Abstract
Pig is an important food source and an excellent system to model human diseases. Careful characterization of the swine skeletal muscle stem cells (satellite cells) will shed lights on generation of swine skeletal muscle disease model and efficient production of porcine meat for the food industry. Paired box protein 7 (Pax7) is a highly conserved transcription factor shared by satellite cells from various species. However, the sequence of Pax7 has not been characterized in pig. The lack of method to isolate highly purified satellite cells hinders the thorough characterization of the swine satellite cells. Here we found molecular markers for swine satellite cells and revealed that the porcine satellite cells were heterogeneous in various pieces of skeletal muscle. We further developed a method to isolate highly purified satellite cells directly from porcine muscles using fluorescence-activated cell sorting. We next characterized the proliferation and differentiation abilities of isolated satellite cells in vitro; and found that long-term culturing of satellite cells in vitro led to stemness loss.
Highlights
Higher seeding cell density yielded myotubes with elevated MyHC expression level and higher percentage of myotube nuclei (≥3 nuclei) in total nuclei (Figures 4i and j). These results suggest that high seed cell density facilitates pig satellite cell differentiation
We further examined the ability of pig satellite cells to repair of the cells isolated from pig skeletal muscles by fluorescence-activated cell sorting (FACS) sorting, we performed immunofluorescent staining of MyoD and Pax[7]
Few myofibers generated from pig satellite cells could be detected when these cells were expanded in vitro for 5 passages (Figures 6c and d), suggesting that pig satellite cells lost their stemness and abilities to repair muscle injury in vivo after prolonged in vitro expansion. These results are consistent with the morphology and differentiation potential changes described above (Figures 4 and 5), suggesting that pig satellite cells lost their stem cell features after being expanded in vitro for 5 passages. These results suggest that pig satellite cells lost their stemness and differentia- 1000 × g for 5 min at 4 °C and incubated with the erythrocyte lysis buffer tion abilities after in vitro expansion, a feature shared by both (ACK) buffer for 5 min on ice
Summary
Pigs have many advantages in modeling human diseases due to their similar anatomic and physiological features to human beings.[13,14] For example, the swine DMD model recapitulates human symptoms better than mouse model. The severe progressive dystrophic changes of skeletal muscles, impaired mobility, muscle weakness, and a much shorter life span are common symptoms in human DMD patients.[15] These symptoms can only be recapitulated in pig DMD model,[16] but not mouse DMD model,[17] supporting the notion that pigs can model human diseases better than rodents. Pax[7] can directly regulate MyoD and Myf[5] to modulate satellite cell maintenance and proliferation.[21,22,23,24] The full-length CDS of
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