The de novo formation of intramuscular fat tissue by the insertion of myogenic and adipogenic cell lineage determination genes into satellite cell genome

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As the first stage of this investigation, the procedure for isolating, purifying, and culturing skeletal muscle satellite cells (satellite cells) from Japanese Black cattle and Holstein cattle was established. In this procedure, satellite cells were separated from cell debris and other cells by a series of differential centrifugation after minced muscle samples were digested by protease. The crude satellite cell preparation was further purified by removing fibroblasts with an aid of differential adhesion to the plastic bottom of culture dishes. By this procedure satellite cells were prepared at better than 95% purity, determined by the immunostaining with monoclonal antibody for desmin. For satellite cell culture, the DMEM/F-12 medium with 10% fetal bovine serum and antibiotics/antimycetics was found suitable for cell proliferation. In this medium, satellite cells attached to the culture dish bottom, took up the spindle-shape, and proliferated. After 3-5 days, cells reached confluence … More and became ready for the cell passage. When the culture medium was replaced with a serum-free medium for the confluent cells, satellite cells underwent differentiation in 1-2 days to form myotubules. In myotubules, the production of myosin was detected by the immunostaining with monoclonal antibody MF-20, proving that satellite cells completed the terminal differentiation as the myogenic cell. As described above, satellite cells spontaneously differentiated to myogenmic lineage under a normal condition. In the second stage of the investigation, the stability of the satellite cell lineage was put on a question and its plasticity was evaluated. With a hypothesis that satellite cells were able to differentiate to cell lineages other than myogenic cells, experiments were conducted. When spindle-shaped satellite cells were transfected with peroxisome proliferators-activated receptors γ (PPARγ)by the lipofection method and the cell culture was continued, satellite cells accumulated lipid droplets, detected by Oil-Red-O staining. This was the first report to demonstrate that satellite cells were capable of being directed the fat cell lineage. When PPARγ was inserted to satellite cells which initiated the terminal differentiation by fusion to form multinucleated cells, lipid accumulation, as well as myosin expression was detected in multinucleated cells. These observations were the direct evidence that satellite cells possessed pluripotency and functioned as adult stem cells. Furthermore, these observations provided a new insight to the concept of cell differentiation. Less