Diaphragm Satellite Cells Isolation by Optimized MACS and the Effect of Mechanical Ventilation on their Proliferation and Differentiation Characteristics through MyoD and Myogenin Pathways

Abstract

Objective: In the present study, we aimed to establish a feasible method to isolate single diaphragm satellite cells from C57 mice, and clarify the effect of mechanical ventilation (MV) on the proliferation and differentiation of diaphragm satellite cells. Moreover, the underlying molecular mechanism was also explored.Methods: After the dissection of the diaphragm, enzymolysis, and specific antibody selection, single diaphragm satellite cells were harvested from C57 mice receiving 6 h of MV or not with optimized magnetic-activated cell sorting (MACS) approach. The cells were stained with BrdU or labeled with the differentiation antibody MYH3, followed by observation using fluorescence microscopy. The cells were counted from randomly selected visual fields, and the proliferation or differentiation characteristics of the control and MV groups were compared by IMAGE software. Besides, the expressions of MyoD and myogenin were detected by quantitative real-time PCR (qRT-PCR). Results: The single diaphragm satellite cells were successfully purified through MACS using a set of optimized parameters. Generally speaking, 1.5×105 cells could be harvested from a single diaphragm. Upon MV, the proliferation rate of diaphragm satellite cells was decreased from 88.74% to 81.92%, while the differentiation rate was increased from 17.94% to 27.58%. Moreover, the expressions of MyoD and myogenin were significantly up-regulated upon MV. Conclusions: In our current work, an efficient method was successfully established to isolate single diaphragm satellite cells. After MV, the differentiation rate of diaphragm satellite cells tended to increase, and the expressions of MyoD and myogenin were up-regulated. Collectively, our findings provided valuable insights into further research and clinical target treatment.