A novel method to isolate mesenchymal stem cells from the human umbilical cord.

Objective: To establish a method to isolate human umbilical cord mesenchymal stem cells (HUCMSCs) in vitro.Methods: MSCs were isolated from human umbilical cord with three-enzyme method,digested for 3 hours and diluted with PBSA,then,centrifugated to obtain umbilical cord mesenchymal stem cells in vitro.The surface markers of the passage 3 (P3) cells were identified by flow cytometry;The P3 cells were induced into adipocytes,chondrocytes and osteocytes by chemical-inductive method.After 2~4 weeks,the differentiated cells were stained by oil red,Safranin'O and alizarin red and observed under inverted microscope to examine the effects of induction.Results: The isolation of human umbilical cord mesenchymal stem cells was fast and efficient ;As the cells were subcultured,the morphology of P3 cells was gradually uniform.The P3 cells expressed CD90,CD73 ,CD44 and CD105,which are known to characterize MSCs,but expressed no cell markers for hematopoietic stem cells or Endothelial cells such as CD45,CD34,CD19,CD14 and human leukocyte antigen HLA-DR,which also lacks in MSCs ;When exposed to lineage-specific differentiation media,HUCMSCs could differentiated into adipogenic,chondrogenic,osteogenic,and neuronal lineages.Accumulation of adipose lipids within differentiated HUCMSCs was confirmed by staining these cells with Oil red;Chondrogenesis was demonstrated by sulfated proteoglycans stained with saffranin' O and osteogenesis by calcium deposition in extracellular matrix stained with Alizarin Red;Conclusion: Three-enzyme digestion and PBSA dilution could fast and efficiently isolate mesenchymal stem cells from human umbilical cord.