Jozef Bartunek

Cardiovascular Center, Aalst, Belgium

Faculty of Biomedical Engineering, TU Eindhoven, NL

What’s New?

On Adult Stem Cells?

JB is a member of an institution which is a founding member of Cardio3 Biosciences

5th International Symposium on Stem Cell Therapy & Applied Biotechnology Madrid, April 2008

STEM CELLSTEM CELL

THERAPY THERAPY

“MECHANISMS AT WORK”• Target organ – heart• Remote Homing• Arrhythmias• Coronary vasculature

CELL PRODUCT• Type• Processing• Function and number

CELL DELIVERY

PATIENT• Disease • Risk Profile • Bone Marrow

Adult Stem Cells for Cardiac RepairFramework towards the optimization

Two Clinically Applied Cell Types

2000 2006

MenaschéAHA

AnversaNature

StrauerCirculation

GalinanesAHA

DiacrinDib

MagicMenasche

ZeiherCirculation

SteinhoffLancet

Steinhoff

SiminiakEHJ

TseLancet

TX-Brazil/HoustonPerin

MacLellanUCLA

BioheartSerruys

Myoblast

Bone Marrow Stem Cells

BoostLancet

Repair-AMI,ASTAMIJanssens

SeismicSerruys

2000 2008

Navarra

Abdel-Latif, Arch Intern Med 2007

Adult Stem Cells Therapy for Cardiac Repair

Overall clinical safety demonstrated over 1 to 2 years period

Adult Stem Cells for Cardiac RepairCell Type

Cardiac RepairCardiac Repair

Endothelial progenitor cellsCD34+, CD133+, CD31+, VEGFR 2+, VE-cadherin+

Hematopoetic stem cells C-kit+, CD34+, CD133+

CD31+

Mesenchymal stem cellsCD34-, CD45-,

Adherent, CD90+

Mononuclear BM cellsCD45+, CD14+

~ 1-2% CD34/CD133+

Skeletal Myoblasts

Stem Cell Type and Therapeutic Effect

• BM mononuclear fraction delivers functional response – No cell type omitted– Effect ~ cellular cross

talk between multiple cell types

Cell type

Potential of enriched Potential of enriched (hematopoietic) (hematopoietic)

cell populations should be cell populations should be InvestigatedInvestigated

-2.0

-1.5

-1.0

-0.5

0.0

CPC BMC

Regional Wall Motion

SD

/ch

ord

* *

Follow-up

Baseline

Shachinger V, JACC 2004

Similar functional effectsEPC vs MNC

Kawamoto A, Circulation 2006

CD34+ superior to MNC

Selected/Enriched Adult Stem Cells for Cardiac Repair

Coronary

Myocardial

Function PlannedCardio-myogenic

Autologous

Limited?

C-kit, Sca1, MDR1, Isl1,

Resident cardiac stem cells

Coronary

Myocardial

Perfusion Rand-DB

Ongoing

Pluripotency

Angiogenic

AutologousCD49D, Stro-1, CD166, CD44

Adipose-derived stem cells

Rand-DB Ongoing

Function

Perfusion

Multipotency

Paracrine

“Off-shelf” use

Myocardial

Systemic

Allogeneic

Autologous

CD166, Stro-1, CD44, CD106

BM mesenchymal stem cells

Coronary

Myocardial

Delivery

Perfusion

Function

Pre-clinical experience

Rand-DB Ongoing

Angiogenic

Paracrine

Autologous

Allogeneic (umbilical cord)

CD133, CD34

CD31

Hematopoietic stem cells

Clinical Studies

PotentialSourceMarkers

• Double-blinded, placebo controlled, randomized study

• Direct comparison of autologous BM MNC and autologous culture expanded BM MSCs in the canine model of the chronic myocardial infarction

• Multimodality functional and morphological assessment including, echocardiography, MRI and invasive pressure-volume loops

• Histology and gene expression analyses

Week - 2

Week +8

9 weeks

16 weeksWeek +16 : (-) Echocardiography

(-) MRI

Week +9 : (-) Echocardiography

(-) Pressure-volume loops

(-) MRI

Week +17 : (-) Sacrifice

(-) Histopathology

(-) Macro-morphometry

Week 0 : (-) Bone-marrow collection (BMNC)

(-) Cells injection

(-) Echocardiography

Week -11 : (-) Bone-marrow collection

(-) Coronary ligation

Baseline

(-) Echocardiography

Week -1 : (-) Echocardiography

(-) Pressure-volume loops

(-) MRI

Stem Cell Type and Therapeutic EffectDirect Comparison MSCs vs BMNCs

Mathieu M, McEntee K, Erasmus, ULB Brussels, ACC 2008

Mathieu M, Mc Entee K, Erasmus, ULB Brussels, ACC 2008

Stem Cell Type and Therapeutic EffectMSCs vs BMNCs

Superior effects of MNC vs MSCs in chronic MI:Superior effects of MNC vs MSCs in chronic MI:- Model specific?Model specific?- Disease/remodelling specific?Disease/remodelling specific?- Implications for the clinical trials?Implications for the clinical trials?

• Mice model of the myocardial infarction (cryoinjury, coronary ligation)• Injection of 2x105 MSCs (passage 3) or total BM from EGFP+ transgenic mice• Follow up from 29 to 268 days.

Stem Cell Type and Therapeutic EffectWord of Caution on Safety?

Breitbach M et al, Blood 2007Osteocalcin, Cy3 EGFP labeled stem cells

Damaged adult heart may not be able to recapitulate Damaged adult heart may not be able to recapitulate necessary millieu to stimulate myocardial specification resulting necessary millieu to stimulate myocardial specification resulting

in the limited efficacy or unwanted signalling/differentiation in the limited efficacy or unwanted signalling/differentiation of “naive” or plastic stem cells of “naive” or plastic stem cells

Adult Stem Cell Therapy

Olson, Nat Medicine 2004; Chien, Nature 2004; Wang, J Thorac Cardiovasc Surg 2001, Yoon, Circulation 2004, Breitbach, Blood 2007

Goals:- to improve cell function- to increase engraftment- to increase integration - to increase cell survival- to guarantee safety

From Non-modified Adult Stem Cells to“Second Generation Stem Cell Products”

Strategies:- Pharmacological

pretreatment- Genetic engineering- Tissue engineering- …..

“Second Generation Stem Cell Products” Increased efficacy after pretreatment with eNOS Enhancer

Sasaki et al, PNAS 2006

Improved Cell Function Enhanced Neovascularization

“Second Generation Stem Cell Products” SDF 1-engineered Skeletal Myoblasts

Enhanced neovascularization and functional improvementAskari et al., JACC 2004

SKMB AdSDF-1

+-

++

Vasc

ular

Den

sity

(ves

sels

/mm

2 )

0

10

20

30

40

50

“Second Generation Stem Cell Products” SCF-engineered MSCs

• Stem cell factor binds to c-kit and leads to bone marrow stem cells activation and mobilization• SCF is upregulated in the myocardial tissue after the infarction • Hypothesis: Upregulation of SCF in MSCs leads to more efficient myocardial repair

Fazel S et al, AHA 2007

“Second Generation Stem Cell Products” SCF-engineered MSCs - Word of Caution?

Stem cell therapy with engineered stem cells may enhance functional effects. However, outcome may be hindered by uncontrolled effect on proliferation

and growth with risk of tumor formation if both techniques are combined in “plastic” cells.

Fazel S et al, AHA 2007

““clues” from embryonic development?clues” from embryonic development?

“Second Generation Stem Cell Products” Cardiomyogenic Specification?

Selected Cardiomyogenic Growth Factors Pretreatment

“Second Generation Stem Cell Products” Cardiomyogenic Specification?

Dog model of chronic myocardial infarction- Injection of culture expanded MSCs- Injection of modified MSCs (coctail of growth factors including

BMP2, IGF-1 and bFGF) - Follow-up up to 12 weeks including histology

Non-modified vs Modified BMSCs in a Chronic Dog MI Model

Regional LV function and Cell Retention

0

1

2

3

4

5

% DiI + myosin+ cells/mm2

ModifiedBMSCs(n=5)

Non-modifiedBMSCs(n=3)

p=0.037

Bartunek et al, AJP 2007

% LV Wall Thickening

BL 2 4 wks 8 wks 12 wksBL 110

20

30

40

50%

*

Non-modified MSC, n=4Non-modified MSC, n=4Modified MSC, n=7Modified MSC, n=7

20October 2006Confidential

Problems encountered:

• Only cytoplasmic expression, but no nuclear translocation

of cardiac markers

• “Terminal’ cardiac commitment (functional excitation-

contraction coupling) in vitro was not achieved

• Suboptimal reproducibility when treatment applied to

mesenchymal stem cells from cardiac patients

“Second Generation Stem Cell ProductsSelected Cardiomyogenic Growth Factors Pretreatment

“Second Generation Stem Cell ProductsHigh Throughput Genomic and Proteomic Technology

A. Behfar & A. Terzic

Behfar A et al , J Exp Med 2007; Arell et al Stem Cells 2008; Nelson TJ et al, Stem Cells 2008; Faustino RS et al , Genome Biol 2008; Behfar A et al, Nat Clin Pract Cardiovasc Med 2006

Candidate effectors of cardiac differentiation were Candidate effectors of cardiac differentiation were identified using the comparative proteomics and genomics identified using the comparative proteomics and genomics

on the secretome of murine visceral endoderm-like cellson the secretome of murine visceral endoderm-like cells in response to TNF-in response to TNF-αα

Behfar et al, J Exp Med 2007; Arell et al , Stem Cells 2008

“Second Generation Stem Cell ProductsHigh Throughput Genomic and Proteomic Technology

The cardiogenic coctail secured guided differentiation of mouse embyronic stem The cardiogenic coctail secured guided differentiation of mouse embyronic stem cells into cardiopoietic cellscells into cardiopoietic cells

Mouse ESCs Guided mouse ESCs

Cardiogenic Cocktail

Behfar et al, 2007, J Exp Med 204: 405-420

“Second Generation Stem Cell ProductsHigh Throughput Genomic and Proteomic Technology

Guided cardiopoietic BM mesenchymal stem cellsGuided cardiopoietic BM mesenchymal stem cells: : - upregulation and nuclear translocation of cardiac transcription factors, upregulation and nuclear translocation of cardiac transcription factors, - sarcomeric organization and expression of the gap junction protein connexin 43sarcomeric organization and expression of the gap junction protein connexin 43- rhythmic calcium transients. rhythmic calcium transients.

“Second Generation Stem Cell ProductsCardiogenic Coctail in Adult BM-MSCs from Cardiac Patients

Behfar & Terizc, personnal communication

“Second Generation Stem Cell ProductsGuided Cardiopoietic BM-MSCs in the Mice Model of the Chronic MI

Guided cardiopoietic BM mesenchymal stem cells:Guided cardiopoietic BM mesenchymal stem cells: - superior effects on functional improvement in the chronically infarcted myocardium superior effects on functional improvement in the chronically infarcted myocardium as compared to nonmodified MSCsas compared to nonmodified MSCs- paralleled by the superior effects on neovascularization and cardiac differentiationparalleled by the superior effects on neovascularization and cardiac differentiation- no toxicity observedno toxicity observed Behfar & Terzic, personnal communication

Cardio³ BioSciences C-Cure™ I Clinical TrialProtocol Number: C3BS-C-07-02

EudraCT Number: 2007-007699-40

Sponsor: Cardio3 Biosciences, Braine L’alleud, Belgium

Goal:

To test the safety and efficacy of guided, autologous bone marrow-derived cardiopoietic mesenchymal stem cells

in ischemic cardiomyopathy

Stage B(n = 195)

Stage A(n = 45)

• A multicenter, prospective, open-label, sequential design with 2 parallel arms• Blinded core lab analyses• 2:1 randomization

Guided cellsn = 30

Control groupn = 15

Guided cellsn =130

Ischemic Heart Failure

2

1

~12 sites ~ 25-30 sites

Control groupn = 65

Cardio³ BioSciences C-Cure™ I Clinical Trial

Primary and Secondary Endpoints

Baseline 1-mo 3-mo 6-mo 9-mo 1-yr 18-mo 2-yrs

LVEF (Muga)LVEF (Muga) LVEF and volumes (Echo)

6-min walking distance Quality of Life Humoral Markers Spiroergometry Clinical events Arrhythmias Resource utilization

Major inclusion criteria

• Patient has history of previous myocardial infarction

• Patient on optimal and stable medical management

(state-of-the-art treatment conform the guidelines)

• Patient has congestive heart failure

NYHA class II or III and LV ejection fraction ≥ 15% and ≤ 40%

Treatment Procedure

• All patients (Phase A) will receive an ICD

• All patients under optimal medical management (CRT allowed if implanted > 6 months before randomization)

• Mesenchymal stem cells culture expanded and treated with the cardiogenic coctail

• Cardiomyogenic specification confirmed by the presence of the cardiac specific markers according to the release criteria

• NOGA-guided cells injection into the dysfunctional and viable myocardium

Trial Management

• Prinicipal Investigators: J. Bartunek, A. Terzic

• Steering Committee: J. Bartunek, A. Terzic, W. Wijns, M. Tendera, S. Henry

• Clinical Event Committee: W. Shen, M. Bertrand, G. Breithardt, H. Klein

• DSMB: S. Waldman, D. Meldrum, J. Thijssen

• Trial monitoring: Cardio3Biosciences, Belgium

• Independent core lab analyses: MUGA, Echocardiography, ECG/ICD

Trial Sites/Investigators – Phase A

Country Site Local PI MEC Status Regulatory Status/Country

Belgium Liege V.Legrand Approved Revision Pending

UZ Antwerpen C. Vrints Submitted

Genk M. Vrolix Submitted

Aalst M. Vanderheyden Approved

Germany Munchen W. Franz Submitted Submitted

Hamburg K-H. Kuck Submitted

Aachen R. Hoffmann Submitted

Switzerland Zurich F. Eberli Approved Submitted

Lugano T. Moccetti Submitted

Lausanne E. Eeckhout Planned

Netherlands Maastricht J. Waltenberger Submitted Planned 05/08

India Ahmedabad K. Parikh Planned Planned 05/08

Interplay between BM and heart after injury Multipotency of BM stem cells Variety of stem cell types Promising functional effects on cardiac repair in experimental setting

Limited effects of naive stem cells Fate of cells: survival, transdifferentiation and integration Optimal cell type? Mechanism? Timing? Delivery? Homing? What Patient?

“New age” in cardiac interventions: “The” fundamental solution for the myocyte loss Heart failure treatment and prevention

“Hype” effect Large scale randomized trials could be negative Intellectual conflict of interest Unforeseen safety problems Proliferation of uncontrolled small trials

Adult Stem Cells Therapy SWOT 2008

Cardiovascular Center, Aalst

Jozef Bartunek*, Marc Vanderheyden, William Wijns, Guy Heyndrickx,

King’s College London, UK

Jonathan Hill

Mayo Clinic, Rochester, MN

Andre Terzic

Atta Behfar

Cardio3 Biosciences, Brussel

Vincienne Gaussin

Sophie Henry

Christian Homsy

Aurrore de Lavareille

Naima Mazouz

Phillipe Willemsen

*Biomedical Engineering, TU Eindhoven, NL Birgit Faber Carlijn Van Bouten Anthal Smits Mark Post

ULB Brussels, Belgium Kathleen McEntee Myreille Mathieu

University of Gent, Belgium Bart Vandekerkhove Frank Timmermans

Ike W. Lee