Hüseyin Ince MD, PhDUniversity of Rostock

Department for Internal Medicine, Cardiology

hueseyin.ince@med.uni-rostock.de

Hüseyin Ince MD, PhDUniversity of Rostock

Department for Internal Medicine, Cardiology

hueseyin.ince@med.uni-rostock.de

Preclinical session

4th International Symposium on Stem Cell Therapy and Applied Cardiovascular Biology

Madrid (Spain), April 26 – 27, 2007

What is stem cell therapy?

What is stem cell therapy?

Stem cells in cardiomyopathy

Stem cells in cardiomyopathy

Ultrastructural findings of idiopathic dilated cardiomyopathy (IDCM):

Myocyte atrophy and myofilament loss

Main epicardial coronary arteries are shorter and smaller

Microvascular density is reduced in the epicardium

Moreover, this defective vascularization is associated with reducedmyocardial expression of vascular -catenin, an important angiogenic regulator

This study shows that both vasculogenesis and angiogenesis arealtered in IDCM

Antoni-Bayes Genís

Simultaneous autologous transplantation of co-cultured stem cells (SC) and skeletal myoblasts (SM) in an experimental model of Chagas disease

Seven Wistar rats received autologous transplant of 5,4x106 and 8,0x106 cells into the LV wall

Control group (n=8) received culture medium

After 4 weeks, cell transplantation significantly improved EF and reduced LVEDV

No change has been observed in the control group.

Conclusion: the co-transplant of SC and SM is functionally effective in Chagas disease (at least in Wistar rats)

LC Guarita-Souza

The Multicenter Randomized Cell Therapy Trial in Cardiopathies(MiHeart) is composed of four independent clinical trials each one dealing with a specific cardiomyopathy:

Chagasic cardiomyopathy

Dilated cardiomyopathy

Acute myocardial infarction

Chronic ischemic heart disease

MiHeartMiHeart

All trials are multicenter, randomized, double-blind and placebo All trials are multicenter, randomized, double-blind and placebo controlled.controlled.

In each trial 300 patients will be enrolled.In each trial 300 patients will be enrolled.

Additionally, half of the patients will receive the autologous bone marrow Additionally, half of the patients will receive the autologous bone marrow cells while the other half will receive placebo (saline with 5% autologous cells while the other half will receive placebo (saline with 5% autologous serum). serum).

The method for cell delivery is intramyocardial for the chronic ischemic The method for cell delivery is intramyocardial for the chronic ischemic heart disease and intracoronary for all others.heart disease and intracoronary for all others.

Primary endpoint for all studies will be the difference in ejection fraction Primary endpoint for all studies will be the difference in ejection fraction six and twelve months after intervention in relation to the basal ejection six and twelve months after intervention in relation to the basal ejection fraction. fraction.

MiHeartMiHeart

Incidence of arrhythmias and conduction disturbancesIncidence of arrhythmias and conduction disturbances Number of ventricular extra systoles in six months and one year.Number of ventricular extra systoles in six months and one year. Number of sustained ventricular tachycardia episodes in six months and Number of sustained ventricular tachycardia episodes in six months and one year.one year. Number of non sustained ventricular tachycardia episodes in six months Number of non sustained ventricular tachycardia episodes in six months and one year.and one year. Incidence of atrial fibrillation in six months and one year.Incidence of atrial fibrillation in six months and one year.

New-onset of atrioventricular or intraventricular conduction disturbances.New-onset of atrioventricular or intraventricular conduction disturbances.

Need for artificial pacemaker implantation.Need for artificial pacemaker implantation.

MiHeart saftey endpointsMiHeart saftey endpoints

SDF-1 (ligand of CXCR4 receptor) is a crucial factor involved in progenitor cellmobilization and homing

There is an increase of SDF-1 production and release from ischemicmyocardium generating the SDF-1 gradient towards the heart in AMI

In patients with acute myocardial infarction the absolute number ofCD34+CXCR4+, CD34+c-kit+ and c-met+ cells was significantly higher incomparison to patients with stable angina and healthy subjects

Homing of tissue committed stem cells is also dependenton leukemia inhibitory factor (LIF) - LIF receptor and hepatocyte growth factor (HGF) - c-met axis

Wojciech Wojakowski

G-CSF in AMI G-CSF in AMI

• Langendorff perfusion model w/wo 300 ng/ml G-CSF

• early contraction with G-CSF in reperfusion

• improved hemodynamics

• less myocardial necrosis

Direct effects of G-CSF on cardiac function after ischemia reperfusioninjury

Firstline-AMI

Harada et al., Nature Medicine 2005;11:305-11

Firstline-AMI

G-CSF group(n = 25)

Control group(n = 25)

p

Infarct related artery, LAD RCA CFX

1393

1384

Onset of AMI to PCI, min 293 ± 115 299 ± 123 ns

PCI plus stent, % 100 100 ns

Abciximab (adjusted dose), % 100 100 ns

Clopidogrel (300 mg loading) 100 100 ns

TIMI III flow post PCI, % 100 100 ns

Creatine kinase max, U/l 3706 ± 1818 3660 ± 2204 ns

Creatine kinase MB max, U/l 385 ± 240 361 ± 204 ns

Time from PCI to first G-CSF injection, min 89 ± 35 - na

AMI = acute myocardial infarction;

PCI = percutaneous coronary intervention;

Angiographic and AMI related characteristicsAngiographic and AMI related characteristics

Ince et al. Circulation 2005; 112:3097-3106

30

35

40

45

50

55

60

Baseline 35 days 4 months

LVEF (%)

30

35

40

45

50

55

60

Baseline 35 days 4 months

Low dose DobutamineRest

LVEF (%)

G-CSF (n=25)

Control (n=25)

48 4

51 4

46 4

54 8

43 5

47 5

54 5

56 4

51 5

59 7

51 8

53 5

p < 0.002 p < 0.001 p < 0.002

p < 0.001

Functional echocardiographic parameters

Firstline-AMI

LVEF

Ince et al. Circulation 2005; 112:3097-3106

6 months angiographic results

0

2

4

6

8

10

12

14

16

18

20

6m binary restenosis rate (%)0,5

0,52

0,54

0,56

0,58

0,6

0,62

0,64

6m late lumen loss (mm)1,9

1,92

1,94

1,96

1,98

2

6m MLD (mm)

16

20

0.60 0.230.61 0.18

1.96 0.50

1.95 0.38

ns ns

ns

Firstline-AMI

G-CSF (n=25)

Control (n=25)

Restenosis parameters (in segments)

Restenosis MLDLate lumen loss

Ince et al. Circulation 2005; 112:3097-3106

G-CSF and RestenosisG-CSF and Restenosis

Bone marrow mobilization strategies after the MAGIC trial.

No risk of restenosis

Bone marrow mobilization strategies after the MAGIC trial.

No risk of restenosis

G-CSF(n=60)

Control(n=33)

p

post PCI

MLD, (mm)2.73±0.49 2.61±0.36 0.25

Stenosis, (%)12.3±9.5 10.3±8.5 0.32

QCA (median 6 months)

MLD, (mm)2.27±0.61 2.04±0.44 0.07

Stenosis, (%)19.5±9.8 20.9±11.0 0.51

LLL, (mm)0.46±0.37 0.57±0.28 0.11

Delta-EF after G-CSF in AMI

-4

-2

0

2

4

6

8

10

1 2 3 4 5 6 7

G-CSF

Control

3D-Säule 3

G-CSF

Control

FIRSTLI

NE-AM

I

Circul

ation

200

5

Küthe

et a

l.

Am H

eart

J 20

05Val

gim

igli e

t al.

EHJ 20

06Kom

uro

et a

l.

Circul

ation

200

5de

Lez

o et

al.

Rev E

sp C

ario

l 20

05 STEMM

I

Circul

ation

200

6

REVIVAL-

2

JAM

A 200

6

Harada et al., Nature Medicine 2005;11:305-11

• G-CSF improves cardiac function

• Dose and time sensitive effects

Firstline-AMI G-CSF early after MIG-CSF early after MI

Myoblasts in CHD Myoblasts in CHD

BiopsyBiopsy

GMP Cell ExpansionGMP Cell Expansion

10 g10 g

Skeletal Myoblast Transplantation : Steps of the Procedure

Felipe Prosper

Two months after induction of myocardial infarction (MI), Goettingen miniature pigs underwent autologous SkM transplant either by direct surgical injection (n=6) or percutaneous access (n=6)

Control animals received media alone (n=4)

Animals received a median of 407.55±115x106 BrdU labeled autologousSkM

Functional analysis was performed by 2D echocardiography

Myoblast transplant was associated with a significant increase in LVEF (p<0.01), increased vasculogenesis, decreased fibrosis (p<0.05) as compared with control animals No differences were found between groups receiving SkM by percutaneous or surgical access

Myoblast Tx : Phase I Surgical Trials

Author No. Pts No. Cells CABG LV Function FU

+ cells Global Cell-Tx

Herreros 12 221 x 106 + 3 mo.

Eur Heart J 2003 EF : 35% to 53% at 3 mo. WMSI : 2.64 to 1.64

Siminiak 10 4 x 105 -5 x 107 + 12 mo.Am Heart J 2004 EF : 35% to 42% at 4 mo.

4/10 segments improved

Dib 24 1 x 107 -3 x 108 + NA 45 mo.

Circulation 2005 EF : 28% to 36% at 2 yrs

Myoblast Tx : Phase I Interventional Trials

Author No. Pts No. Cells LV Function FU

Global Cell-Tx

Smits 5 220 x 106 6 mo.JACC 2003

EF : 36% to 41%

Ince 6 220 x 106 12 mo.JEVT 2004

EF : 24% to 32%

Lack of coupling between grafted myoblasts and host

cardiomyocytes

Embryonic myosin

Connexin 43

No action potentials

Léobon et al. PNAS, 2003;100:7808-11.

Patricia LemarchandPossible Mechanisms of Myoblast-Related Arrhythmias

Impulse propagationResting statePeak of action potentials

Slowing of conduction,wave break & reentry

Abraham et al. Circ Res 2005;97:159-67.

Delayed cardiacrepolarization

Stretch activation bycontracting myotubes

Itabayashi et al.Cardiovasc Res 2005;67:561-70.

Bigger et al. New Engl J Med 1997;337:1569-75.

Actuarial incidence of first discharges : 50% at 1 year and 57% at 2 years

Probability of Discharge of a First Shock in the ICD-Implanted Patients of the PATCH Trial

Implantation of Skeletal Myoblasts :Lessons from Phase I Trials

Efficacy DataInability to draw meaningful conclusions from early studies because of :The small sample sizesThe lack of control groupsThe confounding effect of associated CABGThe dissociation between improved LV function and

achievement of true myocardial regeneration

Only randomized double-blind placebo-controlled adequately powered trials will provide a meaningful assessment of the risk-benefit ratio (In MAGIC the high dose cell group demonstrated a significant decrease in LV enddiastolic and endsystolic volumes)

Concept of stent-induced “Aortic Reconstruction”

before stentgraftbefore stentgraft

Occlusion of proximal Entry

Occlusion of proximal Entry

Reconstruction of TLReconstruction of TL

1 year after stentgraft

Diagnosis

Treatment

Antoine LafontAntoine Lafont

Aortic aneurysm formation is associated with matrix metalloproteinase-9 (MMP-9) activity

Gingival healing is embryo-like in contrast to arterial healing

This could be attributed to the gingival fibroblast

Concept of using gingival fibroblast healing properties in arteries

Gingival fibroblasts reduce MMP-9 expression by increasing TIMP-1 synthesis

Vascular transfer of gingival fibroblasts could be a promising approach to treat aortic aneurysms

Aortic aneurysm formation is associated with matrix metalloproteinase-9 (MMP-9) activity

Gingival healing is embryo-like in contrast to arterial healing

This could be attributed to the gingival fibroblast

Concept of using gingival fibroblast healing properties in arteries

Gingival fibroblasts reduce MMP-9 expression by increasing TIMP-1 synthesis

Vascular transfer of gingival fibroblasts could be a promising approach to treat aortic aneurysms

Kai PinkernellKai Pinkernell

Preclinical evaluations of adipose derived stem and regenerative cells (ADRCs) have been carried out in acute and chronic cardiac diseases showing an improvement of cardiac function as well as a reduction in remodeling

The PRECISE trial in patients with chronic myocardial ischemia has been started

The APOLLO trial in patients with AMI is scheduled to follow soon in order to investigate the safety and feasibility of ADRC therapy in patients

Both trials are designed as prospective, double blind, randomized, dose escalating trials

Preclinical evaluations of adipose derived stem and regenerative cells (ADRCs) have been carried out in acute and chronic cardiac diseases showing an improvement of cardiac function as well as a reduction in remodeling

The PRECISE trial in patients with chronic myocardial ischemia has been started

The APOLLO trial in patients with AMI is scheduled to follow soon in order to investigate the safety and feasibility of ADRC therapy in patients

Both trials are designed as prospective, double blind, randomized, dose escalating trials

ConclusionsConclusions

1. Find the best stem cell types or cytokines for repair of cardiovascular disease.

2. Identify cardiovascular stem cells among circulating stem cells; adipose tissue; epithelial cells; bone marrow-derived stem cells and compare their capabilities in cardiovascular repair in relevant experimental animal models.

3. Identify roles of specific stem cell types or cytokines in reparative medicine and in cardiac regeneration and vascular repair.

1. Find the best stem cell types or cytokines for repair of cardiovascular disease.

2. Identify cardiovascular stem cells among circulating stem cells; adipose tissue; epithelial cells; bone marrow-derived stem cells and compare their capabilities in cardiovascular repair in relevant experimental animal models.

3. Identify roles of specific stem cell types or cytokines in reparative medicine and in cardiac regeneration and vascular repair.

ConclusionsConclusions

4. Identify optimal cell numbers and methods of administration of stem cells in cardiovascular repair.

5. Identify clinical problems most susceptible to stem cell or cytokine treatments.

4. Identify optimal cell numbers and methods of administration of stem cells in cardiovascular repair.

5. Identify clinical problems most susceptible to stem cell or cytokine treatments.

PatientsPatients

STEM CELLSSTEM CELLS

AcuteAcute ChronicChronic

AGEAGEGenderGender

EMBRYONICEMBRYONIC ADULTADULT

BM, Blood, UCBBM, Blood, UCBFatFat MuscleMuscle

Mb, Fb, SP cellsMb, Fb, SP cells

Organ specific-HeartOrgan specific-Heart

MIMI IDCMIDCM HFHFAnginaAngina

CM (Parenchyma)CM (Parenchyma) Stroma (MSCs) MNCMNC

EPCs, EPCs, CD34+, CD34+, AC133+AC133+SubsetsSubsets

Comparison of REPAIR-AMI versus ASTAMIComparison of REPAIR-AMI versus ASTAMI

0

10

20

30

40

50

60

70

80

REPAIR ASTAMI

SDF-1 basal SDF-1 basal SDF-1

*

Inva

sio

n (

x10E

3) /1

0E6

BM

C

Healthy Controls CAD-Patients

+

0

20

40

60

80

100

120

140

REPAIR ASTAMI

basal SDF-1 basal

*

*

Inva

sio

n (

x10E

3) /1

0E6

BM

C

+

Invasion activityInvasion activity

Phase I Trial Objectives To determine the safety and tolerability of 3 different

intravenous doses of Provacel compared to placebo in subjects with AMI

To evaluate the effect of Provacel on exploratory efficacy endpoints

Design Multi center study, randomized, double-blind, placebo-

controlled, dose escalation study 48 adult subjects with AMI (powered to show safety and

preliminary efficacy) 3 dose escalated cohorts and 1 safety cohort at the highest

tolerated dose in a 2:1 ratio of active to placebo

Phase I Trial Objectives To determine the safety and tolerability of 3 different

intravenous doses of Provacel compared to placebo in subjects with AMI

To evaluate the effect of Provacel on exploratory efficacy endpoints

Design Multi center study, randomized, double-blind, placebo-

controlled, dose escalation study 48 adult subjects with AMI (powered to show safety and

preliminary efficacy) 3 dose escalated cohorts and 1 safety cohort at the highest

tolerated dose in a 2:1 ratio of active to placebo

Phase I Study Design