1. Cell Transplantation for the failing myocardium: A UK perspective Tony Gershlick U.H. Leicester BCIS meeting Bristol Sept 2005 Cell Transplantation for the failing myocardium: A UK perspective Tony Gershlick U.H. Leicester BCIS meeting Bristol Sept 2005

2. Cardiomyocytes do not regenerate after birth - respond to mitotic signals by cell hypertrophy rather than by cell hyperplasia. Loss of cardiomyocytes leads to regional contractile dysfunction- dead cardiomyocytes in infarcted ventricular tissues are progressively replaced by fibroblasts to form scar tissues. Some evidence heart is NOT a post-mitotic end organ Cardiomyocytes do not regenerate after birth - respond to mitotic signals by cell hypertrophy rather than by cell hyperplasia. Loss of cardiomyocytes leads to regional contractile dysfunction- dead cardiomyocytes in infarcted ventricular tissues are progressively replaced by fibroblasts to form scar tissues. Some evidence heart is NOT a post-mitotic end organ Impact of failed therapy AMI – time 1 million UK heart failure 5% all admissions 1-2% health budget Impact of failed therapy AMI – time 1 million UK heart failure 5% all admissions 1-2% health budget

3. Therapy for AMI – Lysis v PPC Impact of time/treatment 1 million UK heart failure 5% all admissions 1-2% health budget Therapy for AMI – Lysis v PPC Impact of time/treatment 1 million UK heart failure 5% all admissions 1-2% health budget Heart failure -strategies-  Biventricular pacing  Surgical cardiomyoplasty  Left ventricular assist device  Artificial hearts  Heart transplantation  Cell transplantation ? Medical treatment

4. Stem cells for myocardial dysfunction Questions that (should) concern us in the UK  Do we (the cardiological/scientific community) have a good idea about Stem cells for myocardial dysfunction Questions that (should) concern us in the UK  Do we (the cardiological/scientific community) have a good idea about  Do we have the infra structure to be involved ?  Do we have the infra structure to be involved ?  Are the trials of sufficient quality to provide the answers to the questions being asked ?  What dose/concentration  Does the background science justify trial initiation  Are the trials relevant to our current practice ?  Is it safe ?  Which studies is the UK involved in?  Which in our (my) opinion is the likely way forward ?  W hich in our (my) opinion is the likely way forward ?  Which delivery technique  Which cells

5. Question 1 Is the science sound ? Question 1 Is the science sound ? Stem cells for myocardial dysfunction Questions that (should) concern us in the UK

6. Primative BM stem cells Skeletal muscle satellite cells Endothelial progenitor cells Intra myocardial primitive satellite cells

7. Transplanted adult bone marrow cells repair myocardial infarcts in mice. Orlic D, Kajstura J, Chimenti S, Bodine DM, Leri A, Anversa P. The left coronary artery was ligated and 5 hours later Lin- c-kit+ bone marrow cells obtained from transgenic male mice expressing enhanced green fluorescent protein - injected into the healthy myocardium adjacent to the site of the infarct. 9 days the damaged hearts were examined for regenerating myocardium. A band of new myocardium was observed in 12 surviving mice. The developing myocytes were small and resembled fetal and neonatal myocytes. Y chromosome - positive for EGFP, Y chromosome, and several myocyte-specific proteins including cardiac myosin, and the transcription factors GATA-4, MEF2, and Csx/Nkx2.5. The cells were also positive for connexin 43, a gap junction/intercalated disc component Transplanted adult bone marrow cells repair myocardial infarcts in mice. Orlic D, Kajstura J, Chimenti S, Bodine DM, Leri A, Anversa P. The left coronary artery was ligated and 5 hours later Lin- c-kit+ bone marrow cells obtained from transgenic male mice expressing enhanced green fluorescent protein - injected into the healthy myocardium adjacent to the site of the infarct. 9 days the damaged hearts were examined for regenerating myocardium. A band of new myocardium was observed in 12 surviving mice. The developing myocytes were small and resembled fetal and neonatal myocytes. Y chromosome - positive for EGFP, Y chromosome, and several myocyte-specific proteins including cardiac myosin, and the transcription factors GATA-4, MEF2, and Csx/Nkx2.5. The cells were also positive for connexin 43, a gap junction/intercalated disc component Primative BM stem cells Bone marrow transplantation –plenty of pre-clinical data

8. Primitive cardiomyogenic cells from bone marrow Clin Invest, March 1999, Volume 103, Number 5, 697-70 Shinji Makino 1, Fibroblast like 5-azacytidine ball-like or stick-like appearance spontaneously beating 2/52 form myotube-like structures beating cells were connected and formed myotube-like structures

9. Peripheral blood mononuclear cells Endothelial progenitor cells 1. Heamopoietic markers CD 34 + CD 133 + 2. Endothelial markers EGF -2 Endothelial progenitor cells 1. Heamopoietic markers CD 34 + CD 133 + 2. Endothelial markers EGF -2

10. EPC transdifferentiate in-vitro into cardiomyocytes Intracellular gap junctional coupling between rat cardiomyocytes and human EPCs was demonstrated Badorff et al, Circulation 2003;107:1024-1032 Human EPCs transplanted into rat infarct model day 7 Therapeutic Potential of Ex Vivo Expanded Endothelial Progenitor Cells for Myocardial Ischemia Atsuhiko Kawamoto Circulation. 2001;103:634 A A B B Angiogenic potential / neo-vascularisation

11. Myoblasts are satellite cells and exist in a quiescent state. They have built in resistance to ischemia Myoblasts are satellite cells and exist in a quiescent state. They have built in resistance to ischemia

12. Skeletal myoblasts after MI -sheep model- Control Baseline Follow-up Grafted Baseline Follow-up P- valueLVEF(%)43±433±350±248±50.006 WMS8.9±1.413±2.27.7±0.95.4±1.4<0.01 Ghostine et al, Circulation 2002; [suppl I]:I-131-I-136

13. grafted skeletal myotubes replacing scar fibrosis Myotubes with prominent normal Z bands in myofilaments and peripheral multiple nuclei in myotubes Myoblast grafts can survive and show a switch to slow- twitch fibres. Myoblast grafts can survive and show a switch to slow- twitch fibres. Hagege et al, Lancet 2003;361:491-92 Do not become myocytes

14. fat fibrous infarct 4 months Grafted cells Fast Slow 30% isoforms beta myosin chain 1 year *

15. Science :Good In-vitro, ex-vivo, animal data for BMC, EPC, Myoblast all have potential Which will be clinically applicable ? Science :Good In-vitro, ex-vivo, animal data for BMC, EPC, Myoblast all have potential Which will be clinically applicable ?

16. Autologous bone marrow cells Circulating progenitor cells Skeletal myoblasts Manipulation of in-vivo cells Autologous bone marrow cells Circulating progenitor cells Skeletal myoblasts Manipulation of in-vivo cells Ischaemia (AMI) Chronic Heart Failure Ischaemia (AMI) Chronic Heart Failure What is actually happening ?

17. Stem Cell Therapy MNC from bone marrow Untreated bone marrow Untreated bone marrow CD34/CD133 only a few % CD34/CD133 only a few % Just the cells isolated (2oo mls bone marrow) Give all MNC 50 mls bone marrow Predominantly myocytes Endothelial cells & myocytes Endothelial progenitor cells Endothelial Peripheral blood Give as is / grow them up

18. Autologous bone marrow cells Circulating progenitor cells Skeletal myoblasts Manipulation of in-vivo cells Autologous bone marrow cells Circulating progenitor cells Skeletal myoblasts Manipulation of in-vivo cells Ischaemia (AMI) Chronic Heart Failure Ischaemia (AMI) Chronic Heart Failure What is actually happening ?

19. Questions that (should) concern us in the UK  Is the basic science sound Questions that (should) concern us in the UK  Is the basic science sound  Are the trials of sufficient quality to provide the answers to the questions being asked ?  Are the trials relevant to our current practice ?

20. MNC MNC v CPC MNC MNC

21. Circulation, December 10, 2002 20 patients with reperfused acute myocardial infarction received bone marrow-derived stem cells (n=9) CPC (n=11)250mls Non-randomized matched reference group Trial end-point: LVEF assessed by Echo and PET-Scan @ 4 months follow-up and at one year

24. LVEF 51.3% 59.5% 8.2% Improved myocardial viability in the stem cells group TOPCARE-AMI Would they have got better 2 nd to PCI anyway?

26. The Lancet, July 10, 2004 First randomized clinical trial after successful percutaneous coronary intervention (PCI): 30 patients control group, 30 patients bone-marrow cell group Primary endpoint: global left-ventricular ejection fraction (LVEF) change from baseline to 6 months’ follow-up, determined by cardiac MRI

27. BOOST Mean in-stent restenosis in the infarct-related artery, expressed as a percentage of luminal diameter, was 32% (SD 20) in the control group and 33% (23) in the bone-marrow-cell group (p=0·88). Four patients from the control group and seven from the bone- marrow-cell group presented with an in-stent restenosis of at least 50% (p=0·28).

28. Problems with the acute ischaemic trials Problems with the acute ischaemic trials Unanswered clinical trial questions Unselected and selected cell populations not compared 3-5% : (Stem cell selection~ 200 mls BM) Retention 3% No dose ranging studies Variable numbers of cells transplanted – both CPC and BMC Trials don’t tell us which cells Benefit did not correlate with absolute numbers of CD 34 cells Timing ? Early environment cyto-toxic 3- 7 days ? longer Engraftment

30. Cell Transplant. 2004;13(1):7-13

31. 128 mls 50 mls / 250 mls

32. Problems with the acute clinical trials Problems with the acute clinical trials EF increases small Who is likely to benefit ? No clear messages re type cells, nos. and delivery Small number subjects -pilot studies ( Mortality/ morbidity end point (1000 s patients) Small number subjects -pilot studies ( Mortality/ morbidity end point (1000 s patients) No adequate controls (Bone marrow acquisition?, coronary manipulation ?) All P PCI Mode of Delivery “REPAIR AMI” AHA

33. BOOST

38.  Appropriate trials ?  Who most likely to benefit ?  Cell type ?  Appropriate trials ?  Who most likely to benefit ?  Cell type ? o Which of these questions can be answered ? o What is the UK involvement o Can we add to the Clinical science? o Which of these questions can be answered ? o What is the UK involvement o Can we add to the Clinical science?

39. UHL/ St Barts/ The London Anthony Mather Martin Rothman John Martin Eric Alton Kings College Hospital Jonathan Hill AJ Shah St Marys/ Brompton Nic Peters Philip Poole-wilson UH Leicester Jan Kovac Manual Galinanes Nilesh Samani Tony Gershlick UK Centres Involved in Stem Cell Therapy

40. Single Centre Trial @ UHL

41. Failure of thrombolysis Successful R-PCI Echo d 3 EF < 45% Baseline MRI SC or Heparinised plasma d 4-5 P EP 4 mo MRI Failure of thrombolysis Successful R-PCI Echo d 3 EF < 45% Baseline MRI SC or Heparinised plasma d 4-5 P EP 4 mo MRI Poor LV Setting lysis + RESCUE-PCI Late phase Poor LV Setting lysis + RESCUE-PCI Late phase

42. Clinical ‘stem cell’ trials - Barts and the London NHS Trust/UCLH Randomised control trials using autologous bone marrow derived mononuclear cells in patients with: –Heart failure 2 o IHD - commenced 8/05 - 300 patients –Dilated cardiomyopathy - recruiting - 200 patients –AMI t x with primary angioplasty - funding sort - 200 patients PIs: A Mathur, M Rothman, J Martin Randomised control trials using autologous bone marrow derived mononuclear cells in patients with: –Heart failure 2 o IHD - commenced 8/05 - 300 patients –Dilated cardiomyopathy - recruiting - 200 patients –AMI t x with primary angioplasty - funding sort - 200 patients PIs: A Mathur, M Rothman, J Martin

43.  Appropriate trials ?  Who most likely to benefit ?  Cell type ?  Appropriate trials ?  Who most likely to benefit ?  Cell type ? Which of these questions can be answered, What is the UK involvement Which of these questions can be answered, What is the UK involvement

44. Myocardial infarct Age location NYHA class Cell transplantation No of cells No of inj (X10 6 ) Graft location LVEF Pre TX post TX 2 yearsPosteriorIII-IV82033LAD/D12035 6 yearsAnteriorIII87050OMX22527 9 and 3yAnteriorII62057LAD/OM3141 4 monthsPosterolater al II95031LAD/IM2245 7y and 5+3 months AnteriorIII-IV115042RCA/IM34EPD 5 yearsPosteriorIII88036LAD/IM2630 14 monthsAnteriorIII98034RCA/IM2832 3 monthsAnteriorII110035RCA/D12529 19y and 1 month AnteriorII50027OM/PLA /PDA 1825 13 yearsAnteriorIII84031OM/IM2425 Mean 11 mo NYHA 2.7 …. 1.6 p<0.0001 EF 24%…..32% p<0.02 )

45. Muscular Biopsy Myoblast cells liberated through Enzymatic Digestion intercellular matrices

47. LVEF Alternative Therapies For the Treatment of Ischemic Cardiomyopathy Journal of the American College of Cardiology, Vol. 42, No. 12, 2003, Smits, Serruys et al. Patient Study. Study sponsored by Bioheart, Inc. 3 month, P=0.009, n=5; 6 month, P= 0.23, n=5. NYHA LVEF/NYHA IMPROVEMENT AT SIX MONTHS % Change

48. SAFETY: Multi center phase 1 trial

49. ICD or life vest if EF > 30 ICD EF > 20% < 40 % 46 patients 3 UK centres Glenfield, Leicester St Marys (Nic Peters) Royal Brompton (Philip Poole-Wilson) ICD EF > 20% < 40 % 46 patients 3 UK centres Glenfield, Leicester St Marys (Nic Peters) Royal Brompton (Philip Poole-Wilson)

50. Enrollment Began June 2005. Randomization ICD patients: 30MyoCell™,16 Treatment Arm (Myocell TM 150-800 x 10 6 ) 30 ICD patients - Control Arm (Standard Medical Therapy) 16ICD patients Baseline Evaluation Visit 1 (Week Screening: 46 ICD patients Bioheart Percutaneously Delivered Myoblasts EU Phase II Trial (SEISMIC™) : Standard medical therapy -6) ICD EF > 20% < 40 % 8 European Centres 3 UK : Glenfield, (AHG,JK) Leicester St Marys (Nic Peters) Royal Brompton (Philip Poole-Wilson ICD EF > 20% < 40 % 8 European Centres 3 UK : Glenfield, (AHG,JK) Leicester St Marys (Nic Peters) Royal Brompton (Philip Poole-Wilson

51. Bioheart US Phase I Trial (MYOHEART™) Primary Inclusion Criteria Prior MI involving anterior, lateral, posterior or inferior walls > 12 weeks old at time of implant Patients with prior placement of an ICD > 30 days prior to implant LVEF > 20% and < 40% by MUGA at screening NYHA Class II or III Ablitiy to walk at least 300 meters during 6-minute walk test Need for revascularization ruled out by coronary angiogram or noninvasive stress test within 6 months of screening Target region wall thickness of > 6 mm by echocardiography Age > 30 and < 80 years old Prior MI involving anterior, lateral, posterior or inferior walls > 12 weeks old at time of implant Patients with prior placement of an ICD > 30 days prior to implant LVEF > 20% and < 40% by MUGA at screening NYHA Class II or III Ablitiy to walk at least 300 meters during 6-minute walk test Need for revascularization ruled out by coronary angiogram or noninvasive stress test within 6 months of screening Target region wall thickness of > 6 mm by echocardiography Age > 30 and < 80 years old

52. SEISMIC Safety Endpoints Primary: –Defined Serious Adverse Events (SAEs) Secondary: –Routine Clinical laboratory test results –Holter monitoring, 12-lead ECG data –Overall patient survival –Assessment of the safety of the use of the MyoCath™, by Adverse Event assessment Primary: –Defined Serious Adverse Events (SAEs) Secondary: –Routine Clinical laboratory test results –Holter monitoring, 12-lead ECG data –Overall patient survival –Assessment of the safety of the use of the MyoCath™, by Adverse Event assessment

53. SEISMIC Efficacy Endpoints Primary: –Change in LVEF at 3 and 6 months by MUGA compared with baseline Secondary: –QOL Assessments (6-min. walk, NYHA class) –Hospitalization, readmissions or the need for medical treatment outside of hospitalizations –Echocardiography (global contractility, wall thickness and coronary perfusion improvements) Primary: –Change in LVEF at 3 and 6 months by MUGA compared with baseline Secondary: –QOL Assessments (6-min. walk, NYHA class) –Hospitalization, readmissions or the need for medical treatment outside of hospitalizations –Echocardiography (global contractility, wall thickness and coronary perfusion improvements)

55. Restenosis

56. ?

57. UK Infra structure

58. Questions that (should) concern us in the UK  Do we (the cardiological/scientific community) have a good idea about Questions that (should) concern us in the UK  Do we (the cardiological/scientific community) have a good idea about  Do we have the infra structure be involved  Do we have the infra structure be involved  Are the trials of sufficient quality to provide the answers to the questions being asked ?  What dose/concentration  Does the background science justify trial initiation  Are the trials relevant to our current practice ?  Is it safe ?  Which studies is the UK involved in?  Which in our (my) opinion is the likely way forward ?  Which delivery technique  Which cells

59. Questions that (should) concern us in the UK  Is the basic science sound Questions that (should) concern us in the UK  Is the basic science sound  Are the trials of sufficient quality to provide the answers to the questions being asked ?  Are the trials relevant to our current practice ? Maybe

60. British Collaborative on Stem Cell Research and the Heart Objective: To form a collaborative group of basic scientists and clinicians involved in research in stem cells and the heart, in order to perform joint research. Four meetings held in UCL, average attendance 50 people. Three groups formed: stem cells and vectors animal models clinical trials Objective: To form a collaborative group of basic scientists and clinicians involved in research in stem cells and the heart, in order to perform joint research. Four meetings held in UCL, average attendance 50 people. Three groups formed: stem cells and vectors animal models clinical trials

61. British Collaborative on Stem Cell Research and the Heart A “writing group” has met to define what clinical and basic research needs to be done. Joint grants will be applied for. A document will be circulated shortly. The clinical group will peer review protocols with an emphasis on safety and necessity of studies. The group has a close connection to the European Society of Cardiology Task Force on Stem Cells and the Heart A “writing group” has met to define what clinical and basic research needs to be done. Joint grants will be applied for. A document will be circulated shortly. The clinical group will peer review protocols with an emphasis on safety and necessity of studies. The group has a close connection to the European Society of Cardiology Task Force on Stem Cells and the Heart

62. General Summary and Conclusions  Too soon to draw any conclusions  T oo soon to draw any conclusions  Studies small, all PPCI, ? Safety concerns (restenosis)  Promising  S tudies small, all PPCI, ? Safety concerns (restenosis)  P romising

63. Bone-marrow stem cell Advantages Pluripotent stem cells can develop into cardiomyocytes Easy to isolate and grow well in culture Neovascularization can also occur at the site of the scar Can improve myocardial function Limitations New program of cell differentiation program is requiredNew program of cell differentiation program is required Efficiency of differentiation into adult cardiomyocytes may be limitedEfficiency of differentiation into adult cardiomyocytes may be limited Some studies difficult to replicateSome studies difficult to replicate Small trialsSmall trials LV improves anywayLV improves anyway UK PPCI makes applicability difficultUK PPCI makes applicability difficultLimitations New program of cell differentiation program is requiredNew program of cell differentiation program is required Efficiency of differentiation into adult cardiomyocytes may be limitedEfficiency of differentiation into adult cardiomyocytes may be limited Some studies difficult to replicateSome studies difficult to replicate Small trialsSmall trials LV improves anywayLV improves anyway UK PPCI makes applicability difficultUK PPCI makes applicability difficult Trials to date uncontrolled small numbers un-blinded Post AMI patient

64. Skeletal myoblasts Advantages Cells proliferate in vitro Ischemia resistant Transplanted cells can differentiate into slow-twitch myocytes, enabling cellular cardiomyoplasty Reduce dilatation and improve cardiac function Can use adult cells Limitations Likely do not develop new cardiomyocytes in vivo Electrical coupling to surrounding myocardial cells is probable but not definite Long-term stability of differentiated phenotype is unknown Arrhythmic potential Limitations Likely do not develop new cardiomyocytes in vivo Electrical coupling to surrounding myocardial cells is probable but not definite Long-term stability of differentiated phenotype is unknown Arrhythmic potential Chronic HF AMI patient

65. Transplantation of progenitor Cells AMI patients Intracoronary infusions are safe and feasible Increase in global LVEF Improvement in wall motion abnormalities Reduction in LVESV Complete normalization of CFR(< so in BMC) Increases in myocardial viability Intracoronary infusions are safe and feasible Increase in global LVEF Improvement in wall motion abnormalities Reduction in LVESV Complete normalization of CFR(< so in BMC) Increases in myocardial viability May need cytokines like G–CSF and this may be a problem

66. How long will the transplanted cells survive ? Can they be safely delivered PCI/endocardially Are they able to integrate ? Modified ? Do they have the ability to differentiate ? Will the cell transplant influence LV function ? Can we augment/enhance grafting, proliferation and function of stem cell implants ? Is it safe ? How long will the transplanted cells survive ? Can they be safely delivered PCI/endocardially Are they able to integrate ? Modified ? Do they have the ability to differentiate ? Will the cell transplant influence LV function ? Can we augment/enhance grafting, proliferation and function of stem cell implants ? Is it safe ? Stem Cell Tx appears to “work” Issues and questions 1 Stem Cell Tx appears to “work” Issues and questions 1

67. Issues and questions 2 How many cells do we need to transplant ? Which is the best way for delivery ? Should the donor cell chosen depend on the cause of heart failure/ ventricular dysfunction ? Which is the optimal time for cell transplantation ? What trials are needed to provide definitive answers ?  UK is involved, central organisation, ▓ Funding, Regulatory bodies (MHRA,COREC) collaboration, need for multi discipline are all challenges How many cells do we need to transplant ? Which is the best way for delivery ? Should the donor cell chosen depend on the cause of heart failure/ ventricular dysfunction ? Which is the optimal time for cell transplantation ? What trials are needed to provide definitive answers ?  UK is involved, central organisation, ▓ Funding, Regulatory bodies (MHRA,COREC) collaboration, need for multi discipline are all challenges

68. Cell transplantation is an exciting development Many questions NEED SUFFICIENTLY POWERED RANDOMISED CONTROLLED TRIALS Something (UK) interventional cardiologists need to be involved in Watch this space - new era for interventional cardiology Cell transplantation is an exciting development Many questions NEED SUFFICIENTLY POWERED RANDOMISED CONTROLLED TRIALS Something (UK) interventional cardiologists need to be involved in Watch this space - new era for interventional cardiology “EPC > Bone marrow > myoblasts “ 1.True quantity of effect 2. That it is the SCT that is having any effect 3.Safe 1.True quantity of effect 2. That it is the SCT that is having any effect 3.Safe

70. The Future of Stem Cells and the Heart Need for double blind randomised trials in myocardial infarction and heart failure of autologous bone marrow cells. These should be agreed nationally to avoid duplication. The procedure should not be given as a treatment to individual patients before results of above trials. A second wave of trials will follow using other sources of cells, particular engineered.

71. UK Stem Cell Foundation