E. Cervio, P. Danieli, C. Ciuffreda, F. Pisano, M. Roccio, M. Gnecchi. The authors have no financial disclosures to declare
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1 16 th ISCT Annual Meeting SOLUBLE FACTORS RELEASED BY HUMAN MESENCHYMAL STEM CELLS OF FETAL ORIGIN LEAD TO CARDIOMYOCYTE PROTECTION THROUGH THE INHIBITION OF PRO-APOPTOTIC SIGNALING E. Cervio, P. Danieli, C. Ciuffreda, F. Pisano, M. Roccio, M. Gnecchi The authors have no financial disclosures to declare Fondazione IRCCS Policlinico San Matteo University of Pavia, Italy
2 The problem Heart failure occurring after acute myocardial infarction (AMI) is a leading cause of mortality and long term morbidity in surviving patients Timely reperfusion therapies represent the gold standard treatment for AMI However, reperfusion may itself lead to myocardial injury Identification of new cytoprotective strategies able to prevent reperfusion injury would be desirable
3 Gnecchi M et al. in Heart Development and Regeneration Edited by Rosenthal and Harvey Elsevier (in press)
4 Possible mechanisms of MSC action MSC (Trans)-differentiation or cell fusion (Trans)-differentiation Cardiomyocyte VSMC Endothelial cell Myocyte regeneration Vasculogenesis PARACRINE MECHANISMS
5 Paracrine mechanisms MSC PARACRINE FACTORS Myocardial protection Cardiac metabolism Cardiomyocyte contractility Cardiomyocytes Endothelial cells Smooth muscle Fibroblasts Cardiac stem cells Cardiac regeneration Neovascularization Cardiac remodeling Gnecchi M et al. in Paracrine Mechanism in Adult Stem Cell Signaling and Therapy Circ. Res. 2008;103:
6 Background It has been described that MSC of fetal origin are present in the amniotic membrane of human placenta (A-MSC) A-MSC repair infarcted hearts in experimental models Mechanisms of A-MSC action in heart repair have not been studied in detail In particular, cytoprotective paracrine effects mediated by A-MSC have not been described
7 Goals 1. To isolate and characterize fetal A-MSC 2. To test the cytoprotective effects exerted by conditioned medium from A-MSC (A-MSC- CM) 3. To identify the pathways eventually involved in A-MSC-mediated cardiomyocyte protection
8 Isolation of human A-MSC A-MSC were isolated by a multi-step procedure: Amnion and chorion were manually separated and rinsed Trypsin-EDTA was used to remove epithelial cells from the amniotic membrane
9 Isolation of human A-MSC MSC were then released by digestion with collagenase Finally, cells were plated in culture dishes with specific growth medium and passaged after they reached 80% confluency A-MSC p0 day 14-5x A-MSC p0 AMSC p1-5x A-MSC p1
10 FACS analysis CD90 CD105 CD HLA-ABC CD45 CD34 CD CD133 CD31 HLA-DR CD80
11 Osteopontin Cathepsin K BSP GAPDH Differentiation potential A-MSC p3 Oil Red-O PPAR 2 ADFP GAPDH Alkaline Phosphatase OSTEOCYTES ADIPOCYTES
12 A-MSC are of fetal origin FISH analysis for the Y chromosome
13 Cytoprotective effects Experimental design A-MSC H9c2 Conditioned medium (CM): A-MSC growth medium (36 hrs) Rat neonatal cardiomyocytes (H9c2 cells) were exposed to 6 hrs of hypoxia (0 2 <0.5%) and reoxygenated for 18 hrs in the presence of CM or CTRL-M
14 % of viable cells Cell viability MTS assay p<0.001 vs Basal Normoxia # p<0.001 vs CTRL-M # * 20 0 CTRL-M A-MSC-CM Basal normoxia 6 hrs hypoxia + 18 hrs reoxigenation
15 % TUNEL + nuclei Relative units Apoptosis Assays TUNEL Staining Cleaved Caspase # * 10 * CTRL-M A-MSC-CM 6 hrs hypoxia + 18 hrs reoxygenation Basal normoxia CTRL-M A-MSC-CM 6 hrs hypoxia + 18 hrs reoxigenation * p<0.001 vs CTRL-M # p<0.001 vs Basal normoxia
16 CTRL-M CTRL-M A-MSC-CM A-MSC-CM Pathway activation p-sapk/jnk p-akt p-p38 MAPK p-erk MKP-5 Basal normoxia 6 hrs of hypoxia + 18 hrs reoxygenation Basal normoxia 6 hrs of hypoxia + 18 hrs reoxygenation
17 CTRL-M A-MSC-CM CTRL-M A-MSC-CM Pro- and anti-apototic factors ANTI-APOPTOTIC GENES PRO-APOPTOTIC GENES FasL Bcl-2 TNF-α Stat-3 Bak1 GAPDH GAPDH Basal normoxia 6 hrs of hypoxia + 18 hrs reoxygenation Basal normoxia 6 hrs of hypoxia + 18 hrs reoxygenation
18 Conclusions A-MSC of fetal origin can be isolated and expanded from human placenta A-MSC-CM remarkably protects cardiac myocytes against hypoxia/reoxygenation damage A-MSC-CM leads to the inhibition of SAPK/JNK and p38 MAPK pro-apoptotic pathways and to the concomitant overexpression of anti-apoptotic genes Bcl-2 and Stat3 and downregulation of pro-apoptotic factors FasL, TNF-α and Bak1 A-MSC therapy may represent a novel and powerful approach for cardioprotection in ischemic heart disease
19
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