Osteoblast Differentiation-specific MicroRNAs Expression In Human Bone Marrow-derived Mesenchymal Stem Cells

Background MicroRNAs (miRNAs) are endogenous approximately 20~25nt RNAs that can play important regulatory roles by targeting mRNAs for cleavage or translational repression. Hundreds of miRNA genes have been found in diverse animals. miRNAs regulate a variety of biological processes, including developmental timing, signal transduction, tissue differentiation and maintenance, disease. evidence is mounting that animal miRNAs are more numerous, and their regulatory impact more pervasive, than was previously suspected.Human marrow-derived mesenchymal stem cells(MSCs) are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. MSCs which have been separated and cultured in vitro shows MSCs are suitable for seed cells in tissue engineering.miRNAs regulate gene expression at the posttranscriptional level in many developmental and metabolic processes. Emerging evidence demonstrates that miRNAs also play an essential role in stem cell self-renewal and differentiation. Some miRNAs are specifically expressed in stem cells, control stem cell self-renewal, and differentiation through negatively regulating the expression of certain key genes in stem cells.Objective To describe the accumulation of specific miRNAs in MSCs and MSCs-derived osteoblast, and demonstratede that specific miRNAs expression are precisely regulated during MSCs-derived osteogenesis in vitro. Then to find out some osteoblast differentiation-specific microRNAs, and offer a fundament to thoroughly investigate the function of miRNAs in MSCs-derived osteoblast differentiation.Medthods (1) Bone marrow-derived MSCs were collected by using density centrifugation, and incubated in vitro for primary culture and passage culture. Then the phenotype of MSCs were analyzed using a flow cytometer. (2) MSCs were seeded in osteogenic medium[10-8M dexamethasoney, 0.2mM ascorbic acidy, 10mMβ-glycerolphosphate], and induced to osteoblast differentiation. Then alkaline phosphatase (ALP) staining, Alizarin red S staining and Electron microscopes examing. (3) Using miRNA microarray to detect miRNA expression in MSCs and MSCs-derived osteoblast, and using SAM to bolting different miRNAs between MSCs and MSCs-derived osteoblast. (4) Some specific miRNAs during MSCs-derived osteogenesis in vitro were confirmed through Northern blotting and Real-time PCR.Results (1)Mononuclear cell collected by using density 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 MSCs.(2) The MSCs were cultured in osteogenic medium for 14 days. We can see that the staining of the ALP of the cells in the osteoinductive groups was positive, and osteogenic medium led to the formation of mineralized node visualized by alizarin red S staining. As shown by inverted microscopes and SEM, the morphology of the cells in the osteoinduction groups changed from spindle shaped cells into polygon shaped cells after the same days of culture, and collagen(COL)-like processes were detected under SEM. A large number of the dilated rough endoplasmic reticulua, Golgi apparatus, mitochondria and calcium salts could be seen under TEM. (3)Based on a previously described miRNA microarray technology for miRNA expression, we firstly determined miRNA expression profiles of the human MSCs and MSCs-derived osteoblast, and then compared the expression of miRNAs between the human MSCs and MSCs-derived osteoblast. In 3 samples,for the MSCs-derived osteoblast, 36,14,8 miRNAs that were greatly up regulated, and 24,27,20 miRNAs that were sharply down regulated, but a few specific miRNAs had overlapped in all 3 samples, like hsa-miR-30a-5p,hsa-miR-30c,hsa-miR-210,hsa-miR-31,et al. (4) hsa-miR-30a-5p and hsa-miR-210 were confirmed through Northern blotting and Real-time PCR, which is coincident with the microarray data.Conclusion (1)MSCs can be separated and cultured in vitro by using density centrifugation, and can be induced to differentiate exclusively into the osteocytic lineages.(2)Many miRNAs are expressed in MSCs and MSCs-derived osteoblast, and miRNAs have conspicuous individual variability.(3)Some specific miRNAs which are up or down regulated during MSCs-derived osteogenesis in vitro, may be control MSCs differentiation.