Isolation And Identification Of Equine Umbilical Cord Mesenchymal Stem Cells

Umbilical cord mesenchymal stem cells are easy to isolate,culture,expand and purify due to their wide sources.They still have the characteristics of stem cells after multiple passages,without immune rejection.They have become the best Pluripotent stem cells with clinical application prospects.It has been widely used in cell replacement therapy and gene therapy,but there is few applications on the animal husbandry production.To date,there is a few reports about equine umbilical cord mesenchymal stem cells in China.In this experiment,equine umbilical cord mesenchymal stem cells were collected,separated and cultured,and the biological characteristics of the cells were determined.A series method of equine umbilical cord mesenchymal stem cells separation,cultivation,and identification was established.In this paper,the tissue block separation method is used to prepare mesenchymal stem cells from the part of the horse umbilical cord near the fetus(proximal segment),the middle part of the whole umbilical cord(middle segment)and the part near the placenta(distal segment),and draw the comparison of cell growth curves.The proliferation ability of three parts of umbilical cord mesenchymal stem cells,Real-time fluorescent quantitative PCR was used to detect the expression of pluripotency genes(NANOG,POU5F1,SOX2),surface marker molecules(CD14,CD19,CD34,CD45,CD73,CD79 a,CD90,CD105)and inflammatory factors(IL4,IL6,IL8)in the P1 and P5 generations of the three groups of cells,and the potential of the three groups of cells to differentiate into chondrocytes after chondrogenic induction.To compare the proliferation rate of umbilical cord mesenchymal stem cells isolated from three different parts of the same umbilical cord from the equine female fetus,the expression of pluripotency genes,surface marker molecules and inflammatory factors,and the differences in the cartilage differentiation potential of each group of cells.The results of the study are as follows:1.The three groups of cells(proximal segment,middle segment,and distal segment)obtained by separation and culture all grow adherently,and the three groups of cells are fibrous,vortex,flowing,and clustered.The cells are subcultured to the P5 generation.The cells still maintained good proliferation ability,and the cell morphology of each group did not change significantly.2.The doubling time of the three groups of cells were: 35.4 h(proximal segment),25.5 h(middle segment),34.9 h(distal segment),and the average doubling time was 31.9 h.The comparison and analysis of the doubling time of the middle segment cell was significantly lower than that of the proximal segment and distal segment cells(P<0.05).3.The three groups of cells all express the pluripotency genes NANOG,POU5F1 and SOX2,high expression of surface marker molecules CD34,CD73,CD90,and CD105,low expression of CD14,CD19,CD45 and CD79a;and expression of pro-inflammatory factors IL6 and IL8,but no expression Anti-inflammatory factor IL4.4.All three groups of cells can be induced into chondroblasts,and specific staining can be seen after alcian blue staining,only the distal segment cells detected the relative expression of cartilage-specific genes SOX9,ACAN,and COL2A1,which increased with the induction time,the middle segment cells relative expression of specific genes ACAN and COL2A1 was significantly higher than that of the other two groups(P<0.05).The above results indicate that the three parts from the entire umbilical cord can be used to prepare MSCs.The three groups of cells all express pluripotency genes and mesenchymal stem fine surface marker molecules,express pro-inflammatory factors,and do not express anti-inflammatory factors.Induce differentiation into chondrocytes.In this study,the proliferation ability and cartilage differentiation potential of umbilical cord mesenchymal stem cells derived from the middle segment are superior to those of the proximal and distal mesenchymal stem cells.