Isolation, cultivation and identification of skeletal muscle stem cells derived from human orbicularis oculi muscle

Objective To establish the isolation and culture methods of skeletal muscle stem cells, derived from human orbicularis oculi muscle (OOMSCs), and to identify their multi-directional differentiation potential in vitro. Methods The cellswere isolated from pretarsal orbicularis oculi muscle (OOM), obtained in routine blepharoplasty surgery.The tissue was digested using collagenase type I combined with re-plating methods. Specific cell surface antigen markers were detected using flow cytometry analysis. OOMSCs were cultured under different inductive conditions, to identify their pluripotent differentiation ability. Results OOMSCs exhibited similar fibroblast-like morphology as mesenchymal stem cells with high expression of skeletal muscle-derived stem cell surface markers. OOMSCs were able to differentiate into adipocytes, osteoblasts and chondrocytes in vitro, in the presence of lineage-specific inductive media. Moreover, after myogenic induction, the differentiated cells were fused into multinucleated myotube-like structure, and positive for myogenic-related marks, Pax3, Pax7, Myf5 and MyoD. Conclusions Muscle-derived stem cells can be isolated from human OOM with myogenic differentiation properties, showing further applications potential intissue regeneration and medical therapies of muscle diseases. Key words: Orbicularis oculi muscle; Skeletal muscle-derived stem cells; Isolation and culture; Myogenic differentiation; Neuromuscular diseases