Mesenchymal Stem Cells Adopt a Myofibroblastic Phenotype in Culture: Implications for Cellular Cardiomyoplasty Melanie A. Ngo, Ryan H. Cunnington, Sunil G. Rattan, Yun Li, Melanie M. Durston, Ian M.C. Dixon, Rakesh C. Arora, Darren H. Freed Laboratory of Molecular Cardiology, Institute of Cardiovascular Sciences, St. Boniface Research Centre, University of Manitoba, Winnipeg, Manitoba, Canada OBJECTIVE: The ability of the heart to regenerate is limited. Cell transplantation, in an effort to repair wounded hearts has been proposed and undertaken with variable success. Various studies have shown an increase in heart function with mesenchymal stem cell (MSC) transplantation, but limited differentiation of transplanted cells to mature, functional cardiomyocytes. One explanation for this observation is that MSCs spontaneously differentiate in culture, which limits their capacity for further differentiation in vivo. We examined MSC in vitro and compared their phenotypic and physiological characteristics to cultured cardiac fibroblasts. These observations were made on human and rat cells. METHODS: Rat bone marrow cells were isolated from the long bones using the Caplan method ("Mesenchymal Stem Cells", Arnold I. Caplan, Journal of Orthopaedic Research, pp , 1991). Rat cardiac fibroblasts were isolated using collagen digestion. Human bone marrow cells (from the sternum) and human cardiac fibroblasts (atrial/ventricular tissue) were obtained from patients undergoing open heart surgery. All cells were cultured in standard DMEM F12 with 10% fetal bovine serum conditions. Phenotype characterization and protein expression were determined by immunofluorescent staining and western blotting. Myofibroblasts (MF) exert sustained tonic contraction on scar tissue, and this physiological function was assessed with collagen gel contraction assays on second passage cells (P2). RESULTS: Rat and human bone marrow cells initially expressed the stem cell marker c- kit which decreased over serial passage. In addition, these cells increasingly expressed the myofibroblastic markers alpha smooth muscle actin (α-SMA), the embryonic isoform of smooth muscle myosin (SMemb), procollagen 1A2 and vimentin over serial passage. Collagen gel contraction assays revealed that MSCs contracted to a similar degree as cardiac MF derived from either rats or humans. Human cells treated with TGF  (10 ng/mL), showed increased contractile function compared to non-treated cells (p < 0.05). CONCLUSIONS: Both rat and human MSC of bone marrow origin display phenotypic and physiologic transformation to MF during cell expansion in vitro. These cells demonstrate similar contractile ability between species as well with their respective cardiac derived MF. These data suggest that MSCs differentiate readily in vitro to a myofibroblast phenotype that is unlikely to support further differentiation to cardiomyocytes that will integrate into the host myocardium to improve systolic function. Additional studies are warranted to address this spontaneous differentiation and improve cardiomyocyte differentiation potential. Furthermore, the contribution of MSCs to cardiac fibrosis needs to be investigated. Collagen Gel Contraction After 24 hours Collagen Gel Contraction Assessment Control TGF  treated Human BM Human MF Rat BM Rat MF P1 Human BMSC Positive for Myofibroblast Markers  -SMA SMemb Vimentin P1 Rat BMSC Positive for Myofibroblast Markers  -SMA SMemb Vimentin