Jorg de Bruin

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.

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.

Dual in vivo magnetic resonance evaluation of magnetically labeled mouse embryonic stem cells and cardiac function at 1.5 t

Cell therapy has demonstrated the potential to restore injured myocardium. A reliable in vivo imaging method to localize transplanted cells and monitor their restorative effects will enable a systematic investigation of this therapeutic modality. The dual MRI capability of imaging both magnetically labeled mouse embryonic stem cells (mESC) and their restorative effects on cardiac function in a murine model of acute myocardial infarction is demonstrated. Serial in vivo MR detection of transplanted mESC and monitoring of the mESC‐treated myocardium was conducted over a 4‐week period using a 1.5 T clinical scanner. During the 4‐week duration, the mESC‐treated myocardium demonstrated sustained improvement of the left ventricular (LV) ejection fraction and conservation of LV mass. Furthermore, no significant difference of their restorative effects on the cardiac function was created by the magnetic labeling of mESC. Thus, in vivo MRI enables simultaneous detection of transplanted mESC and their therapeutic effect on the injured myocardium. Magn Reson Med 2006.

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.

In Vivo Visualization of Cardiac Allograft Rejection and Trafficking Passenger Leukocytes Using Bioluminescence Imaging

Background—We investigated the feasibility of bioluminescence imaging (BLI) for the in vivo assessment of cardiac allograft viability and visualization of passenger leukocytes during the course of acute rejection. Methods and Results—Hearts of FVB (H-2q) luciferase-green fluorescent protein transgenic mice (β-actin promoter) or FVB luciferase transgenic mice (CD5 promoter) were heterotopically transplanted into either BALB/c (H-2d) or FVB recipients. Light intensity emitting from the recipient animals was measured daily by in vivo BLI until 12 days after transplantation. Graft beating score (0 to 4) was assessed by daily abdominal palpation until 12 days after transplantation. Inflammatory cell infiltration (CD45 stain) and structural changes of green fluorescent protein-positive cardiomyocytes were followed by immunohistochemistry. All cardiac allografts were acutely rejected by 12 days after transplantation. The intensity of light emitting from cardiac allografts declined 4 days after transplantation and correlated with graft beating scores (R2=0.91, P=0.02). Immunohistochemistry confirmed these results by showing an increase of CD45+ inflammatory cell infiltration and destruction of green fluorescent protein-positive cardiomyocytes in the cardiac allografts during acute rejection. In vivo BLI visualized migration and proliferation of CD5+ passenger leukocytes in both syngeneic and allogeneic recipients. In the allograft recipients, light signal from CD5+ passenger leukocytes peaked at 6 hours and diminished by 12 hours, whereas in the syngeneic recipients, the signal remained high until 10 days after transplantation. Conclusions—BLI is a useful modality for the quantitative assessment of in vivo cardiac graft viability and tracking of passenger leukocytes in vivo during the course of acute rejection.

Postoperative Complications Decrease Long-Term Survival After Thoracic Aneurysm Repair Despite Apparently Successful “Rescue” From Early Mortality

Elective repair of thoracic aortic aneurysms is a prophylactic procedure performed when the risk of aneurysm rupture is judged to outweigh perioperative risk. It has been demonstrated that mortality because of any cause in the 5 years after repair in frail surgical candidates is high, despite the fact that perioperative mortality is relatively favorable and aortic-related death is reduced.1,2 Patient selection, therefore, is vital if survival gains are to be achieved through elective repair of thoracic aneurysms. Preoperative factors are known to affect life expectancy after surgery, but the effect of perioperative complications on subsequent survival has not been reported. Patients undergoing elective thoracic aneurysm repair for thoracic aortic aneurysm from the updated MOTHER (Medtronic Thoracic Endovascular Registry) were studied, which comprises 7 clinical trials and 1 institutional series.3 Subjects with aneurysmal chronic aortic dissection were excluded. Perioperative complications were classified as neurological, cardiac, renal, respiratory, or access vessel-related. For all clinical trials, adverse events were reported via the trial data-collection process and were adjudicated by a review panel according to trial protocol. For the St George’s Vascular Institute cohort, all adverse events were reviewed …