potential of growth factors to enhance cell engraftment and differentiation and to promote functional improvement after transfer of undifferentiated embryonic stem cells into the injured heart. Methods and Results—Green fluorescent protein (GFP)–positive embryonic stem cells derived from 129sv mice were injected into the ischemic area after left anterior descending artery ligation in allogenic (BALB/c) mice. Fifty nanograms of recombinant mouse vascular endothelial growth factor, fibroblast growth factor (FGF), and transforming growth factor (TGF) was added to the cell suspension. Separate control groups were formed in which only the growth factors were given. Echocardiography was performed 2 weeks later to evaluate heart function (fractional shortening [FS]), end-diastolic diameter, and left ventricular wall thickness). Hearts were harvested for histology (connexin 43, α-sarcomeric actin, CD3, CD11c, major histocompatability complex class I, hematoxylin-eosin). Degree of restoration (GFP-positive graft/infarct area ratio), expression of cardiac markers, host response, and tumorigenicity were evaluated. Cell transfer resulted in improved cardiac function. TGF-β led to better restorative effect and a stronger expression of connexin 43, α-sarcomeric actin, and major histocompatability complex class I. TGF-β and FGF retained left ventricular diameter. FS was better in the TGF-β, FGF, and embryonic stem cells–only group compared with left anterior descending artery–ligated controls. Growth factors with cells (TGF-β, FGF) resulted in higher FS and smaller end-diastolic diameter than growth factors alone. Conclusions—Growth factors can promote in vivo organ-specific differentiation of early embryonic stem cells and improve myocardial function after cell transfer into an area of ischemic lesion. TGF-β should be considered as an adjuvant for myocardial restoration with the use of embryonic stem cells.

Adenoviral Human BCL-2 Transgene Expression Attenuates Early Donor Cell Death After Cardiomyoblast Transplantation Into Ischemic Rat Hearts

Background— Cell transplantation for myocardial repair is limited by early cell death. Gene therapy with human Bcl-2 (hBcl-2) has been shown to attenuate apoptosis in the experimental setting. Therefore, we studied the potential benefit of hBcl-2 transgene expression on the survival of cardiomyoblast grafts in ischemic rat hearts. Methods and Results— H9c2 rat cardiomyoblasts were genetically modified to express both firefly luciferase and green fluorescent protein (mH9c2). The cells were then transduced with adenovirus carrying hBcl-2 (AdCMVhBcl-2/mH9c2). Lewis rats underwent ligation of the left anterior descending artery (LAD) to induce a sizable left ventricular (LV) infarct. Hearts were explanted and the infarcted region was restored using collagen matrix (CM) seeded with 1×106 mH9c2 cells (n=9) or AdCMVhBcl-2/mH9c2 cells (n=9). Control animals received CM alone (n=6) or no infarct (n=6). Restored hearts were transplanted into the abdomen of syngeneic recipients in a “working heart” model. Cell survival was evaluated using optical bioluminescence imaging on days 1, 5, 8, 14, and 28 after surgery. The left heart function was assessed 4 weeks postoperatively using echocardiography and magnetic resonance imaging. During 4 weeks after surgery, the optical imaging signal for the AdCMVhBCL2/mH9c2 group was significantly (P<0.05) higher than that of the mH9c2-control group. Both grafts led to better fractional shortening (AdCMVhBcl-2/mH9c2: 0.21±0.03; mH9c2: 0.21±0.04; control: 0.15±0.03; P=0.04) and ejection fraction (AdCMVhBcl-2/mH9c2: 47.0±6.2; mH9c2: 48.7±6.1; control: 34.3±6.0; P=0.02) compared with controls. Importantly, no malignant cells were found in postmortem histology. Conclusion— Transduction of mH9c2 cardiomyoblasts with AdCMVhBcl-2 increased graft survival in ischemic rat myocardium without causing malignancies. Both AdCMVhBcl-2/mH9c2 and mH9c2 grafts improved LV function.