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Keng Ang VII International Symposium on Stem Cell Therapy Madrid, 6-7 May 2010

Understanding the Mechanisms and the Potential of Cell Therapy for the Repair of the Adult Mammalian Heart. Keng Ang VII International Symposium on Stem Cell Therapy Madrid, 6-7 May 2010. Intramuscular injection and cytokine mobilisation of bone marrow cells repair infarct myocardium.

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Keng Ang VII International Symposium on Stem Cell Therapy Madrid, 6-7 May 2010

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  1. Understanding the Mechanisms and the Potential of Cell Therapy for the Repair of the Adult Mammalian Heart Keng Ang VII International Symposium on Stem Cell Therapy Madrid, 6-7 May 2010

  2. Intramuscular injection and cytokine mobilisation of bone marrow cells repair infarct myocardium Orlic D et al Nature 2001;410:701 Orlic D et al PNAS 2001;98:10344

  3. Haematopoietic Stem Cells Do Not Transdifferentiate Into Cardiac Myocytes in Myocardial Infarcts • Murry CE et al. Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature 2004;428:664 • Balsam LB et al. Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature 2004;428:668

  4. 1st Phase Study on BMCs • 14 patients: one or more MI (>3 months) • Elective CABG surgery • Bone marrow: • aspirated from sternum • mixed with serum (1:2 ratio) • injected into scarred areas at end of surgery • 250µl/injection 1cm apart into mid-depth • flow cytometry analysis of nucleated cell count and CD34+/CD117+ cells Galiñanes et al. Cell Transplantation 2004;13:7-13

  5. Dobutamine Stress Echocardiography Galiñanes et al. Cell Transplantation 2004;13:7-13

  6. 2nd Phase Study on BMCs Objectives • 1. To determine whether the transplantation of autologous BMCs into myocardial scar improves systolic function • 2. And whether this improvement, if any, depends on the route of administration: • Intramuscular (IM) • Intracoronary (IC)

  7. Study Design • BMCs preparation & administration: • aspirated from iliac crest at the start of operation; • Separated by density gradient & diluted in autologous serum • Administered IM or IC before cross-clamp release • Investigations: • DSE: Pre-op & 6 month • MRI: Pre-op & 6 month

  8. % Systolic Fractional Thickening in Scarred Segments (Systolic segmental thickness – Diastolic segmental thickness)% FT= ------------------------------------------------------------------------------------------------ x 100Diastolic segmental thickness

  9. % Infarct Volume Infarct volume % Infarct volume = --------------------------------------------------------- x 100% Left ventricular myocardial volume

  10. Left Ventricular Ejection Fraction

  11. Summary Administration of BMCs (IM or IC) into myocardial scar during CABG is safe, but: • Do not improve segmental systolic function • Do not reduce infarct size • Do not influence global LV parameters

  12. Martin-Rendon E et al. Eur Heart J 2008;29:1807

  13. If BMCs cannot differentiate into cardiac tissue, is the observed beneficial effect due to • improvement in cell survival • stimulation of cardiac progenitor cells

  14. Cardioprotection by BMCs • Right atrial appendage from patients undergoing elective cardiac surgery • Slices 300-500µm thickness, 30-50mg weight • Incubation Krebs solution at 37°C • 90-min simulated ischemia/120-min reoxygenation • End-points: • CK release during reoxygenation • Necrosis assessed by PI at the end of reoxygenation • Apoptosis assessed by TUNEL at the end of reoxygenation • Bone marrow was aspirated from the iliac crest of the patients and separated by density gradient

  15. Anti-ischemic Effect of BMC Kubal C et al J Thorac Cardiovasc Surg 2006;132:1112

  16. The Role of PKC Kubal C et al J Thorac Cardiovasc Surg 2006;132:1112

  17. The Role of p38MAPK Kubal C et al J Thorac Cardiovasc Surg 2006;132:1112

  18. Is Protection Against Ischemic Injury Cell Type Specific? Kubal C et al J Thorac Cardiovasc Surg 2006;132:1112

  19. What Is the Most Effective Dose of BMCs-induced Cardioprotection? Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400

  20. How Potent is BMCs-induced Cardioprotection? Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400

  21. Cardioprotective Efficacy of Allogenic BMCs Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400

  22. Does Manipulation of Cells Affect BMCs-induced Cardioprotection? Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400

  23. Does the Time of Administration Influence BMCs-induced Cardioprotection? Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400

  24. Do BMCs Precondition the Myocardium? Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400

  25. Is the Cardioprotection Induced by BMCs Triggered by Secreted Factor(s)? Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400

  26. The Role of IGF-1R in Mediating BMCs-induced Cardioprotection Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400

  27. Summary • BMCs possess potent cardioprotective properties • Protection is triggered by a secreted factor(s) • Protection is mediated by IGF-1R and by activation of the protein kinases PKC and p38MAPK

  28. RCT on the cardioprotective effects of BMCs in patients undergoing CABG 44 elective CABG patients: • randomised to control or BMCs group BMCs group: • BMCs harvested & administered at the end of each cardioplegia dose as an adjunct(49.6±28.7 x 106 cells/injection). Primary end point: • plasma cardiac enzymes (troponin I, CK-MB) during the first 48 hours after CPB. Ang et al. Eur Heart J. 2009;30:2354

  29. Plasma cardiac enzymes Ang et al. Eur Heart J. 2009;30:2354

  30. Mycardial injury before & after CPB 10 mins after starting CPB Pre- CPB Ang et al. Eur Heart J. 2009;30:2354

  31. If BMCs cannot differentiate into cardiac tissue, is the observed beneficial effect due to • improvement in cell survival • stimulation of cardiac progenitor cells

  32. Methodological difficulties in the identification of cardiomyocyte nuclei Confocal microscopy – advocated, but diagnostic accuracy has not been previously tested. • MHC-nLAC mice (ß-GAL is expressed in 100% of myocyte) • membrane marker (WGA) • markers involved in cardiogenesis such as GATA4 Ang et al. Am J Physiol Cell Physiol (2010, in press)

  33. Troponin – Red; DAPI nucleus – Blue

  34. Troponin – Red; DAPI nucleus – Blue; Membrane – White

  35. Troponin – Red; DAPI nucleus – Blue; Membrane – White; Myocyte nucleus - Green

  36. Sensitivity and specificity of myocyte nuclei identification in presence & absence of WGA Abbreviation: WGA – wheat germ agglutinin;CI – Confidence interval

  37. Diagnostic accuracy of myocyte nuclei identification in the presence and absence of WGA Abbreviation: WGA – wheat germ agglutinin; CI – Confidence interval

  38. Diagnostic performance of GATA4 immune-reactivity Sensitivity of 91% & specificity of 88% Positive predictive power of 72% & Negative predictive power of 97% Diagnostic accuracy for myocyte nuclei (89.5%)

  39. Summary: • concerns about the diagnostic accuracy of confocal approaches for the correct identification of cardiomyocyte nuclei events • transgenic models (MHC-nLAC) can be of help for a more accurate identification of cardiomyocyte nuclei

  40. CONCLUSIONS • BMCs induce survival of the myocardium but its potential to stimulate the proliferation of cardiac resident stem cells needs to be elucidated • There is a need to improve & refine the accuracy of the methodological tools used so far for the identification of cardiomyocytes’ nuclei

  41. Acknowledgements University of Leicester • Keng Ang • Vien K Lai • Lincoln Shenje • José Linares-Palomino • Catrin Pritchard • Derek Chin Goodhope H (Birmingham) • Francisco Leyva • Paul Foley University of Indiana • Loren Field • Michael Rubart • Mark Soonpa Bernado Nadal-Ginard (JMLU) Funding: • Bristol-Myer Squibbs • British Heart Foundation • Vietnamese Government

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