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Pediatric Thymic Tissue as a Source of Regulatory T Cells for Cellular Therapy
Abstracts
S67 n¼2; Age, average 42, rang 18-66; P/F at donor hospital, 198.3, 66336; CIT, 10.6 hrs, 3.5-19.5. EVLP was performed using Toronto system (n¼11) and VivoLine (n¼4). Seven lungs (46%) were assessed to be acceptable with 4 lungs (80%) in short CIT group (o8 hrs, 6.3 hr, 3.57, n¼5) and 3 lungs (30%) in long CIT group (48 hrs, 15.0 hr, 919.5, n¼10). There was a statistically significant difference in P/F (365.4 vs 270.9, po0.05). pH and Glu levels were significantly lower in Toronto system. Conclusions: These preliminary data suggest that a high percentage of rejected donor lungs with CIT of less than 8 hrs can be used for clinical LTx following EVLP. With CIT of more than 8 hrs, a smaller but still reasonable percentage of rejected donor lungs meet criteria for transplantation following EVLP. 162 Endothelin-1 and Big-Endothelin-1 as Potential Biomarkers in Clinical Ex Vivo Lung Perfusion T.N. Machuca, M. Cypel, Y. Zhao, J.C. Yeung, Y.-M. Chun, R. Zamel, M. Chen, M.K. Hsin, T. Saito, Z. Guan, H. Grasemann, M. dePerrot, T.K. Waddell, M. Liu, S. Keshavjee. University of Toronto, Toronto, ON, Canada.
anti-inflammatory and cytoprotective effects as well as activation of mitochondrial biogenesis in lungs. 161 Ex Vivo Lung Perfusion of Rejected Human Donor Lungs; Are Donor Lungs with Prolonged Cold Ischemic Time Re-Conditioned by EVLP? T. Okamoto,1,2 D. Wheeler,1 P. Shen,2 S. Keshavamurthy,2 A. Rafael,2 K.R. McCurry.1,2 1Pathobiology, Lener Research Institute, Cleveland Clinic, Cleveland, OH; 2Thoracic and Cardiovascular Surgery, Cleveland Clinic, Cleveland, OH. Purpose: Ex vivo lung perfusion (EVLP) has the potential to increase the number of donor lungs available for lung transplantation (LTx). While the current maximum cold ischemic time (CIT) for donor lungs in clinical LTx is around 8 hrs, there are no data regarding the potential use of rejected donor lungs with CIT longer than 8 hrs after EVLP. Methods and Materials: Between 1/2012 to 11/2012, brain dead/DCD donor lungs (n¼15) were obtained when they were rejected for clinical LTx. Procurement procedures were the same as for clinical LTx. Lungs with severe pulmonary infection, severe lung contusion, and severe bullous emphysema were excluded. CIT was defined as the time between cross clamping and the start of EVLP. Both the Toronto style system (acellular, 4 hr) and VivoLines (cellular, 2 hr) were randomly used. Assessment for potential clinical use was established on ABG, fluid in airway, and airway/vascular parameters. A case of fluid in airway, PaO2/FiO2 [sup]([/sup]P/F) ratio o350, or PA pressure 420 was considered unsuitable for clinical use. Results: Donor lungs (n¼15) were rejected due to low P/F, abnormal CXR or other reason. Donor characteristics were: BD n¼13, DCD
Purpose: Normothermic ex vivo lung perfusion (EVLP) is a preservation technique that allows reassessment and improvement of donor lungs prior to transplantation. We hypothesized that the endothelin-1 (ET-1) axis is associated with donor lung performance during EVLP and recipient outcomes after transplantation. Methods and Materials: We assessed levels of ET-1, big ET-1 and endothelin converting enzyme 1 (ECE-1) in the perfusates of donor lungs enrolled in a clinical trial EVLP. The trial included lungs from high-risk brain death donors (BDD) and lungs from donation after cardiac death (DCD). They were divided into three groups: I. Control: bilateral transplantation with good early outcomes (absence of PGD grade 3); II. PGD3: bilateral lung transplantation with PGD grade 3 whithin 72 hours; III. Declined: lungs rejected following EVLP. Single-lung transplants and patients bridged with extracorporeal life support were excluded. Results: There were 25 cases in group I, 7 in group II and 16 in group III. At 1 hour of EVLP, perfusates of declined lungs had significantly higher levels of ET-1 (3.1⫾2.1 vs 1.8⫾2.3 pg/ml, p¼0.01) and big ET1 (15.8⫾14.2 vs 7.0⫾6.5, p¼0.001) compared to control lungs. At the 4 hours of EVLP, declined lungs also had higher levels of ET-1 (2.7⫾2.2 vs 1.3⫾1.1 pg/ml, p¼0.007) and big ET-1 (31.7⫾17.4 vs 19.4⫾9.5 pg/ml, p¼0.007) compared to controls. In BDD lungs the ET-1 axis did not show significant differences between groups. However for DCD cases, groups II and III had higher ET-1 and big ET-1 levels at 4 hr perfusion when compared to group I (group II vs I: ET-1 p¼0.03, big ET-1 p¼0.01; group III vs I: ET-1 p¼0.007, big ET-1 p¼0.003). There were no differences in ECE-1 levels between groups. Conclusions: In DCD lungs ET-1 and big ET-1 in perfusate predicted outcomes after lung transplantation. They were also associated with non-utilization of lungs after EVLP and thus could represent useful biomarkers to improve the accuracy of selection of donor lungs. 163 Pediatric Thymic Tissue as a Source of Regulatory T Cells for Cellular Therapy E. Dijke,1,2 A. McMurchy,3 M. Levings,3 I. Larsen,1,2 I. Rebeyka,1,2 D. Ross,2,4 L. West.1,2,4 1Pediatrics, University of Alberta, Edmonton, AB, Canada; 2Alberta Institute for Transplant Sciences, University of Alberta, Edmonton, AB, Canada; 3Surgery, University of British Columbia, Vancouver, BC, Canada; 4Surgery, University of Alberta, Edmonton, AB, Canada. Purpose: Infant heart transplant (HTx) recipients have better graft survival than older recipients. Nonetheless, due to need for lifelong therapy, infants carry a heavier immunosuppressive burden. Cellular
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The Journal of Heart and Lung Transplantation, Vol 32, No 4S, April 2013
therapy using regulatory T cells (Tregs) to suppress graft-directed immune responses would greatly benefit these infants. A major challenge is generating a large quantity of stable, suppressive Tregs. During infant cardiac surgery, thymectomy is usually performed to gain exposure of the retrosternal operative field. We studied the potential of thymic tissue as a source of CD25þFOXP3þ Tregs. Methods and Materials: Thymuses (n¼8) were obtained during pediatric cardiac surgery; thymocytes were recovered through mechanical dissociation. FOXP3þ cells were sorted by automated magnetic cell separation of CD25þ cells and expanded with aCD3, IL-2, rapamycin and CD32þ L-cells; CD25- cells were used as controls. FOXP3 and intracellular cytokine staining were done to define characteristics. Suppressive capacity was determined by co-culturing expanded cells with aCD3/CD28-stimulated peripheral blood mononuclear cells (PBMC) and analyzing proliferative responses by Cell Proliferation ELISA. Results: FOXP3þ cell frequency within total thymocytes ranged from 2.2 to 3.2%. Isolated CD25þ cells were 72% positive for FOXP3 (median, range: 60-81%). After two weeks of culture, we observed a 4 to 48-fold expansion of CD25þ cells with 495% viability; 0.4 to 25-fold expansion was observed for control cells with 49–88% viability. Expanded CD25þ cells were 490% FOXP3þ and produced no IL-2 or IFN-g, whereas control cells were o14% FOXP3þ and 58–65% produced IFN-g. Moreover, in contrast to controls, expanded CD25þFOXP3þ cells were potent suppressors, efficiently suppressing proliferating PBMC 450% at a 1:10 ratio of Tregs:PBMC. Conclusions: Highly suppressive FOXP3þ Tregs can be expanded from CD25þ thymocytes isolated from pediatric thymic tissue, indicating that explanted thymuses may be a source of Tregs for cellular therapy for infant HTx recipients. 164 Kidney-Induced Cardiac Allograft Tolerance across a Full MHC-Barrier in Miniature Swine S.G. Michel,1 M.L.L. Madariaga,1 M. Tasaki,1 V. Villani,1 G.M. LaMuraglia II,1 E.A. Farkash,2 J.S. Allan,1 D.H. Sachs,1 K. Yamada,1 J.C. Madsen.1 1Transplantation Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA; 2 Department of Pathology, Massachusetts General Hospital, Boston, MA. Purpose: We have demonstrated previously that isolated hearts transplanted into class I disparate miniature swine treated with a 12-day course of CyA all developed cardiac allograft vaszculopathy and rejected within 55 days. In contrast, hearts co-transplanted with donor-specific kidneys survived indefinitely without CAV. Here, we asked whether tolerance can be induced in heart/ kidney recipients across a full MHC barrier, which is more clinically relevant. Methods and Materials: Heart/kidneys (n¼5) or hearts alone (n¼3) were transplanted heterotopically into the abdomen of MGH inbred miniature swine across a full MHC mismatch. Immunosuppression consisted of a 12-day course of tacrolimus. Open biopsies were taken on days 30, 60, and 100 to rule out rejection. CML and MLR assays were performed to determine responsiveness toward donor antigen. Results: All control animals rejected their isolated hearts within 40 days. In contrast, all heart and kidney recipients survived for 4100 days with no evidence of rejection on serial cardiac biopsies. Heart/ kidney recipients were hyporesponsive or unresponsive toward donor antigen by CML/MLR assays and had no detectable alloantibody by flow cytometry. Conclusions: Induction of long-term cardiac allograft tolerance is possible across a full MHC- mismatched barrier in large animals cotransplanted with a kidney from the same donor. Elucidating the renal element responsible for cardiac allograft tolerance could provide mechanistic information that could lead to protocols aimed at inducing tolerance in recipients of isolated heart allografts.
165 Transmyocardial Revascularization Enhances Mesenchymal Stem Cell Engraftment in Infarcted Hearts through SCF–c-Kit and SDF-1–CXCR4 Signaling Axes U. Shahzad,1,2 G. Li,2 T.M. Yau.1,2 1Department of Cardiovascular Surgery, University of Toronto, Toronto, ON, Canada; 2Division of Cardiovascular Surgery, Peter Munk Cardiac Centre, University Health Network, Toronto, ON, Canada. Purpose: We investigated the roles of stem cell factor(SCF)–c-kit and stromal derived factor-1(SDF-1)–chemokine receptor type 4(CXCR4) stem cell signaling axes in transmyocardial revascularization (TMR)enhanced engraftment of transplanted mesenchymal stem cells (MSC) in infarcted hearts. Methods and Materials: 3 weeks after LAD ligation, female Lewis rats underwent 10-channel needle TMR, followed by daily IV injections of 1 million male donor MSC for 5 days, either wild type (WT) or with knockdown (K/D) of c-kit or CXCR4, accomplished via a shRNAþplasmid in a lentiviral vector. Experimental groups included: WT MSC with or without TMR, c-kit K/D MSC with or without TMR, and CXCR4 K/D MSC with or without TMR (N¼6/group). Results: In vitro cell surface expression of c-kit (N¼3) was reduced from 14⫾0.7% of WT MSC to 1.6⫾0.4% c-kit K/D MSC, and CXCR4 (N¼3) was reduced from 39⫾10% WT MSCs to 3.7⫾0.7% CXCR4 K/ D MSC after 1 week (po0.05). The number of MSCs that had homed into infarct was affected by both TMR and donor cell type at 3 days(TMR, cell type, & interaction, po0.05) and 1 week (TMR & cell type, po0.05) after the last MSC injection, with greater MSC engraftment with TMR and with WT MSC. At 3 days, TMR significantly upregulated in vivo transcription of c-kit (TMR, po0.05), SCF (TMR & cell type, po0.05), CXCR4 (TMR & cell type, po0.05), and SDF-1 (TMR & cell type, po0.05). At 1 week, we saw similar declines in expression of c-kit (cell type, po0.05), SCF (TMR, po0.05), CXCR4 (TMR & cell type, po0.05), and SDF-1 (TMR, po0.05). At 1 week, TMR improved LVEF (N¼5) when WT MSCs were infused, but K/D of either c-kit or CXCR4 completely abrogated this TMR-mediated augmentation of MSC reparative effect (TMR & cell type, po0.05). Conclusions: Downregulation of either c-kit or CXCR4 in MSC decreased engraftment of circulating MSC and inhibited the reparative effects of TMR. Hence, both SCF–c-kit and SDF-1– CXCR4 signaling axes are required for TMR-augmented repair of the infarcted heart. 166 Hearts from Donations after Circulatory Death (DCD) Donors – Assessment in a Porcine Transplant Model Utilising Transmedics Organ Care System for Organ Perfusion Preservation A. Iyer,1,2 L. Gao,1 A. Doyle,1 G. Kumarasinghe,1,2 A. Jabbour,1,2 M. Hicks,1 P. Jansz,2 K. Dhital,2 P. Macdonald.1,2 1Heart Transplantation Lab, Victor Chang Cardiac Research Institute, Sydney, Australia; 2Heart & Lung Transplant Program, St Vincent’s Hospital, Sydney, Australia.
S67 n¼2; Age, average 42, rang 18-66; P/F at donor hospital, 198.3, 66336; CIT, 10.6 hrs, 3.5-19.5. EVLP was performed using Toronto system (n¼11) and VivoLine (n¼4). Seven lungs (46%) were assessed to be acceptable with 4 lungs (80%) in short CIT group (o8 hrs, 6.3 hr, 3.57, n¼5) and 3 lungs (30%) in long CIT group (48 hrs, 15.0 hr, 919.5, n¼10). There was a statistically significant difference in P/F (365.4 vs 270.9, po0.05). pH and Glu levels were significantly lower in Toronto system. Conclusions: These preliminary data suggest that a high percentage of rejected donor lungs with CIT of less than 8 hrs can be used for clinical LTx following EVLP. With CIT of more than 8 hrs, a smaller but still reasonable percentage of rejected donor lungs meet criteria for transplantation following EVLP. 162 Endothelin-1 and Big-Endothelin-1 as Potential Biomarkers in Clinical Ex Vivo Lung Perfusion T.N. Machuca, M. Cypel, Y. Zhao, J.C. Yeung, Y.-M. Chun, R. Zamel, M. Chen, M.K. Hsin, T. Saito, Z. Guan, H. Grasemann, M. dePerrot, T.K. Waddell, M. Liu, S. Keshavjee. University of Toronto, Toronto, ON, Canada.
anti-inflammatory and cytoprotective effects as well as activation of mitochondrial biogenesis in lungs. 161 Ex Vivo Lung Perfusion of Rejected Human Donor Lungs; Are Donor Lungs with Prolonged Cold Ischemic Time Re-Conditioned by EVLP? T. Okamoto,1,2 D. Wheeler,1 P. Shen,2 S. Keshavamurthy,2 A. Rafael,2 K.R. McCurry.1,2 1Pathobiology, Lener Research Institute, Cleveland Clinic, Cleveland, OH; 2Thoracic and Cardiovascular Surgery, Cleveland Clinic, Cleveland, OH. Purpose: Ex vivo lung perfusion (EVLP) has the potential to increase the number of donor lungs available for lung transplantation (LTx). While the current maximum cold ischemic time (CIT) for donor lungs in clinical LTx is around 8 hrs, there are no data regarding the potential use of rejected donor lungs with CIT longer than 8 hrs after EVLP. Methods and Materials: Between 1/2012 to 11/2012, brain dead/DCD donor lungs (n¼15) were obtained when they were rejected for clinical LTx. Procurement procedures were the same as for clinical LTx. Lungs with severe pulmonary infection, severe lung contusion, and severe bullous emphysema were excluded. CIT was defined as the time between cross clamping and the start of EVLP. Both the Toronto style system (acellular, 4 hr) and VivoLines (cellular, 2 hr) were randomly used. Assessment for potential clinical use was established on ABG, fluid in airway, and airway/vascular parameters. A case of fluid in airway, PaO2/FiO2 [sup]([/sup]P/F) ratio o350, or PA pressure 420 was considered unsuitable for clinical use. Results: Donor lungs (n¼15) were rejected due to low P/F, abnormal CXR or other reason. Donor characteristics were: BD n¼13, DCD
Purpose: Normothermic ex vivo lung perfusion (EVLP) is a preservation technique that allows reassessment and improvement of donor lungs prior to transplantation. We hypothesized that the endothelin-1 (ET-1) axis is associated with donor lung performance during EVLP and recipient outcomes after transplantation. Methods and Materials: We assessed levels of ET-1, big ET-1 and endothelin converting enzyme 1 (ECE-1) in the perfusates of donor lungs enrolled in a clinical trial EVLP. The trial included lungs from high-risk brain death donors (BDD) and lungs from donation after cardiac death (DCD). They were divided into three groups: I. Control: bilateral transplantation with good early outcomes (absence of PGD grade 3); II. PGD3: bilateral lung transplantation with PGD grade 3 whithin 72 hours; III. Declined: lungs rejected following EVLP. Single-lung transplants and patients bridged with extracorporeal life support were excluded. Results: There were 25 cases in group I, 7 in group II and 16 in group III. At 1 hour of EVLP, perfusates of declined lungs had significantly higher levels of ET-1 (3.1⫾2.1 vs 1.8⫾2.3 pg/ml, p¼0.01) and big ET1 (15.8⫾14.2 vs 7.0⫾6.5, p¼0.001) compared to control lungs. At the 4 hours of EVLP, declined lungs also had higher levels of ET-1 (2.7⫾2.2 vs 1.3⫾1.1 pg/ml, p¼0.007) and big ET-1 (31.7⫾17.4 vs 19.4⫾9.5 pg/ml, p¼0.007) compared to controls. In BDD lungs the ET-1 axis did not show significant differences between groups. However for DCD cases, groups II and III had higher ET-1 and big ET-1 levels at 4 hr perfusion when compared to group I (group II vs I: ET-1 p¼0.03, big ET-1 p¼0.01; group III vs I: ET-1 p¼0.007, big ET-1 p¼0.003). There were no differences in ECE-1 levels between groups. Conclusions: In DCD lungs ET-1 and big ET-1 in perfusate predicted outcomes after lung transplantation. They were also associated with non-utilization of lungs after EVLP and thus could represent useful biomarkers to improve the accuracy of selection of donor lungs. 163 Pediatric Thymic Tissue as a Source of Regulatory T Cells for Cellular Therapy E. Dijke,1,2 A. McMurchy,3 M. Levings,3 I. Larsen,1,2 I. Rebeyka,1,2 D. Ross,2,4 L. West.1,2,4 1Pediatrics, University of Alberta, Edmonton, AB, Canada; 2Alberta Institute for Transplant Sciences, University of Alberta, Edmonton, AB, Canada; 3Surgery, University of British Columbia, Vancouver, BC, Canada; 4Surgery, University of Alberta, Edmonton, AB, Canada. Purpose: Infant heart transplant (HTx) recipients have better graft survival than older recipients. Nonetheless, due to need for lifelong therapy, infants carry a heavier immunosuppressive burden. Cellular
S68
The Journal of Heart and Lung Transplantation, Vol 32, No 4S, April 2013
therapy using regulatory T cells (Tregs) to suppress graft-directed immune responses would greatly benefit these infants. A major challenge is generating a large quantity of stable, suppressive Tregs. During infant cardiac surgery, thymectomy is usually performed to gain exposure of the retrosternal operative field. We studied the potential of thymic tissue as a source of CD25þFOXP3þ Tregs. Methods and Materials: Thymuses (n¼8) were obtained during pediatric cardiac surgery; thymocytes were recovered through mechanical dissociation. FOXP3þ cells were sorted by automated magnetic cell separation of CD25þ cells and expanded with aCD3, IL-2, rapamycin and CD32þ L-cells; CD25- cells were used as controls. FOXP3 and intracellular cytokine staining were done to define characteristics. Suppressive capacity was determined by co-culturing expanded cells with aCD3/CD28-stimulated peripheral blood mononuclear cells (PBMC) and analyzing proliferative responses by Cell Proliferation ELISA. Results: FOXP3þ cell frequency within total thymocytes ranged from 2.2 to 3.2%. Isolated CD25þ cells were 72% positive for FOXP3 (median, range: 60-81%). After two weeks of culture, we observed a 4 to 48-fold expansion of CD25þ cells with 495% viability; 0.4 to 25-fold expansion was observed for control cells with 49–88% viability. Expanded CD25þ cells were 490% FOXP3þ and produced no IL-2 or IFN-g, whereas control cells were o14% FOXP3þ and 58–65% produced IFN-g. Moreover, in contrast to controls, expanded CD25þFOXP3þ cells were potent suppressors, efficiently suppressing proliferating PBMC 450% at a 1:10 ratio of Tregs:PBMC. Conclusions: Highly suppressive FOXP3þ Tregs can be expanded from CD25þ thymocytes isolated from pediatric thymic tissue, indicating that explanted thymuses may be a source of Tregs for cellular therapy for infant HTx recipients. 164 Kidney-Induced Cardiac Allograft Tolerance across a Full MHC-Barrier in Miniature Swine S.G. Michel,1 M.L.L. Madariaga,1 M. Tasaki,1 V. Villani,1 G.M. LaMuraglia II,1 E.A. Farkash,2 J.S. Allan,1 D.H. Sachs,1 K. Yamada,1 J.C. Madsen.1 1Transplantation Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA; 2 Department of Pathology, Massachusetts General Hospital, Boston, MA. Purpose: We have demonstrated previously that isolated hearts transplanted into class I disparate miniature swine treated with a 12-day course of CyA all developed cardiac allograft vaszculopathy and rejected within 55 days. In contrast, hearts co-transplanted with donor-specific kidneys survived indefinitely without CAV. Here, we asked whether tolerance can be induced in heart/ kidney recipients across a full MHC barrier, which is more clinically relevant. Methods and Materials: Heart/kidneys (n¼5) or hearts alone (n¼3) were transplanted heterotopically into the abdomen of MGH inbred miniature swine across a full MHC mismatch. Immunosuppression consisted of a 12-day course of tacrolimus. Open biopsies were taken on days 30, 60, and 100 to rule out rejection. CML and MLR assays were performed to determine responsiveness toward donor antigen. Results: All control animals rejected their isolated hearts within 40 days. In contrast, all heart and kidney recipients survived for 4100 days with no evidence of rejection on serial cardiac biopsies. Heart/ kidney recipients were hyporesponsive or unresponsive toward donor antigen by CML/MLR assays and had no detectable alloantibody by flow cytometry. Conclusions: Induction of long-term cardiac allograft tolerance is possible across a full MHC- mismatched barrier in large animals cotransplanted with a kidney from the same donor. Elucidating the renal element responsible for cardiac allograft tolerance could provide mechanistic information that could lead to protocols aimed at inducing tolerance in recipients of isolated heart allografts.
165 Transmyocardial Revascularization Enhances Mesenchymal Stem Cell Engraftment in Infarcted Hearts through SCF–c-Kit and SDF-1–CXCR4 Signaling Axes U. Shahzad,1,2 G. Li,2 T.M. Yau.1,2 1Department of Cardiovascular Surgery, University of Toronto, Toronto, ON, Canada; 2Division of Cardiovascular Surgery, Peter Munk Cardiac Centre, University Health Network, Toronto, ON, Canada. Purpose: We investigated the roles of stem cell factor(SCF)–c-kit and stromal derived factor-1(SDF-1)–chemokine receptor type 4(CXCR4) stem cell signaling axes in transmyocardial revascularization (TMR)enhanced engraftment of transplanted mesenchymal stem cells (MSC) in infarcted hearts. Methods and Materials: 3 weeks after LAD ligation, female Lewis rats underwent 10-channel needle TMR, followed by daily IV injections of 1 million male donor MSC for 5 days, either wild type (WT) or with knockdown (K/D) of c-kit or CXCR4, accomplished via a shRNAþplasmid in a lentiviral vector. Experimental groups included: WT MSC with or without TMR, c-kit K/D MSC with or without TMR, and CXCR4 K/D MSC with or without TMR (N¼6/group). Results: In vitro cell surface expression of c-kit (N¼3) was reduced from 14⫾0.7% of WT MSC to 1.6⫾0.4% c-kit K/D MSC, and CXCR4 (N¼3) was reduced from 39⫾10% WT MSCs to 3.7⫾0.7% CXCR4 K/ D MSC after 1 week (po0.05). The number of MSCs that had homed into infarct was affected by both TMR and donor cell type at 3 days(TMR, cell type, & interaction, po0.05) and 1 week (TMR & cell type, po0.05) after the last MSC injection, with greater MSC engraftment with TMR and with WT MSC. At 3 days, TMR significantly upregulated in vivo transcription of c-kit (TMR, po0.05), SCF (TMR & cell type, po0.05), CXCR4 (TMR & cell type, po0.05), and SDF-1 (TMR & cell type, po0.05). At 1 week, we saw similar declines in expression of c-kit (cell type, po0.05), SCF (TMR, po0.05), CXCR4 (TMR & cell type, po0.05), and SDF-1 (TMR, po0.05). At 1 week, TMR improved LVEF (N¼5) when WT MSCs were infused, but K/D of either c-kit or CXCR4 completely abrogated this TMR-mediated augmentation of MSC reparative effect (TMR & cell type, po0.05). Conclusions: Downregulation of either c-kit or CXCR4 in MSC decreased engraftment of circulating MSC and inhibited the reparative effects of TMR. Hence, both SCF–c-kit and SDF-1– CXCR4 signaling axes are required for TMR-augmented repair of the infarcted heart. 166 Hearts from Donations after Circulatory Death (DCD) Donors – Assessment in a Porcine Transplant Model Utilising Transmedics Organ Care System for Organ Perfusion Preservation A. Iyer,1,2 L. Gao,1 A. Doyle,1 G. Kumarasinghe,1,2 A. Jabbour,1,2 M. Hicks,1 P. Jansz,2 K. Dhital,2 P. Macdonald.1,2 1Heart Transplantation Lab, Victor Chang Cardiac Research Institute, Sydney, Australia; 2Heart & Lung Transplant Program, St Vincent’s Hospital, Sydney, Australia.