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. 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 1 st Phase Study on BMCs Galiñanes et al. Cell Transplantation 2004;13:7-13

5. Dobutamine Stress Echocardiography

6. 2 nd 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 63 eligible patients undergoing CABG Control No BMCs IM BMCs → mid-depth of scar (500µl/injection) IC BMCs → via graft Supplying scar 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 100 Diastolic 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. 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 Summary

28. 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 10 6 cells/injection). Primary end point: plasma cardiac enzymes (troponin I, CK-MB) during the first 48 hours after CPB. RCT on the cardioprotective effects of BMCs in patients undergoing CABG 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 Pre- CPB 10 mins after starting 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

37. Sensitivity and specificity of myocyte nuclei identification in presence & absence of WGA OrientationObserver Sensitivity (%) [CI]Specificity (%) [CI] without WGAwith WGAwithout WGAwith WGA Transverse 145.1 [37.7 - 52.8]64.6 [57.1 - 71.5]90.1 [87.6 - 92.1]98.4 [97.1 - 99.1] 248.8 [41.2 - 56.4]68.9 [61.5 - 75.5]89.5 [86.9 - 91.6]99.0 [97.9 - 99.5] 354.3 [46.6 - 61.7]72.6 [65.3 - 78.8]94.4 [92.4 - 95.9]98.4 [97.1 - 99.1] Longitudinal 128.9 [21.8 - 37.3]59.4 [50.7 - 67.5]83.4 [80.0 - 86.3]94.0 [91.7 - 95.7] 234.4 [26.7 - 43.0]56.3 [47.6 - 64.5]84.5 [81.2 - 87.4]95.5 [93.4 - 97.0] 339.8 [31.8 - 48.5]61.7 [53.1 - 69.7]88.5 [85.5 - 90.9]95.3 [93.2 - 96.8] Overall 138.0 [32.6 - 43.7]62.3 [56.6 - 67.7]87.1 [85.1 - 88.9]96.5 [95.3 - 97.4] 242.5 [36.9 - 48.2]63.4 [57.7 - 68.7]87.3 [85.3 - 89.0]97.4 [96.4 - 98.2] 348.0 [42.3 - 53.7]67.8 [62.2 - 72.9]91.7 [90.1 - 93.2]97.0 [95.9 - 97.8] Abbreviation: WGA – wheat germ agglutinin;CI – Confidence interval

38. Diagnostic accuracy of myocyte nuclei identification in the presence and absence of WGA Abbreviation: WGA – wheat germ agglutinin; CI – Confidence interval OrientationObserverDiagnostic accuracy without WGA (%) [CI] Diagnostic accuracy with WGA (%) [CI] Transverse 181.4 [78.6 - 83.8]91.8 [89.7 - 93.4] 281.5 [78.7 – 84.0]93.1 [91.2 - 94.6] 386.5 [84.0 - 88.7]93.3 [91.4 - 94.8] Longitudinal 172.9 [69.4 - 76.2]87.4 [84.6 - 89.7] 274.9 [71.5 – 78.0]88.0 [85.3 - 90.2] 379.1 [75.8 – 82.0]88.9 [86.3 - 91.0] Overall 177.6 [75.4 - 79.6]89.8 [88.2 - 91.2] 278.6 [76.4 - 80.6]90.8 [89.3 - 92.2] 383.2 [81.3 – 85.0]91.4 [89.8 - 92.7]

39. 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%)

40. 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

41. 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

42. 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