Isolation,culture and biological characterization of horse umbilical cord-derived mesenchymal stem cells

【Objective】 The in vitro isolation and culture system of horse umbilical cord-derived mesenchymal stem cells(UC-MSCs) has been established to study its proliferative ability, differentiation ability and biological characteristics, and provide a theoretical basis for promoting the application of UC-MSCs in the treatment of horse racing sports injury.【Method】 Horse UC-MSCs were obtained by using type I collagenase digestion, and cell proliferation in vitro was determined by drawing growth curve and calculating population doubling time. Cell surface markers(CD29, CD44, CD45, CD73, CD90 and CD105) were identified by flow cytometry and RT-PCR amplification. The differentiation potential was detected by inducing adipose formation and osteogenesis differentiation in vitro.【Result】 The results revealed the isolated primary horse UC-MSCs was a round suspension cell with strong refraction. After 10 d of culture, the cell fusion reached 90%, and the cell proliferation rate increased after passage. After passage to the P3 generation, the cells grew in a vortex shape with a uniform long fusiform shape, and the growth trend was in accordance with the law of Logistic growth curve, showing a typical S shape. The results of flow cytometry showed that horse UC-MSCs highly expressed CD29, CD44 and CD90, and the expression rates were 98.45%, 97.08% and 96.56%, respectively, which were highly positive. But CD45 was not expressed. The results of RT-PCR showed that horse UC-MSCs expressed MSCs surface marker genes such as CD29, CD44, CD73, CD90 and CD105, but did not express CD45 gene. Adipogenesis of horse UC-MSCs was induced in vitro with insulin, IBMX, rosiglitazone and dexamethasone as main reagents. Small lipid droplets were found on the 10^th day of induction, and a large number of lipid droplets formed on the 18^th d of induction.Ascorbic acid, sodium β-glycerophosphate and dexamethasone were used as the main reagents for osteogenic induction in vitro. Calcium nodule formation was observed on the 7^th d of induction.【Conclusion】 The results show that the purity of the horse UC-MSCs obtained by I-type collagen enzyme digestion is high and has good in vitro proliferation and multi-directional potential;master the effective freezing mode of horses UC-MSCS, providing quality seed cells in the treatment of horse.