INTRODUCTION AND OBJECTIVES: Although BM-MSCs were shown to differentiate into multiple cell types including smooth muscle cells (SMC), factors necessary to induce SMC differentiation are not well delineated. The purpose of our study was to examine the role of culture conditions on BM-MSC SMC differentiation and to investigate the ability of Sca-1 cell sorted BM-MSC to differentiate into SMCs.

METHODS: BM-MSC were isolated from CD1TM female mice age 5-6 weeks, expanded in stem cell media (Mesencult) and passage (P) 3 cells were identified by functional assays and phenotypic surface markers including Sca-1, CD44, CD29, CD45, CD31, and CD34. Immunofluorescence and western blot analysis of the expression of SMC markers: á-smooth muscle actin (á-SMA), calponin, and smooth muscle myosin heavy chain (MHC) were used to evaluate SMC differentiation potential. The effect of serum and growth factors, recombinant human TGF-â1, and recombinant human PDGF-BB on BM-MSC SMC differentiation was evaluated. In addition, sorted Sca-1 positive cells from BM-MSC cultures were evaluated for SMC differentiation potential. Bladder SMCs were used as controls.

RESULTS: In stem cell culture conditions, BM-MSC exhibited a similar phenotype to bladder SMC expressing all three SMC proteins. Low serum concentrations significantly reduced SMC differentiation potential and proliferation rate. A positive correlation was found between serum concentration, cell proliferation and SMC differentiation. Growth factor treatment (TGF-â1, and TGF-â1+PDGF-BB) modulated BM-MSC SMC differentiation in higher serum concentrations (10% and 20%), and led to more elongated, fusiform bladder SMC like shape. Sca-1 selected cells showed marked decrease in potential for SMC differentiation as shown by reduced expression of SMC markers.

CONCLUSIONS: SMC differentiation increases in stem cell media and higher serum concentrations correlating with higher cell proliferation. Exogenous addition of TGF-â1 +PDGF-BB to serum increased SMC differentiation potential morphologically and phenotypically. Sca-1 sorted BM-MSC showed greatly reduced ability for SMC differentiation in contrast to previous suggestions. These observations suggest that SMC differentiation requires the coordinated promotion of serum factors and exogenous growth factors on a preferential mixed cell BM-MSC population.

Source of Funding: None