Cardiovascular Stem Cell Therapies after Myocardial Infarction: Can We Regenerate the Human Heart? Vincent J. Pompili, MD, FACC Professor of Internal Medicine Director of Interventional Cardiovascular Medicine Director Cardiovascular Cell-Based Therapies

Vincent J. Pompili, MD, FACC

Professor of Internal Medicine

Director of Interventional Cardiovascular Medicine

Director Cardiovascular Cell-Based Therapies

Regenerative Medicine and Cardiovascular Disease

Regenerative Medicine?

Cell Therapy-Regenerative Medicine

UCB Blood Marrow Muscle Adipose Embryonic

The Stem Cell Dogma… Circ Res, 2005; 96: 151 – 63.

Improved clinical outcome after intracoronary administration of bone marrow-derived progenitor cells in acute myocardial infarction: final results of the REPAIR-AMI trial Volker Schächinger, Sandra Erbs, Albrecht Elsässer, Werner Haberbosch, Rainer Hambrecht, Hans Hölschermann, Jiangtao Yu, Roberto Corti, Detlef G. Mathey, Christian W. Hamm, Tim Süselbeck, Nikos Werner, Jürgen Haase, Jörg Neuzner, Alfried Germing, Bernd Mark, Birgit Assmus, Torsten Tonn, Stefanie Dimmeler, and Andreas M. Zeiher for the REPAIR-AMI investigators Eur Heart J 2006; doi:10.10.1093/eurheartj/ehl388

Background Acute Chronic Myocardial Heart Infarction Failure infarct chronic expansion LV- dilatation p = 0.014 Proof-of-concept 6 Intracoronary infusion of bone marrow-derived 4 progenitor cells (BMC) 3-7 days after AMI 2 enhances recovery of 3.0 ± 0.7 5.5 ± 0.7 LV contractile function 0 Placebo BMC N Engl J Med after 4 months (LV angiogram) 2006; 355:1210 n = 92 n = 95

Study Flowchart - Double-blind, placebo-controlled, randomized multicenter trial - Patients with acute MI (STEMI) 204 sucessfully revascularized (stent PCI) Bone marrow aspiration (all patients) Placebo BMC & Randomization ( = intention-to-treat) n = 103 n = 101 -attempted (day 3-7) n = 101 n = 101 -performed n = 98 n = 101 Complete LV angio analysis at 4 months n = 92 n = 95 4 months follow up 103 101 Lost to 12 months follow up n = 3 12 months follow up 100 101 Dead n = 6 n =2 Eur Heart J 2006; doi:10.10.1093/eurheartj/ehl388

12 months clinical follow up Placebo BMC Per patient analysis p value n = 103 n = 101 number of patients Death (n) 6 2 0.28 - Cardiac (n) (AMI, myocard. rupture, sudden death, heart failure) 4 2 - Cardiovascular (n) (stroke) 1 - Non-cardiovascular (n) (cancer) 1 Myocardial reinfarction (n) 6 0 0.029 Rehospitalization for heart failure (n) 3 0 0.25 Revascularization (n) 37 22 0.026 -T T arget vessel revascularization (n) 26 16 0.097 --Stent thrombosis (n) 3 1 0.62 - Non-target revascularization (n) 16 7 0.052

12 months clinical follow up - Kaplan Meier Analysis - - Death, MI, Rehospitalization for heart failure - 100 BMC 90 Placebo 80 p = 0.006 (log rank) 70 60 0 0 100 200 300 360 days # exposed Placebo 103 93 89 85 79 to risk BMC 101 99 99 98 85 V. Schächinger et al., Eur Heart J 2006; doi:10.10.1093/eurheartj/ehl388

12 months clinical follow up - Kaplan Meier Analysis - - Death, MI, Revascularization - 100 90 BMC 80 70 60 p = 0.010 (log rank) Placebo 0 0 100 200 300 360 days # exposed Placebo 103 91 68 63 55 to risk BMC 101 97 80 77 66 V. Schächinger et al., Eur Heart J 2006; doi:10.10.1093/eurheartj/ehl388

Summary Intracoronary infusion of bone marrow-derived mononuclear cells in patients with reperfused acute myocardial infarction  is safe does not accelerate atherosclerosis or restenosis  is associated with significant reduction of the > combined endpoint death, MI or rehospitalization for heart failure > predefined endpoint death, MI or revascularization within 12 months after therapy.

does not accelerate atherosclerosis or restenosis

 is associated with significant reduction of the > combined endpoint death, MI or rehospitalization for heart failure

Clinical implications Intracoronary infusion of bone marrow-derived progenitor cells, improving left ventricular function, may also reduce cardiovascular events after revascularized acute myocardial infarction Large-scale, prospective, clinical endpoint trials are warranted to confirm the effects on mortality and morbidity in patients with acute myocardial infarctions

Alternate Pathway to Hematopoietic Stem Cell Development

Hematopoietic Stem Cell Lineage Hemangioblasts 133+ 34+ 31+ Hematopoietic Stem Cells Endothelial Cells Cell Differentiation Pathway Early Stem Cells Terminally Differentiated Cell Surface Markers

Cell populations 133+ vs. 34+ selection using CliniMacs or Isolex systems 133+/34-/31- 133+/34+/31- 133- /34+/31- 133- /34+/31+ Further Differentiation 133+ selection 34+ selection Cell Differentiation Pathway Hemangioblasts Hematopoietic Stem Cells Endothelial Cells 31+ are MHC +

Isolation of CD133 cells from UCB or marrow Y UCB MNC Anti-CD133 antibody with magnetic bead (Miltenyi Biotec) Negative fraction - contains all cells but labeled CD133 cells Positive fraction - contains all labeled CD133 cells

vWF staining via photo phase contrast microscopy 40x. Brown perinuclear stain = vWF particles (immunoperoxidase conjugated to secondary Ab’s reacting with perinuclear vWF)

vWF staining via photo phase contrast microscopy 40x.

Brown perinuclear stain = vWF particles (immunoperoxidase conjugated to secondary Ab’s reacting with perinuclear vWF)

In Vitro Differentiation of CD133+ into Vascular Endothelium Cells acLDL uptake pattern Lectin staining acLDL + lectin stain (40x confocal microscope) A B C

acLDL uptake pattern Lectin staining acLDL + lectin stain

(40x confocal microscope)

Angiogenic Factors Secreted from CD133 Unstimulated Cells

A. HUVECS alone B. HUVECS plus CD +133 Organotypic culture systems to study HUVEC-stromal cell interactions. Stromal cells were grown to confluency prior to being seeded with HUVEC alone (A) or with equivalent numbers of HUVECs and EPCs. After 2 weeks, cultures were fixed and permeabilized with 60% acetone/PBS and stained with anti-CD31-FITC antibody. EPCs cells enhances both the formation of tubules and stimulates the proliferation of HUVECs Field 2 Field 3 Field 1 Field 2 Field 3 Field 1

A. HUVECS alone B. HUVECS plus CD +133

Organotypic culture systems to study HUVEC-stromal cell interactions. Stromal cells were grown to confluency prior to being seeded with HUVEC alone (A) or with equivalent numbers of HUVECs and EPCs. After 2 weeks, cultures were fixed and permeabilized with 60% acetone/PBS and stained with anti-CD31-FITC antibody. EPCs cells enhances both the formation of tubules and stimulates the proliferation of HUVECs

UCB CD133 + Cells and Vascular Flow Control CD133 + Pre-ligation Post-ligation 28 days Finney M, Joseph M, Martin J, Hedrick D, Swan J, Kadereit S, Kozik M, Emancipator S, Haynesworth S, Fu P, Laughlin M, Pompili VJ, Comparison of Umbilical Cord Blood versus Marrow Derived Endothelial Precursor Cells in Mediating Neovascularization in Response to Vascular Ischemia. Biology of Blood and Marrow Transplantation. 2006 12(5): 585-593

Clinical Development SEACOAST Trial Safety and Efficacy of Autologous, Intra Coronary Stem Cell Injections in Total Coronary Artery Occlusions Single center NIH sponsored Phase I safety Dose escalation of CD 133+ Cell Direct coronary infusion into collateral circ. of occluded vessel Endpoints Primary -safety, major adverse cardiac events Secondary - ETT, perfusion, cardiac function Warren Sherman, M.D. – Columbia University

Safety and Efficacy of Autologous, Intra Coronary Stem Cell Injections in Total Coronary Artery Occlusions

Single center

NIH sponsored

Phase I safety

Dose escalation of CD 133+ Cell

Direct coronary infusion into collateral circ. of occluded vessel

Endpoints

Primary -safety, major adverse cardiac events

Secondary - ETT, perfusion, cardiac function

Warren Sherman, M.D. – Columbia University

SEACOAST Trial Inclusion Criteria One region of chronically ischemic myocardium perfused by a coronary artery which is now 100% occluded and not revascularizable by conventional percutaneous methods. Well-established collateral vessels at least 1.5-mm luminal diameter by coronary angiography to the chronically ischemic myocardium. Evidence of viable myocardium in the area supplied by collateral conduits demonstrated by nuclear (sestamibi) stress imaging. LVEF >45% per 2D echocardiogram. Patient must experience class II – IV angina as defined by the Canadian Cardiovascular Society (CCS).

One region of chronically ischemic myocardium perfused by a coronary artery which is now 100% occluded and not revascularizable by conventional percutaneous methods.

Well-established collateral vessels at least 1.5-mm luminal diameter by coronary angiography to the chronically ischemic myocardium.

Evidence of viable myocardium in the area supplied by collateral conduits demonstrated by nuclear (sestamibi) stress imaging.

LVEF >45% per 2D echocardiogram.

Patient must experience class II – IV angina as defined by the Canadian Cardiovascular Society (CCS).

SEACOAST 18 Month Follow up Clinical Outcomes Improvement in Ischemia Improvement in EF Seattle Angina Questionnaire

Clinical Outcomes

Improvement in Ischemia

Improvement in EF

Seattle Angina Questionnaire

Upcoming Trials

Magellan ™ Rapid Bedside Stem Cell Processing System FDA Approved Device Existing business in Sternal Closure Small marrow volume yields a stem cell rich dose Quick (15min process)

FDA Approved Device

Existing business in Sternal Closure

Small marrow volume yields a stem cell rich dose

Quick (15min process)

MNC Yield with Magellan ™

Rapid Bedside Fractionation of Marrow 510K approval: BK030040 platelet rich plasma can be mixed with autograft and/or allograft bone prior to application to an orthopedic site as deemed necessary by the clinical use requirements. BK040068 for the fractionation of bone marrow. The fractionated unit can be made from a mixture of blood and bone marrow.

510K approval: BK030040 platelet rich plasma can be mixed with autograft and/or allograft bone prior to application to an orthopedic site as deemed necessary by the clinical use requirements.

BK040068 for the fractionation of bone marrow. The fractionated unit can be made from a mixture of blood and bone marrow.

STEM-PREP intra-myocardial delivery

N=3 N=5 N=2 N=4 N=3 Magellan® vs. Ficol Study

Evaluation of the Magellan® Autologous Platelet Separator and STEM PREP Kit Baseline Angiogram

Evaluation of the Magellan® Autologous Platelet Separator and STEM PREP Kit Occlusion

Quantification of Cardiac Function Evaluation of the Magellan® Autologous Platelet Separator and STEM PREP Kit

Evaluation of the Magellan® Autologous Platelet Separator and STEM PREP Kit Ejection Fraction

Evaluation of the Magellan® Autologous Platelet Separator and STEM PREP Kit Infarct Size without CTO animals

Evans Blue staining was used to determine the area at risk (AAR) for infarction during the ischemic episode. Evaluation of the Magellan® Autologous Platelet Separator and STEM PREP Kit A 1% solution of 2,3,5-triphenyltetrazolium chloride (TTC) to identify the infarcted region. Double Staining Technique

Area at risk outlined in green Infarct zone outlined in red AAR 810.85 mm 2 Infarct 501.06 mm 2 % AAR infarcted 61.8% Evaluation of the Magellan® Autologous Platelet Separator and STEM PREP Kit Quantification of Infarct Size

Evaluation of the Magellan® Autologous Platelet Separator and STEM PREP Kit Infarct Size

Evaluation of the Magellan® Autologous Platelet Separator and STEM PREP Kit Infarct Size without CTO animals

State of Ohio RCP Plan Programs Magellan STEM-PREP for CLI - Year 1 Objective: 1 st Patient Enrolled – Q1 ‘10 - Status: Recruiting Site/Investigator Magellan VANCO-PREP for Infection Control - Year 1 Objective: 1 st Patient Enrolled – Q1 ’10 - Status: Recruiting Site/Investigator Magellan STEM-PREP with Cardiac TMR - Year 1 Objective: 1 st Patient Enrolled – Q1 ‘10 - Status: Recruiting Site/Investigator Magellan STEM-PREP/CD with Coronary Infusion - Year 1 Objective: 1 st Patient Enrolled – Q1 ‘10 - Status: Recruiting Site/Investigator

Magellan STEM-PREP for CLI - Year 1 Objective: 1 st Patient Enrolled – Q1 ‘10 - Status: Recruiting Site/Investigator

Magellan VANCO-PREP for Infection Control - Year 1 Objective: 1 st Patient Enrolled – Q1 ’10 - Status: Recruiting Site/Investigator

Magellan STEM-PREP with Cardiac TMR - Year 1 Objective: 1 st Patient Enrolled – Q1 ‘10 - Status: Recruiting Site/Investigator

Magellan STEM-PREP/CD with Coronary Infusion - Year 1 Objective: 1 st Patient Enrolled – Q1 ‘10 - Status: Recruiting Site/Investigator

Cell Therapy-Regenerative Medicine

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