Betacellulin (BTC), a ligand of the epidermal growth factor receptor, has been shown to promote growth and differentiation of pancreatic b-cells and to improve glucose metabolism in experimental diabetic rodent models. Mesenchymal stem cells (MSCs) have been already proved to be multipotent. Recent work has attributed to rat and human MSCs the potential to differentiate into insulin-secreting cells. Our goal was to transfect rat MSCs with a plasmid containing BTC cDNA to guide MSC differentiation into insulin-producing cells. Prior to induction of cell MSC transfection, MSCs were characterized by flow cytometry and the ability to in vitro differentiate into mesoderm cell types was evaluated. After rat MSC characterization, these cells were electroporated with a plasmid containing BTC cDNA. Transfected cells were cultivated in Dulbecco's modified Eagle medium high glucose (H-DMEM) with 10 mM nicotinamide. Then, the capability of MSC-BTC to produce insulin in vitro and in vivo was evaluated. It was possible to demonstrate by radioimmunoassay analysis that 10 4 MSC-BTC cells produced up to 0.4 ng=mL of insulin, whereas MSCs transfected with the empty vector (negative control) produced no detectable insulin levels. Moreover, MSC-BTC were positive for insulin in immunohistochemistry assay. In parallel, the expression of pancreatic marker genes was demonstrated by molecular analysis of MSC-BTC. Further, when MSC-BTC were transplanted to streptozotocin diabetic rats, BTC-transfected cells ameliorated hyperglycemia from over 500 to about 200 mg=dL at 35 days post-cell transplantation. In this way, our results clearly demonstrate that BTC overabundance enhances glucose-induced insulin secretion in MSCs in vitro as well as in vivo.

The aim of this study was to investigate the effect of aging and timing of left ventricular ischemic injury on the availability and functionality of stem cells. We studied young and aged male inbred Lewis rats that were used as donors of bone marrow mononuclear cells (BM-MNCs), divided in four experimental groups: controls, sham operated, 48 h post-myocardial infarction (MI), and 28 days post-MI. In vitro studies included flow cytometry analysis, hematopoietic colony-forming capacity, and invasion assays of migration capacity. BM-MNCs from these groups were transplanted in female rats after MI induction. Late engraftment was evaluated by real-time PCR of the SRY chromosome. Percentage of CD34 + /CD45 +low cells was similar among different experimental groups in young rats, but was significantly higher in aged animals (p < 0.001), particularly 28 days post-MI. KDR + /CD34 + cells were increased 48 h after MI and decreased 28 days post-MI in young animals, while they were profoundly reduced in the aged group (p < 0.001). Triple staining for CD44 + /CD29 + /CD71 + cells was similar in different groups of aged rats, but we observed an intense increase 48 h post-MI in young animals. Colony-forming units and cytokine-induced migration were significantly attenuated 28 days after the MI. Late engraftment in infarcted transplanted female hearts was present, but considerably heterogeneous. Finally, recovery of left ventricular systolic function in transplanted female recipients was significantly influenced by donors' BM-MNCs groups (p < 0.01). We have demonstrated that aging and timing of myocardial injury are factors that may act synergistically in determining stem cell availability and function. Such interaction should be considered when planning new cell therapy strategies for acute and chronic ischemic heart disease in the clinical arena.