Human fetal mesenchymal stem cells protect cardiac myocytes against hypoxia/reoxygenation injury

Background: Myocardial reperfusion injury represents a major complication of the reperfusive therapies used to treat acute infarction. Consequently, the identification of new cytoprotective strategies able to prevent reperfusion injury is urgently needed. We and others have shown that adult mesenchymal stem cells (MSC) limit infarct size in rodents mainly through cytoprotective paracrine mechanisms. More recently, the existence of fetal MSC in the human placenta has been described but it is unknown whether these cells can mediate cardiomyocyte protection. Methods: MSC were isolated from the placental amniotic membrane (A-MSC) of women delivering male newborns. We performed FACS analysis and FISH staining for the Y-chromosome to determine the immunophenotype and to confirm the fetal origin of the cells, respectively. The expression of several known cytoprotective factors was verified by RT-PCR. Rat neonatal cardiomyocytes (H9c2) 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 A-MSC. H9c2 viability was evaluated by MTS assay and cleaved Caspase 3 was quantified by colorimetric assay and Western blotting. The anti-apoptotic protein Bcl-2 was analyzed in H9c2 cells by Western blotting. Results: A-MSC were successfully isolated from 15 amniotic membranes. At passage three, the A-MSC displayed the antigen profile typical of MSC and were positive for the Y chromosome. Furthermore, the A-MSC expressed several known cytoprotective factors, such as EPO, HGF, IGF-1, FGF2, VEGF, BMP2, PDGF-b, SFRP2, TGF-B, and thymosin B4. The hypoxia/reoxygenation protocol reduced by 68% the H9c2 viability (p<0.05 vs basal conditions). The A-MSC-CM remarkably increased cell viability by 62% compared with CTRL-M (p<0.05%). The colorimetric assay documented that in H9c2 fed with CRTL-M the amount of cleaved Caspase 3 was increased by 36% after hypoxia/reoxygenation (p<0.05) and that the A-MSC-CM significantly reduced the level of cleaved Caspase 3 (- 70% vs CTRL-M, p