keng ang vii international symposium on stem cell therapy madrid, 6-7 may 2010

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

Intramuscular injection and cytokine mobilisation of bone marrow cells repair infarct myocardium

Orlic D et al Nature 2001;410:701Orlic D et al PNAS 2001;98:10344

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

• 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

1st Phase Study on BMCs

Galiñanes et al. Cell Transplantation 2004;13:7-13

Before Surgery

6 weeks 10 months 2 years

After Surgery

1

2

3

4

LV s

eg

me

nta

l WM

SI

Rest Low Dobutamine Peak Dobutamine

Before Surgery

6 weeks 10 months 2 years

After Surgery

Before Surgery

6 weeks 10 months 2 years

After Surgery

**

**

* *

BM Transplant Alone (n=11 segments)

Bypass Graft Alone (n=13 segments)

BM Transplant + Bypass Graft (n=10 segments)

Galiñanes et al. Cell Transplantation 2004;13:7-13

Dobutamine Stress Echocardiography

2nd 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)

Study Design63 eligible patients

undergoingCABG

Control IM IC

BMCs → mid-depth

of scar(500µl/injection)

BMCs →via graft

Supplying scarNo BMCs

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

% Systolic Fractional Thickening in Scarred Segments

(Systolic segmental thickness – Diastolic segmental thickness)% FT= ------------------------------------------------------------------------------------------------ x 100

Diastolic segmental thickness

LOW DOSE DSE

-20

-15

-10

-5

0

Control IM IC

Treatment group

%F

TPreop Postop

REST

-20

-15

-10

-5

0

Control IM IC

Treatment group

%F

T

Preop Postop

% Infarct Volume

0

10

20

30

40

50

60

70

80

Control IM IC

Treatment group

%In

farc

t v

olu

me

Preop Postop

Infarct volume% Infarct volume = --------------------------------------------------------- x 100% Left ventricular myocardial volume

Left Ventricular Ejection Fraction

0

5

10

15

20

25

30

35

40

45

Control IM IC

Treatment group

%

Preop Postop

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

Martin-Rendon E et al. Eur Heart J 2008;29:1807

If BMCs cannot differentiate into cardiac tissue, is the observed

beneficial effect due to

improvement in cell survival stimulation of cardiac progenitor cells

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

Anti-ischemic Effect of BMC

0

0.5

1

1.5

CK

re

lea

se (

IU/m

g w

et

tiss

ue

)

control bmc

*

0

5

10

15

Ce

ll n

ecr

osi

s (%

of

ae

rob

ic)

control bmc

*0

5

10

15

Ce

ll a

po

pto

sis

(%o

f a

ero

bic

)

control bmc

*

Kubal C et al J Thorac Cardiovasc Surg 2006;132:1112

CK

rele

ase

(IU

/mg

wet

tiss

ue)

0

0.5

1

1.5

*

Control- BM +BM - BM +BM

Chelerythrine

The Role of PKC

Kubal C et al J Thorac Cardiovasc Surg 2006;132:1112

Cell

necro

sis

(% o

f aero

bic

)

-10

0

10

20

30

40

*

Control- BM +BM - BM +BM

Chelerythrine

Cell

ap

op

tosi

s (%

of

aero

bic

)

0

10

20

30

40

*

Control- BM +BM - BM +BM

Chelerythrine

The Role of p38MAPK

CK

rele

ase

(IU

/mg

wet

tis

sue)

0

0.5

1

1.5

2

*

Control- BM +BM - BM +BM

SB203580

Kubal C et al J Thorac Cardiovasc Surg 2006;132:1112

Cell

necro

sis

(% o

f aer

ob

ic)

-10

0

10

20

30

40

50

*

Control- BM +BM - BM +BM

SB203580

Cell

ap

op

tosi

s (%

of

aero

bic

)

-10

0

10

20

30

40

*

Control- BM +BM - BM +BM

SB203580

Is Protection Against Ischemic Injury Cell Type Specific?

0

0.5

1

1.5

en

doth

elia

l cells

*

CK

re

lea

se (

IU/m

g w

et

tiss

ue

)

kera

tin

ocyte

s

con

trol

bm

c

Kubal C et al J Thorac Cardiovasc Surg 2006;132:1112

What Is the Most Effective Dose of BMCs-induced Cardioprotection?

Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400

How Potent is BMCs-induced Cardioprotection?

Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400

Cardioprotective Efficacy of Allogenic BMCs

Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400

Does Manipulation of Cells Affect BMCs-induced Cardioprotection?

Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400

Does the Time of Administration Influence BMCs-induced

Cardioprotection?

Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400

Do BMCs Precondition the Myocardium?

Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400

Is the Cardioprotection Induced by BMCs Triggered by Secreted Factor(s)?

Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400

The Role of IGF-1R in Mediating BMCs-induced Cardioprotection

Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400

• 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

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 106 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

Plasma cardiac enzymes

Ang et al. Eur Heart J. 2009;30:2354

Mycardial injury before & after CPB

Pre- CPB 10 mins after starting CPB

Ang et al. Eur Heart J. 2009;30:2354

If BMCs cannot differentiate into cardiac tissue, is the observed

beneficial effect due to

improvement in cell survival stimulation of cardiac progenitor cells

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)

Troponin – Red; DAPI nucleus – Blue

Troponin – Red; DAPI nucleus – Blue; Membrane – White

Troponin – Red; DAPI nucleus – Blue; Membrane – White; Myocyte nucleus - Green

Sensitivity and specificity of myocyte nuclei identification in presence & absence of WGA

Orientation ObserverSensitivity (%) [CI] Specificity (%) [CI]

without WGA with WGA without WGA with WGA

Transverse  

1 45.1 [37.7 - 52.8] 64.6 [57.1 - 71.5] 90.1 [87.6 - 92.1] 98.4 [97.1 - 99.1]

2 48.8 [41.2 - 56.4] 68.9 [61.5 - 75.5] 89.5 [86.9 - 91.6] 99.0 [97.9 - 99.5]

3 54.3 [46.6 - 61.7] 72.6 [65.3 - 78.8] 94.4 [92.4 - 95.9] 98.4 [97.1 - 99.1]

Longitudinal  

1 28.9 [21.8 - 37.3] 59.4 [50.7 - 67.5] 83.4 [80.0 - 86.3] 94.0 [91.7 - 95.7]

2 34.4 [26.7 - 43.0] 56.3 [47.6 - 64.5] 84.5 [81.2 - 87.4] 95.5 [93.4 - 97.0]

3 39.8 [31.8 - 48.5] 61.7 [53.1 - 69.7] 88.5 [85.5 - 90.9] 95.3 [93.2 - 96.8]

Overall  

1 38.0 [32.6 - 43.7] 62.3 [56.6 - 67.7] 87.1 [85.1 - 88.9] 96.5 [95.3 - 97.4]

2 42.5 [36.9 - 48.2] 63.4 [57.7 - 68.7] 87.3 [85.3 - 89.0] 97.4 [96.4 - 98.2]

3 48.0 [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

Diagnostic accuracy of myocyte nuclei identification in the presence and absence of WGA

Abbreviation: WGA – wheat germ agglutinin; CI – Confidence interval

Orientation Observer Diagnostic accuracy without WGA (%) [CI] Diagnostic accuracy with WGA (%) [CI]

Transverse  

1 81.4 [78.6 - 83.8] 91.8 [89.7 - 93.4]

2 81.5 [78.7 – 84.0] 93.1 [91.2 - 94.6]

3 86.5 [84.0 - 88.7] 93.3 [91.4 - 94.8]

Longitudinal  

1 72.9 [69.4 - 76.2] 87.4 [84.6 - 89.7]

2 74.9 [71.5 – 78.0] 88.0 [85.3 - 90.2]

3 79.1 [75.8 – 82.0] 88.9 [86.3 - 91.0]

Overall  

1 77.6 [75.4 - 79.6] 89.8 [88.2 - 91.2]

2 78.6 [76.4 - 80.6] 90.8 [89.3 - 92.2]

3 83.2 [81.3 – 85.0] 91.4 [89.8 - 92.7]

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%)

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

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

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