Kai Pinkernell, MD4th Int. Symp. Stem Cell Therapy & CV Biology

Madrid, Spain26th of April 2007

Adipose Derived Stem Cell Therapy in Cardiovascular Disease.

AdipogenicOsteogenic ChondrogenicNeurogenic

Adipose Tissue Derived Stem Cells- The Concept

GalC AP Oil Red-O Alcian Blue

Zuk et al. Microbiology of the Cell 2003Zuk et al. Tissue Engineering 2001

- Easily accessible- Large amounts available

Liposuction ofAdipose Tissue

How to Obtain Adipose Derived Cells?

Washing media

Liquid fat

Collagen/Adipocytes/Debris

Cell pellet/erythrocytes

Adipose Derived Cells after Enzyme Digestion +

Centrifugation

Right picture from Tulane University

• Adipocytes

• Adult stem cells

• Endothelial cells

• Vasc. smooth muscle cells

• Fibroblasts

• Pericytes

• ……..

Adipose Derived Regenerative Cells (ADRCs)

Unique Population of Regenerative Cells

• Adipocytes

• Adult stem cells

• Endothelial cells

• Vasc. smooth muscle cells

• Fibroblasts

• Pericytes

• ……..

Adipose Tissue

How do ADRCs compare to other cell sources?

Cultured Adipose and Bone Marrow - CD Comparison

ADSC

MSC

ADSC

MSC

CD29

CD29

CD44

CD44

CD71

CD71

CD90

CD90

CD45

CD45

CD31

CD31CD105

CD105 SH3

SH3

CD34

CD34

Expression of Cell Surface Markers – Comparing ADSCs vs. BMCs

Fraser et al. Nat Med Clin Prac CV Med 2006;3,Suppl 1:S33-7

Expression of Cell Surface Markers –ADSCs Over Time in Culture

Fraser et al. Nat Med Clin Prac CV Med 2006;3,Suppl 1:S33-7

0

20

40

60

80

100

120

CD 34 CD 45 CD 11b CD 105Epitope

Perc

ent

24 hours 120 hours

* *<1%

*none

detected

CD34bright : 56.5% CD31bright : 6.8%

‘Fresh’ ADRCs CD Marker Expression

Representative of freshly isolated ADRCs; analysis using FACSDiva

Alfonso et al.

Proposed Mechanisms for ADCs –

Angiogenesis

ADRCs (top row) in a tube formation assay using Antibodies against CD31(red), von-Willebrandt factor (vWF, green) and DAPI (blue) as a nuclear stain. Fibroblasts (bottom row) were used as a control.

‘Fresh’ ADCs Tubule Formation

ADRC

Fibro-blasts

Angiogenesis: ADRC Participate in Vessel Formation

CD31

SMA

Von VIII

SMM

M. ZuhGel Plug (0.1 mL, 0.5 Mio.) of RosaLacZ ADRCs into collagen gel (no growth factors) in SCID mice, 3 weeks later.

Angiogenic Potential of ADCs

Miranville et al. Circ 2004; 110;349-355

Mouse Hindlimb Ischemia Model

24 hour ischemia, then i.v. infusion of 0.5 Mio human cells; murine athymics

Miranville et al. Circ 2004; 110;349-355

Plasticity of Human Adipose Lineage Cells Toward Endothelial Cells

Planat-Benard, et al., Circulation (2004)

CD31 CD31

Proposed Mechanisms for ADCs –

Differentiation

IHC – cardiac Troponin-T

100 m

day 20

Proposed Mechanisms – Paracrine Effects

Paracrine Factor Release

Rehmann et al. Circ.2004;109:1292-1298

Paracrine Factor Release

Rehmann et al. Circ.2004;109:1292-1298

Anti-Apoptotic Effect – in vivo

0

2

4

6

8

10

12

14

Saline ADC treatedAp

op

toti

c C

ard

iac

Myo

cyte

s/H

igh

Po

wer

F

ield

p < 0.01

TUNEL positive cells, 48 hours post AMI induction in rats.

In vivo PRE-Clinical Work- Hindlimb Ischemia

In vivo PRE-Clinical Work- Cardiac functional

39

4244

41

3735

0

10

20

30

40

50

60

AMI 4 wk 8 wk

[%]

55

3835

55

3943

0

10

20

30

40

50

60

70

Baseline AMI 8 weeks

[%]

Control

ADC

Porcine AMI Model - Tulane

*p=0.023

*

**p=0.037

**

Angiographic LVEF Perfusion (Cardiolite®)

• 16 pigs, 3 hour LAD infarction• Immediate Delivery of 1.5 Mio cells/kg

Porcine AMI Model - UCLA

30

35

40

45

50

55

60

Post-MI 6 Month

Eje

ctio

n F

ract

ion

%

Control

Cell Infusion

40

45

50

55

60

Post-MI 6 Month

Eje

ctio

n F

ract

ion

%

Control

Cell Infusion

Trans-thoracic Echocardiography Cineangiography

p=0.01 p=0.16

• 13 pigs, 1 hour LAD infarction• Delayed Delivery of 40-140 Mio cells at 48 hours

Watanabe et al. Am J Cardiol 2004; 94(suppl 6A):188E.

Rat AMI Model (UCLA)

Echocardiographic Dobutamine Responsiveness LVEF (Tip Catheter)

60

70

80

90

Pre 12 wk

[%]

SalineADCs

p<0.05

0

4000

8000

12000

16000

20000

baseline 3ng 6ng 15ng 30ng

Dobutamine Levels

Pre

ssu

re [

mm

Hg

]

p<0.05 for all levels of dobutamine challange

Strem et al. Circ, 2005; 112(17)

In vivo PRE-Clinical Work- Cardiac structural

Porcine AMI Model – Cultured ADSCs

Control ratio: 0.48 Adipose Cell ratio: 0.77

Control ADC - treated

Tulane University

Wall thickness infarcted myocardium / wall thickness healthy myocardium

0,58

0,750,69

0,00

0,20

0,40

0,60

0,80

1,00

sham ftMSC bmMSC

ns

ns

p < 0,02

Tulane University

Control ADC BMC

Porcine AMI Model – Cultured ADSCsLV Wallthickness

0

5

10

15

20

Infarct Area Border Zone Myocard

Wa

ll T

hic

kn

es

s [

mm

]

Control

ADC

Porcine AMI Model – Fresh ADRCsLV Wallthickness

(*p<0.05)

*

*

In vivo PRE-Clinical Work- Cardiac structural (histology)

Immunohistochemistry

• Persistence of labeled, transplanted Cells for 4, 8, 12 weeks and longer

• Co-staining with– Endothelial Markers– Smooth muscle cells markers– Low positivity with cardiac markers

Increase in Capillary Density

Tulane University

200x

630x

Porcine AMI Model – Cultured ADSCs

Box-Plot

Control ADC BMC

Tulane University

0

20

40

60

80

100

120

140

Infarct area Border Zone Myocardium

[co

un

ts/0

.1 m

m2

]

Control

ADC

Porcine AMI Model – Fresh ADRCs

(*p<0.05)*

Summary of Benefits of ADCs in Cardiovascular Diseases

Hindlimb Models• Increase in perfusion

Cardiac Disease Models• No arrhythmias or other cell related

mortality/morbidity• Improvement in function• Increased perfusion• Increased wall thickness

Cardiac Clinical Trials

Objective:

• Safety and feasibility in patients with non-revascularizable ischemia

• Center: Universitario Gregorio Marañon in Madrid, PI’s Fernandez-Avilés and Perin (Texas Heart Institute)

Trial design & inclusion:

• ADRCs delivered by intramyocardial injection via NOGA™

• Prospective, randomized, placebo-controlled, double-blind, dose escalation trial

• Measures to include ejection fraction, perfusion, max vO2, & 6-minute walk

• Up to 36 patients

• 6 month follow up

• Currently enrolling

PRECISE: Chronic Myocardial Ischemia Trial

Objective:

• Safety and feasibility in STEMI patients

• Center: Thoraxcenter, Erasmus University Rotterdam, PI Patrick Serruys

Proposed trial design & inclusion:

• Intra-coronary delivery of ADRCs

• Prospective, randomized, placebo-controlled, double-blind, dose escalation trial

• Measures to include ejection fraction, perfusion & wall motion

• Up to 48 patients

• 6-month follow-up

• Begin enrolling Q2 2007

APOLLO: Acute Myocardial Infarction Trial