Isolation, Culture, Identification, and Bioenergetics Metabolism of Equine Skeletal Muscle Satellite Cells

Abstract

Equine skeletal muscle satellite cells (EMSCs) are muscle stem cells in horses, responsible for the postnatal growth, repair, and homeostasis of skeletal muscles. EMSCs are an attractive model for horses to investigate the mechanisms of muscle growth and spontaneously fuse to form differentiated muscle fiber types through activating a battery of muscle-specific genes. Previous reports on the successful isolation and culture of skeletal muscle satellite cells mostly used skeletal muscles of young animals. With the high value of horses, skeletal muscle samples of foals are very difficult to obtain. The present study describes protocols for enriching the satellite cell fraction from the semitendinosus of a 2-year-old Mongolian horse to isolate the EMSCs. Optimized culture conditions with gelatin layering accelerated the adhesion speed of EMSCs. The identification of EMSCs was carried out through multiple dimensions including cell morphology, myogenic induction, differential adhering, and molecular signatures. In particular, the Seahorse Extracellular Flux analyzer was utilized for evaluating the bioenergetics metabolism of EMSCs by measuring the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). The present study provides reference for the isolation, purification, identification, and bioenergetics metabolism characteristics of EMSCs, which would be useful for studying the molecular mechanisms for muscle development, muscle fiber type differentiation, and recovery from muscle injury in horses.

Keywords

• equine
• muscle satellite cells
• isolation
• bioenergetics metabolism