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Myocardial restoration with embryonic stem cell bioartificial tissue transplantation
The Journal of Heart and Lung Transplantation Volume 23, Number 2S
Purpose: Embryonic stem cells (ESC) have been regarded immune priviledged at an early differentiation state. In the present study we seek to prove a significant allogeneic host response and evaluate the expression pattern of host cytokines following ESC transfer. Procedures: 129sv(H-2b)- derived, GFP⫹, undifferentiated ESC were injected into injured mouse myocardium of Balb C (H-2d) recipients (250,000 cells/injection). Spleenes and sera were harvested at various time points, (n ⫽ 5 per Group), (Group 1: 1 week post cell injection, Group 2: 2 weeks, Group 3: 4 weeks). Flow cytometry was used to assess the humoral antibody response to donor ESC. ELISPOT was utilized to identify and quantify CD-4 T-cell cytokine expression for IFN-␥, Il-2 (Th-1 pathway) and Il-4, Il-5 (Th-2 pathway). Recipient spleenocyte stimulation was compared to stimulation with syngeneic cells, Concavalin A (positive controls) and ESC sonicates (cells fragmented by ultrasound). Results: There was significant humoral host immune response to the donor ESC and it increased over time (percentage of cells responding to antigen by Flow Cytometry: 2.58 ⫾ 1.74 at 1w, 14.57 ⫾ 3.85 at 2w, 48.2 ⫾ 9.49 at 4w). ELISPOT demonstrated strong donor specific IFN-␥ expression as early as 1 week following cell implantation and increased over time. IFN-␥ reached significantly higher counts in the allogeneic stimulator group compared to the syngeneic stimulator group (438 ⫾ 20 vs. 216 ⫾ 16 at 1w, 469 ⫾ 34 vs. 196 ⫾ 19 at 2 weeks and 730 ⫾ 95 vs. 175 ⫾ 17 at 4 weeks post implantation). The Il-2 production was also hyperbolic and parallel to IFN-␥ but at lower counts. Il-4 production was low at 2 weeks and peaked significantly at 4 weeks. Conclusion: Administration of ESC triggers a significant donor specific Th1 T cell- and humoral response. These results refute the theory of non-immunogenicity of ESC in vivo. 353 MYOCARDIAL RESTORATION WITH EMBRYONIC STEM CELL BIOARTIFICIAL TISSUE TRANSPLANTATION T. Kofidis,1 J.L. de Bruin,1 G. Hoyt,1 T. Yamane,2 R.-J. Swijnenburg,1 D.R. Lebl,1 M. Tanaka,1 I.L. Weissman,2 R.C. Robbins,1 1Cardiothoracic Surgery/Falk Research Center, Stanford University Medical School, Stanford, CA; 2Department of Immunology, Stanford University Medical School, Stanford, CA Purpose: The optimal cell-matrix combination for robust and sustained myocardial restoration has not been identified. We use stem cells (ESC) as the substrate of bioartificial myocardial tissue and evaluate the potential for engraftment and the facilitation of functional recovery. Procedures: Collagen type I was populated with early GFP-positive mouse embryonic stem cells. The mixture was left to consolidate (48 hrs). An intramural left ventricular pouch was fashioned after ligation of the LAD in a rat heterotopic heart transplant model. The compound (0.125 ml) was implanted within the pouch. Echocardiography was performed prior to animal sacrifice to assess fractional shortening (FS) in groupI: infarcted rats which received cell-matrix transfer (n ⫽ 5), group II: matrix recipients alone (n ⫽ 5), group III: infarcted and transplanted controls (n ⫽ 5) and group IV: rats received an uninjured and untreated heart. Hearts were stained for GFP and cardiac markers (connexin 43, a-sarcomeric actin). Tissue remodeling was evaluated by trichrome and H&E stains. Results: GFP-positive ESC displayed remarkable robustness, building stable intramyocardial grafts and retaining myocardial structure. The bioartificial structure incorporated in the surrounding area without distorting myocardial geometry, thereby preventing ventricular wall thinning (anterior wall thickness in group I:1.4 ⫾ 0.1, group II: 1.0 ⫾ 0.1, group III: 0.9 ⫾ 0.2 and group IV: 1.3 ⫾ 0.2). The inoculated cells expressed connexin 43 and a-sarcomeric actin in vivo. Fraction shortening and contractility was better in ESC-treated animals (group I: 21.5 ⫾ 3.5%, group II: 12.4 ⫾ 2.8%, group III: 8.2 ⫾ 2.9% and group
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IV: 23.2 ⫾ 4.2%). Cellular atypia and nuclear polymorphism were only minor but still present. Conclusion: Embryonic stem cells are an efficient alternative substrate for myocardial tissue engineering, prevent myocardial wall thinning and improve contractility following implantation into injured myocardium in a three-dimensional matrix. 354 OVEREXPRESSION OF HUMAN Bcl-2 ATTENUATES GRAFT CORONARY ARTERY DISEASE BY MODULATING LOCAL Th1 IMMUNE RESPONSE BUT NOT HOST IMMUNE RESPONSE IN MURINE CARDIAC ALLOGRAFTS M. Tanaka,1 R.D. Terry,1 S. Nakae,2 D.T. Cooke,1 T. Kofidis,1 R.C. Robbins,1 1Department of Cardiothoracic Surgery; 2Department of Pathology, Stanford University, Stanford, CA The allo-immune response is key to development of graft coronary artery disease (GCAD). We hypothesized that Bcl-2 overexpression in donor hearts would reduce GCAD by modulating this response. Method: Donor hearts of FVB (H-2q) transgenic mice overexpressing human Bcl-2 (Tg) and of FVB wild-type (WT) mice were transplanted into C57BL/6 (H-2b) mice. In one series (n ⫽ 6, Tg and WT groups), acute rejection was assessed at 8 day. In a chronic rejection series (n ⫽ 8, Tg and WT groups given i.p. cyclosporine A 20 mg/kg/day), GCAD indices were determined at 30 day. We examined: intragraft T cell subsets (CD4⫹, CD8⫹), B cells (B220⫹), macrophages (Mac-1⫹), dendritic cells (CD11c⫹) (immunostaining); IFN-␥, MCP-1, IP-10, Mig, Fas ligand, ICAM-1, VCAM-1 levels (ELISA); caspase-3 activity (ELISA); and apoptosis (TUNEL) in both series and IL-4 and -10 (ELISA) in the first. In a third series (n ⫽ 3, Tg and WT groups), host spleen cell response was assessed by flow cytometry and mixed lymphocyte reaction at 1, 3, 5, 7 day. Results: At 8 day, infiltration of CD8⫹ and CD11c⫹ cells was significantly decreased in the Tg group vs WT group; CD4⫹, Mac-1⫹, B220⫹ cells did not differ. All immune cell counts were decreased in the Tg group at 30 day. IFN-␥, MCP-1, IP-10, Mig, Fas ligand, ICAM-1, VCAM-1 levels; caspase-3 activity; apoptosis were significantly reduced in the Tg group at 8 and 30 day; there was no difference in IL-4 or -10 at 8 day. Total cell number, CD4⫹, CD8⫹ T cell count, proliferation profile, TNF-␣ or IFN-␥ production did not differ. Luminal narrowing (Tg vs WT: 17 ⫾ 11% v 46 ⫾ 12%, p ⬍ 0.001), intima/media ratio (Tg v WT: 0.20 ⫾ 0.12 v 0.60 ⫾ 0.15, p ⬍ 0.001), % diseased vessels (Tg v WT: 42 ⫾ 23 v 98 ⫾ 7%, p ⬍ 0.001), and apoptosis (p ⬍ 0.001) differed at 30 day. Conclusions: Bcl-2 overexpression in cardiac allografts reduced Th1 but not Th2 immune response in the acute and chronic phases of graft rejection without affecting host spleen cell response. This reduction correlated with a decrease in GCAD. 355 BOTH AFTER HEART AND BONE MARROW TRANSPLANTATION THE HEART HARVEST BONE MARROW DERIVED CARDIOMYOCYTES R.A. De Weger,1 I. Verbrugge,1 L.F. Verdonck,3 N. De Jonge,2 M. Van Oosterhout,1 D.F. Van Wichen,1 Y. De Souza,4 F.G. Gmelig Meyling,4 1Pathology, UMC-U, Utrecht, Utrecht, Netherlands; 2 Heart and Lung Institute, UMC-U, Utrecht, Utrecht, Netherlands; 3 Hematology, UMC-U, Utrecht, Utrecht, Netherlands; 4 Immunology, UMC-U, Utrecht, Utrecht, Netherlands Introduction: Several studies report the generation of cardiomyocytes from bone marrow derived stem cells. In this study we analyzed the presence of cardiomyocytes of recipient bone marrow origin after heart transplantation (HTx) or of donor origin after Bone Marrow Transplantation (BMT).
Purpose: Embryonic stem cells (ESC) have been regarded immune priviledged at an early differentiation state. In the present study we seek to prove a significant allogeneic host response and evaluate the expression pattern of host cytokines following ESC transfer. Procedures: 129sv(H-2b)- derived, GFP⫹, undifferentiated ESC were injected into injured mouse myocardium of Balb C (H-2d) recipients (250,000 cells/injection). Spleenes and sera were harvested at various time points, (n ⫽ 5 per Group), (Group 1: 1 week post cell injection, Group 2: 2 weeks, Group 3: 4 weeks). Flow cytometry was used to assess the humoral antibody response to donor ESC. ELISPOT was utilized to identify and quantify CD-4 T-cell cytokine expression for IFN-␥, Il-2 (Th-1 pathway) and Il-4, Il-5 (Th-2 pathway). Recipient spleenocyte stimulation was compared to stimulation with syngeneic cells, Concavalin A (positive controls) and ESC sonicates (cells fragmented by ultrasound). Results: There was significant humoral host immune response to the donor ESC and it increased over time (percentage of cells responding to antigen by Flow Cytometry: 2.58 ⫾ 1.74 at 1w, 14.57 ⫾ 3.85 at 2w, 48.2 ⫾ 9.49 at 4w). ELISPOT demonstrated strong donor specific IFN-␥ expression as early as 1 week following cell implantation and increased over time. IFN-␥ reached significantly higher counts in the allogeneic stimulator group compared to the syngeneic stimulator group (438 ⫾ 20 vs. 216 ⫾ 16 at 1w, 469 ⫾ 34 vs. 196 ⫾ 19 at 2 weeks and 730 ⫾ 95 vs. 175 ⫾ 17 at 4 weeks post implantation). The Il-2 production was also hyperbolic and parallel to IFN-␥ but at lower counts. Il-4 production was low at 2 weeks and peaked significantly at 4 weeks. Conclusion: Administration of ESC triggers a significant donor specific Th1 T cell- and humoral response. These results refute the theory of non-immunogenicity of ESC in vivo. 353 MYOCARDIAL RESTORATION WITH EMBRYONIC STEM CELL BIOARTIFICIAL TISSUE TRANSPLANTATION T. Kofidis,1 J.L. de Bruin,1 G. Hoyt,1 T. Yamane,2 R.-J. Swijnenburg,1 D.R. Lebl,1 M. Tanaka,1 I.L. Weissman,2 R.C. Robbins,1 1Cardiothoracic Surgery/Falk Research Center, Stanford University Medical School, Stanford, CA; 2Department of Immunology, Stanford University Medical School, Stanford, CA Purpose: The optimal cell-matrix combination for robust and sustained myocardial restoration has not been identified. We use stem cells (ESC) as the substrate of bioartificial myocardial tissue and evaluate the potential for engraftment and the facilitation of functional recovery. Procedures: Collagen type I was populated with early GFP-positive mouse embryonic stem cells. The mixture was left to consolidate (48 hrs). An intramural left ventricular pouch was fashioned after ligation of the LAD in a rat heterotopic heart transplant model. The compound (0.125 ml) was implanted within the pouch. Echocardiography was performed prior to animal sacrifice to assess fractional shortening (FS) in groupI: infarcted rats which received cell-matrix transfer (n ⫽ 5), group II: matrix recipients alone (n ⫽ 5), group III: infarcted and transplanted controls (n ⫽ 5) and group IV: rats received an uninjured and untreated heart. Hearts were stained for GFP and cardiac markers (connexin 43, a-sarcomeric actin). Tissue remodeling was evaluated by trichrome and H&E stains. Results: GFP-positive ESC displayed remarkable robustness, building stable intramyocardial grafts and retaining myocardial structure. The bioartificial structure incorporated in the surrounding area without distorting myocardial geometry, thereby preventing ventricular wall thinning (anterior wall thickness in group I:1.4 ⫾ 0.1, group II: 1.0 ⫾ 0.1, group III: 0.9 ⫾ 0.2 and group IV: 1.3 ⫾ 0.2). The inoculated cells expressed connexin 43 and a-sarcomeric actin in vivo. Fraction shortening and contractility was better in ESC-treated animals (group I: 21.5 ⫾ 3.5%, group II: 12.4 ⫾ 2.8%, group III: 8.2 ⫾ 2.9% and group
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IV: 23.2 ⫾ 4.2%). Cellular atypia and nuclear polymorphism were only minor but still present. Conclusion: Embryonic stem cells are an efficient alternative substrate for myocardial tissue engineering, prevent myocardial wall thinning and improve contractility following implantation into injured myocardium in a three-dimensional matrix. 354 OVEREXPRESSION OF HUMAN Bcl-2 ATTENUATES GRAFT CORONARY ARTERY DISEASE BY MODULATING LOCAL Th1 IMMUNE RESPONSE BUT NOT HOST IMMUNE RESPONSE IN MURINE CARDIAC ALLOGRAFTS M. Tanaka,1 R.D. Terry,1 S. Nakae,2 D.T. Cooke,1 T. Kofidis,1 R.C. Robbins,1 1Department of Cardiothoracic Surgery; 2Department of Pathology, Stanford University, Stanford, CA The allo-immune response is key to development of graft coronary artery disease (GCAD). We hypothesized that Bcl-2 overexpression in donor hearts would reduce GCAD by modulating this response. Method: Donor hearts of FVB (H-2q) transgenic mice overexpressing human Bcl-2 (Tg) and of FVB wild-type (WT) mice were transplanted into C57BL/6 (H-2b) mice. In one series (n ⫽ 6, Tg and WT groups), acute rejection was assessed at 8 day. In a chronic rejection series (n ⫽ 8, Tg and WT groups given i.p. cyclosporine A 20 mg/kg/day), GCAD indices were determined at 30 day. We examined: intragraft T cell subsets (CD4⫹, CD8⫹), B cells (B220⫹), macrophages (Mac-1⫹), dendritic cells (CD11c⫹) (immunostaining); IFN-␥, MCP-1, IP-10, Mig, Fas ligand, ICAM-1, VCAM-1 levels (ELISA); caspase-3 activity (ELISA); and apoptosis (TUNEL) in both series and IL-4 and -10 (ELISA) in the first. In a third series (n ⫽ 3, Tg and WT groups), host spleen cell response was assessed by flow cytometry and mixed lymphocyte reaction at 1, 3, 5, 7 day. Results: At 8 day, infiltration of CD8⫹ and CD11c⫹ cells was significantly decreased in the Tg group vs WT group; CD4⫹, Mac-1⫹, B220⫹ cells did not differ. All immune cell counts were decreased in the Tg group at 30 day. IFN-␥, MCP-1, IP-10, Mig, Fas ligand, ICAM-1, VCAM-1 levels; caspase-3 activity; apoptosis were significantly reduced in the Tg group at 8 and 30 day; there was no difference in IL-4 or -10 at 8 day. Total cell number, CD4⫹, CD8⫹ T cell count, proliferation profile, TNF-␣ or IFN-␥ production did not differ. Luminal narrowing (Tg vs WT: 17 ⫾ 11% v 46 ⫾ 12%, p ⬍ 0.001), intima/media ratio (Tg v WT: 0.20 ⫾ 0.12 v 0.60 ⫾ 0.15, p ⬍ 0.001), % diseased vessels (Tg v WT: 42 ⫾ 23 v 98 ⫾ 7%, p ⬍ 0.001), and apoptosis (p ⬍ 0.001) differed at 30 day. Conclusions: Bcl-2 overexpression in cardiac allografts reduced Th1 but not Th2 immune response in the acute and chronic phases of graft rejection without affecting host spleen cell response. This reduction correlated with a decrease in GCAD. 355 BOTH AFTER HEART AND BONE MARROW TRANSPLANTATION THE HEART HARVEST BONE MARROW DERIVED CARDIOMYOCYTES R.A. De Weger,1 I. Verbrugge,1 L.F. Verdonck,3 N. De Jonge,2 M. Van Oosterhout,1 D.F. Van Wichen,1 Y. De Souza,4 F.G. Gmelig Meyling,4 1Pathology, UMC-U, Utrecht, Utrecht, Netherlands; 2 Heart and Lung Institute, UMC-U, Utrecht, Utrecht, Netherlands; 3 Hematology, UMC-U, Utrecht, Utrecht, Netherlands; 4 Immunology, UMC-U, Utrecht, Utrecht, Netherlands Introduction: Several studies report the generation of cardiomyocytes from bone marrow derived stem cells. In this study we analyzed the presence of cardiomyocytes of recipient bone marrow origin after heart transplantation (HTx) or of donor origin after Bone Marrow Transplantation (BMT).