In vitro differentiation of progenitor cells isolated from juvenile pig hearts - expression of relevant gene and protein markers.

Objectives. Heart failure is a significant cause of mortality worldwide, and most current therapies treat only its symptoms. The results of cardiac stem cell research suggest a promising treatment option for heart failure, but there is currently an unmet demand for better research models. We have therefore, for the first time, isolated, expanded and differentiated progenitor cells obtained from juvenile pig hearts to use as a platform for cardiac stem cell research. Design. Progenitor cells were isolated from the left ventricles of porcine hearts using collagenase enzymatic digestion and Percoll((R))-gradient centrifugation. Cells were proliferated in Matrigel((R))-coated wells. Cell differentiation was initiated by applying 5-azacytidine and subsequently controlled by modifying the serum concentration. Western blotting and qPCR were used to determine protein and gene expression, respectively. Results. Cardiac-specific genes, from the following proteins: troponin I-3, and myosin-heavy-chain 7 were stably expressed during proliferation and differentiation. Connexin-43 was upregulated and Actinin alfa 2 was downregulated during differentiation. The immature-cardiomyocyte marker GATA binding protein 4 was stably expressed but with a decrease in expression at day 4 of differentiation. Smooth muscle actin decreased in expression and Von Willebrand factor were stably expressed during differentiation. Smooth muscle protein expression was documented but no expression of cardiac-specific proteins after differentiation was found. Conclusion. The isolated progenitor cells had key cardiac-lineage gene expression characteristics but they did not express cardiac-specific proteins. Smooth muscle protein was expressed confirming commitment to the smooth muscle lineage.