A comprehensive evaluation strategy for chondrogenic differentiation of human mesenchymal stem cells

ZHANG Ke-hua; LIU Jing; NA Tao; YUAN Bao-zhu

Abstract：Objective:To explore a comprehensive assessment methodology and strategy for evaluating chondrogenic differentiation of human mesenchymal stem cells (hMSCs),thus to provide technical support for evaluation of the in vitro biological effectiveness of hMSCs.Methods:Chondrocyte-born microballs were formed by culturing hMSCs in high density in form of droplets in cell culture dish.The microballs were then put in the in vitro environment of chondrogenic differentiation and analyzed by three approaches,i.e.,further forming chondrocyte tissues with typical morphological characteristics,staining intracellular matrix derived from chondrocyte tissues by Alcian blue and Safranin O,and measuring the changes in expression of chondrocyte-specific genes,i.e.ACAN,COL1A2,COL2A1,COL10A1,and MIA,by real-time PCR.At the end,after validating by histological analysis,the capability of chondrogenic differentiation of hMSCs was evaluated by all the three approaches together.Results:The hMSCs with potential of chondrogenic differentiation exhibited the following features:formation of microballs spontaneously after the cells being congregated in high density and further condensation of the microballs with smooth appearance and positive Alcian blue staining in the condition of chondrogenic differentiation;formation of typical chondrocyte intracellular matrix showing positive staining by Alcian blue and arrangement in concentric circles at the periphery of the chondrocyte microballs;dramatic up-regulation of chondrocyte specific genes following the treatment of chondrogenic differentiation.Moreover,hMSCs of different donors and tissue origins exhibited significant difference in each of all three aspects chondrogenic differentiation.Conclusion:The evaluation technology employing three assessment approaches in combination,which covers the morphology,histology and gene expression associated with chondrocyte characteristics,is much more objective and accurate than single approach in evaluating the potential of chondrogenic differentiation of hMSCs.