Isolation And Biological Characteristics Of Mouse Dermal Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a kind of tissue stem cells with the ability to cross-embryonic layer differentiation potential and high self-replication ability, easily isolated, have low immunogenicity and migration ability to tumor. MSCs was originally found in the bone marrow. In the beginning of this century, researchers isolated MSCs from the dermal tissue, and named it for dermis mesenchymal stem cells (DMSCs). DMSCs are easily obtained, have wide variety of sources and are able to promote hematopoietic recovery. So DMSCs were the important seed cells for stem cell therapy and have a wide range of potential clinical application, can not only be used to repair the skin and nerve damage, but also be applied to the anti-skin aging and skin tissue engineering. Based on this, this experiment aims to establish fast isolation and steady expansion system of mice DMSCs in vitro, and explore the muilt-differentiation potential, which will provides theoretical foundation and experimental basis for the clinical application of DMSCs.Two to three-day newborn ICR (institute of cancer researcch, ICR) mice back skin was mechanically seperated and digested by trypsin, then DMSCs were obtained. It's culture and proliferation system were composed of basic medium and different cytokines. The CD44, CD29and CD45expression of DMSCs was identified through immunohistochemical method. The expressions of Sca-1, CD29, CD90, CD34, Oct4, Sox2, c-Myc and Kif4were detected by RT-PCR. DMSCs were induced to differentiation into adipocytes, osteoblasts, neurocytes, cardiocytes and sweat gland cells, and identified by cytochemistry method and RT-PCR. The activity was detected by MTT method after DMSCs were treated by different proportion (20%,40%,60%,80%,100%) fibroblasts conditioned medium of DMSCs. To explore the effect of Reversine on the proliferation and differentiation ability of DMSCs, DMSCs were treated by different concentration of Reversine.The results showed that fast isolation and steady expansion system of DMSCs in vitro were obtained by improving the culture condition. Immunohistochemical identification indicated that DMSCs expressed CD44, weakly expressed CD29and did not express CD45. RT-PCR results showed that DMSCs expressed Sca-1, CD29, CD90, CD34, c-Myc, Kif4and unstablely expressed Oct4. DMSCs were successfully induced to differentiation into adipocytes and osteoblasts.20%,40%and60%fibroblasts conditioned media were able to promote the proliferation of DMSCs. Reversine had not significant effect on DMSCs' proliferation, but could promote DMSCs to trans-differentiate into adipocytes.