Studies On Inducing Human Umbilical Cord Mesenchymal Stem Cells To Differentiate Into Adipocyte

Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow. They can replicate as undifferentiated cells and have the potential to be differentiated to lineages of mesenchymal tissue. Just like bone marrow mesenchymal stem cells, umhilical cord mesenchymal stem cells have the capacity of multi-differentiation. However, UCMSCs can be isolated easily with abundant resources. Thus more and more researchers became interested in the study of UCMSCs.Objective:To establish the method of isolation of human umbilical cord mesenchymal stem cells (hUC-MSC) and to identify their surface markers. To study mesenchymal stem cells differentiation into adipose cell and to investigate the expression of miRNA-27a and miRNA-143during the differentiation. All of these will provide theoretical basis to clarify the regulation mechanisms of MSCs’ adipogenic differentiation, promote the development of adipose tissue engineering and provide evidence to clinical application of soft tissue repair and reconstruction. At the same time, these will provide new ideas and methods for the diagnosis and treatment of obesity, diabetes and metabolic diseases.Methods: The UCMSCs were isolated from neonate umbilical cord, cultured by collagenase digestion and tissue explants adherent methods. The surface markers were identified by flow cytometry. Adipogenic differentiation of the fifth generation of UCMSCs was induced with IB MX、insulin、indomethacin and dexamethasone. The differentiated cells were identified by oil red O immunohistochemistry stain. We also explored the expression of miRNA-27a and miRNA-143and their target genes in UCMSCs group and adipocytes group by real-time quantitative polymerase chain reaction (qRT-PCR).Results:The purified human umbilical cord mesenchymal stem cells showed the morphology of fiber oblasts. Some mesenchymal like cells were shown after2-3days by collagenase digestion. We found colonies after3-5days. The number of UCMSCs was increased rapidly. After5days of primary culture, isolated UCMSCs reached80%confluence.1week after transferring fragments into plate, some mesenchymal like cells with a fusiform or stellate appearance migrated from the explants. After2weeks isolated UCMSCs reached80-90%confluence. When UCMSCs reached80-90%confluence by either of the above two methods, we need to passage them at1:3under the same culture conditions. After passaging, the cells picked from the clones could be passaged and grew like whirlpool as the same morphous as fusiform shape or similar to the fibroblast. After approximately3days, cell culture reached80%confluence. We selected the fifth generation for analysis. The results of flow cytometry analysis revealed that CD44、CD73. CD105、CD166、HLA-ABC were highly expressed on the cell surface, while CD34、CD106were negative expressed. The hUC-MSCs could differentiated into adipocytes by IBMX、insulin indomethacin and dexamethasone. And the differentiation rate was more than90%. The miRNA-27a was down-regulated (0.0095±0.002vsl±0.03) and miRNA-143was up-regulated (17.81±0.88vsl±0.004) during adipogenic differentiation detected by using qRT-PCR.Conclusion: hUC-MSCs could be successfully obtained by collagenase digestion and tissue isolation. The hUC-MSCs had the potent of proliferation and self-renewal. Umbilical cord derived fibroblast-like cells have the same immunophenotype with mesenchymal stem cell. More than90%hUC-MSCs could differentiate into adipocytes by using IBMX、insulin、indomethacin and dexamethasone. During the adipogenic differentiation, the expression of miRNA-27a and miRNA-143were changed, which indicated miRNA-27a and miRNA-143may regulate the adipogenic differentiation of MSCs.