Chondrogenic-related MicroRNAs Expression In Human Bone Marrow Mesenchymal Stem Cells

Objective To find the miRNAs that may regulate the chondrogenesis of human mesenchymal stem cells by detecting the different miRNAs profiles of human BMSCs and BMSCs-derived chondrocytes with microarrays, and offer a fundament to thoroughly investigate the function of miRNAs in BMSCs-derived chondrocytes differentiation.Medthods (1) The human BMSCs were collected from bone marrow by using density gradient centrifugation, and incubated in vitro for primary culture and passage culture. Then the phenotype of BMSCs was checked using a flow cytometer for identification. (2) BMSCs were incubated in chondrogenic medium [1ng/ml TGF-β1, 0.2mM ascorbic acidy , 6.25ug/ml transferrin, 1.25ug/mL bovine serum albumin, 10-8M dexamethasoney, 6.25ng/l insulin], and induced to chondrocyte differentiation. Then Toluidine Blue staining and s-100 protein antigen immunohistochemical staining were performed for chondrocyte identification. (3) Using miRNA microarray to detect miRNA expression profiles of BMSCs and BMSCs-derived chondrocytes, and performing SAM software to bolting differentiated expressed miRNAs between BMSCs and BMSCs-derived chondrocytes. (4) Some differentiated expressed miRNAs betweent BMSCs and BMSCs-derived chondrocytes were validated through Real-time PCR, then the validated miRNAs were predicted their target genes with computational softeware online.Results (1)Mononuclear cell isolated from bone marrow by using density gradient centrifugation was detected by flow cytometer, phenotypes showed CD29, CD44 surface antigens of culture cells was positive, but CD34, CD45 surface antigens was negative, which indicated these cells were human Bone marrow-derived BMSCs.(2) The BMSCs were cultured in chondrogenic medium for 21 days. We can obatain that the stainings of the Toluidine Blue and s-100 protein antigen immunohistochemical of the BMSCs-derived chondrocytes cells were all positive. (3)Based on miRNA microarray technology for miRNA expression, we firstly determined miRNA expression profiles of the human BMSCs and BMSCs-derived chondrocytes, and then compared the expression of miRNAs between the human BMSCs and BMSCs-derived chondrocytes. In the BMSCs-derived chondrocytes of the 2 samples, 26, 7 miRNAs that were significantly up-regulated, and 1, 2 miRNAs that were greatly down-regulated, and 5 up-regulated miRNAs had overlapped in all 2 samples. (4) 4 miRNAs bolt from previous step were confirmed by Real-time PCR, and the result is coincident with the microarray data. Conclusion (1) BMSCs can be isolated from bone marrow by using density centrifugation and cultured in vitro, and can be induced to differentiate exclusively into the chondrocytes. (2) miRNAs profiles expressed in BMSCs and BMSCs-derived chondrocytes from different donors exist conspicuous individual variability. (3) There are several differential expressed miRNAs between BMSCs and BMSCs-derived chondrocytes, and some of them may regulate the chondrogenic pocedure of BMSCs.