Muscle-derived stem cells in tissue engineering: defining cell properties suitable for construct design.

Abstract

The terms construct or tissue equivalent refer to neotissue produced by tissue engineering techniques. The elements forming the construct are scaffolds on which cells are "recreated" to form an engineered-tissue sensitive to certain cell signals. The ability of the cells to expand and differentiate on the scaffold is determined by properties such as fixation, adhesion, proliferation and migration. Among the cell types that seem to be most promising for designing constructs are tissue-residing, or adult, stem cells, which show two main features: a capacity to differentiate into many cell lineages and the power of self-renewal. These features make them good candidates for cell replacement therapies. Here, we report the identification, isolation and culture of muscle stem cells aimed at establishing the ideal culture in terms of defining when the cultured cell population would show optimal characteristics for transfer to the scaffold to obtain a particular construct. Stem cells harvested from the dorsal muscle of white New Zealand rabbits were cultured in vitro and characterized 5 to 14 days after the start of culture. Fibroblasts obtained from the same experimental animal served as controls. The stem cells were examined by light and scanning electron microscopy. For stem cell identification, we used the antibodies anti-m-cadherin, anti-CD34 and anti-Myf-5. The markers of muscle differentiation used were: anti-vimentin, anti-alpha-actin, anti-desmin and anti-myosin. The expression profiles of the different markers of muscle differentiation and TGFbeta1 in the cell cultures were confirmed by Western blotting. Proliferation rates were determined by monitoring tritiated thymidine incorporation. The thymidine incorporation rate was substantially higher for the population of undifferentiated cells than for control fibroblasts obtained from the same animal. During the first five days of culture, most cells were negative for all the markers examined, with the exception of m-cadherin, CD34 and Myf-5, although discrete signs of vimentin expression started to emerge. After 14 days of culture, the adult stem cells showed vimentin (94.2%) and desmin (33.8%) expression yet scarce labeling for myosin (16.2%) and alpha-actin (8.3%). Control fibroblasts showed intense labeling for vimentin (99.3%) and alpha-actin (62.2%), while less than 2% of the population expressed myosin (0.9%) and desmin (1.6%). After two weeks of culture, muscle-derived stem cells show good proliferative and adhesion properties as they initiate differentiation. These conditions seem ideal for obtaining the desired construct.