Donor age negatively influences the cytoprotective paracrine effects exerted by human mesenchymal stem cells

Background: In animal models mesenchymal stem cells (MSC) repair infarcted hearts mainly through cytoprotective paracrine mechanisms. 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 influences the production of cytoprotective factors. Accordingly, we compared the paracrine properties of fetal MSC (F-MSC) with adult MSC. Furthermore, we aimed to elucidate through which signalling pathway MSC lead to cytoprotection. Methods: F-MSC were isolated from human placenta and adult MSC from the bone marrow of young (yBM-MSC; age<65 years) or old (oBM-MSC; age>65) donors. Rat neonatal cardiomyocytes (H9c2) were exposed to hypoxia (6 hrs)/reoxygenation (18 hrs) (H/R) in the presence of control medium (CTRL-M) or conditioned medium from F-MSC (F-CM), yBM-MSC (y-CM) or oBM-MSC (o-CM). H9c2 viability was evaluated by MTS assay. Apoptosis was measured by TUNEL staining and by cleaved Caspase 3 (colorimetric assay and Western blot). We evaluated SAPK/JNK and p38 MAPK activation by Western blot and the expression of pro- and anti-apoptotic genes by RT-PCR. Results: The H/R protocol reduced H9c2 viability by 55% (p<0.001 CTRL-M vs basal condition). Compared with CTRL-M, both F-CM and y-CM increased cell viability (+45% and +33% respectively; p<0.017) while o-CM had no effect. F-CM significantly reduced the number of TUNEL positive cells (-91% vs CTRL-M and -89% vs o-CM; p<0.001). 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 apoptosis (-11% vs CTRL-M, p=ns). Both colorimetric assay and Western blot showed that Caspase-3 activation was prevented by F-CM and y-CM but not by o-CM. The H/R protocol strongly activated both SAPK/JNK and p38 MAPK. This activation was markedly reduced by F-CM while y-CM and o-CM had modest effect on both pathways. Furthermore, compared with CTRL-M and both y-CM and o-CM, F-CM up-regulated the anti-apoptotic genes Bcl-2 and Stat3 and down-regulated the pro-apoptotic genes TNF-α and FasL. Conclusions: We showed that human MSC mediate cardiomyocyte protection by releasing soluble anti-apoptotic factors. However, donor age negatively influences the cytoprotective properties of adult MSC. We also demonstrated that MSC of fetal origin exerts powerful cytoprotective effects via inhibition of different pro-apoptotic signalling pathways. Our data suggest that autologous MSC therapy for ischemic heart diseases may be less effective in elderly patients.