The cardioprotective paracrine effects exerted by human mesenchymal stem cells are negatively influenced by donor age

Background: In experimental animal models mesenchymal stem cells (MSC) repair infarcted hearts mainly through paracrine mechanisms. In particular, MSC produce and release anti-apoptotic factors that lead to cytoprotection. For translational purposes, it would be important to verify if human MSC also mediate cardioprotection. In particular, since ischemic heart diseases occur mainly in elderly, it is essential to establish if donor age negatively influences the production of cytoprotective factors. Accordingly, we have compared the paracrine properties of fetal MSC with adult MSC from young and old patients. Methods: MSC of fetal origin (F-MSC) were isolated from placental amniotic membranes processed immediately after delivery. Adult MSC were collected from bone marrow samples of young (yBM-MSC; donor age < 65 years) or old donors (oBM-MSC; donor age > 65 years). Rat neonatal cardiomyocytes (H9c2 cells) were used to test cytoprotection. H9c2 cells were exposed to 6 hours of hypoxia followed by 18 hours of reoxygenation in the presence of control medium (CTRL-M) or conditioned medium (CM) from F-MSC (F-CM) or yBM-MSC (Y-CM) or oBM-MSC (O-CM). CM was obtained by growing MSC for 36 hours in the absence of serum. H9c2 viability was measured by MTS assay. The rate of apoptosis was quantified by TUNEL staining. We also evaluated cleaved Caspase 3 using both a colorimetric assay and Western blotting. Gene expression profile of several known cytoprotective factors was assessed by RT-PCR. Results: The hypoxia/reoxygenation protocol reduced H9c2 viability by 55% compared with basal condition (p<0.001). Both F-CM and Y-CM prevented cell damage compared with CTRL-M resulting in a significant increase in cell viability by 45% (p<0.017) and 33% (p<0.017), respectively. At the contrary, O-CM had no significant effect on H9c2 viability (p=n.s. vs CTRL-M). After the induction of hypoxia/reoxygenation injury, 35 ± 8% of H9c2 cells tested positive for TUNEL staining. Compared with CTRL-M and O-CM, in the presence of F-CM we observed a relative reduction in the number of TUNEL positive nuclei of 91% (p<0.001) and 89% (p<0.001) respectively. The Y-CM also reduced H9c2 apoptotic nuclei (- 67,5% vs CTRL-M, p<0.01; - 64% vs O-CM, p<0.01). In contrast, O-CM did not prevent H9c2 apoptotic death (-11% vs CTRL-M, p=n.s.). The colorimetric assay documented that the F-CM significantly reduce the level of cleaved Caspase 3 by 50% vs CTRL-M (p< 0.017) and by 42% vs Y-CM (p< 0.017); furthermore, the Y-CM reduced the amount of cleaved Caspase 3 by 33% vs O-CM (p< 0.017). Finally, the oBM-MSC-CM did not reduce cleaved Caspase 3 compared with CTRL-M. In the presence of F-CM Western blot analysis confirmed a marked reduction in Caspase 3 activation, while no striking differences were present between CTRL-M, Y-CM and O-CM. The RT-PCR analysis documented that F-MSC express several known cytoprotective factors such as PDGF-β, BMP2, EPO, FGF2 and VEGF at significantly higher level compared with oBM-MSC (p<0.05) and that VEGF, FGF2 and HGF transcripts were significantly higher in yBM-MSC than in oBM-MSC (p<0.05). Conclusions: We have shown that human MSC can mediate cardiomyocyte protection through the release of soluble anti-apoptotic factors. However, we documented that donor age negatively influences the paracrine cytoprotective properties of adult MSC. Our data suggest that autologous MSC theraphy for ischemic heart disease might be less effective in elderly patients.