Kai Pinkernell, MD 4 th Int. Symp. Stem Cell Therapy & CV Biology Madrid, Spain 26 th of April 2007 Adipose Derived Stem Cell Therapy in Cardiovascular Disease.

Adipogenic Osteogenic Chondrogenic Neurogenic Adipose Tissue Derived Stem Cells - The Concept GalC AP Oil Red-O Alcian Blue Zuk et al. Microbiology of the Cell 2003 Zuk et al. Tissue Engineering Easily accessible - Large amounts available

Liposuction of Adipose 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 CD44 CD71 CD90 CD45 CD31 CD105 SH3 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 Epitope Percent 24 hours 120 hours ** <1% *none detected

CD34 bright : 56.5% CD31 bright : 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. Zuh Gel 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;

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;

Plasticity of Human Adipose Lineage Cells Toward Endothelial Cells Planat-Benard, et al., Circulation (2004) 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:

Paracrine Factor Release Rehmann et al. Circ.2004;109:

Anti-Apoptotic Effect – in vivo TUNEL positive cells, 48 hours post AMI induction in rats.

In vivo PRE-Clinical Work - Hindlimb Ischemia

In vivo PRE-Clinical Work - Cardiac functional

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 Trans-thoracic EchocardiographyCineangiography p=0.01p= pigs, 1 hour LAD infarction Delayed Delivery of 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) Strem et al. Circ, 2005; 112(17)

In vivo PRE-Clinical Work - Cardiac structural

Porcine AMI Model – Cultured ADSCs Control ratio: 0.48Adipose Cell ratio: 0.77 Control ADC - treated Tulane University

Wall thickness infarcted myocardium / wall thickness healthy myocardium ns p < 0,02 Tulane University Control ADC BMC Porcine AMI Model – Cultured ADSCs LV Wallthickness

Porcine AMI Model – Fresh ADRCs LV 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

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 vO 2, & 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 Q APOLLO: Acute Myocardial Infarction Trial