Isolation and Culture of Human Amniotic Mesenchymal Stem Cells(hAMSCs) and Its Differentiation Potential In vitro

Human amniotic mesenchymal stem cells(h AMSCs) are adult stem cells, and have the characteristics of wide source, low immunogenicity and rapid proliferation. The isolation and culture condition of h AMSCs and its differentiation potency of three germ layers were investigated in this study. The amnion was mechanically separated from the placenta obtained by cesarean section after 38~40 weekpregnancy. The h AMSCs were isolated from human amnion digested twice with 0.05% trypsin-EDTA for 30 min and once with collaenase type Ⅰ for 1 h. Therefore, the obtained cells were high purity human amniotic mesenchymal stem cells. The morphololgy of h AMSCs was observed under the microscope. The growth curve of h AMSCs was checked by cell counting. Chromosome analysis was checked, then the flow cytometry,immunofluorescence and reverse transcription-PCR techniques were adopted to identify the surface molecular markers and stem cell characteristics. Further more, h AMSCs were able to differentiate into multi- potentialability when cultured in the special inducing medium. The results showed that high-purified h AMSCs isolated from amnion in the most appropriate culture condition was established. The morphology of h AMSCs was identical with fibroblast and the cells showed active proliferative ability. Results of immunocytochemistry and immunofluorescence showed that they were positive for stage specific embryonic antigens(SSEA- 4) and vimentin and had a strong proliferation and multipotential capacity. By FASC and RT- PCR analysis, the following cell surface antigens including CD29, CD49 d and CD73 expressed in h AMSCs, but negatived for CD34, CD45 and HLA-DR. The stem cell special gene markers such as OCT4, SOX2 and NANOG expressed constantly in these cells, and these cells could differentiate into fat cells, osteoblasts cells, neuron- like cells and insulin secreting cells; special marker genes such as PPARγ2, Osteocalcin, MAP- 2, PDX1 expressed in insulin secreting cells. h AMSCs could be successfully isolated, cultured and proliferation in vitro culture conditions. The results showed that h AMSCs can be separated and maintained a stable genetic characteristics in vitro. h AMSCs not only has the characteristics of mesenchymal stem cells, but also has the potential to differentiate the three germ layers. These results provide experimental basis for clinical applications of tissue engineering and regenerative medicine.