Biological Characteristics Of Adipose-Derived Stromal Cells And Preparation Of Reprogramming Vectors

The research and application of stem cells have exerted an important influence on the field of disease therapy, drug development, pharmacodynamics and toxicity assessment, animal cloning, transgenic animals, developmental biology. In theory, ESCs have unlimited growth and differentiation potential, however, because of the immunological regection and ethical controversy, the application of ESCs has been limited. So the resent research has tended to find other kind of cells to instead of ESCs, for example, the study on somatic stem cells and pluripotent stem cells obtained by nuclear transfer or reprogramming provide a more broad prospects for the stem cells. Because of the extensive sources, convenience of drawing materials and more stem cells, adipose-derived stromal cells have become a research focus of tissue engineering. However, the mechanism of self-renew and multiple differentiation potential in ADSCs is not sure, and the growth and differentiation potential of ADSCs is limited compared with ESCs. In ESCs, Oct3/4, Sox2 and Nanog play an important role in the matainance of pluripotency. So this research focus on the level of these transcriptional factors in ADSCs, and the preparation of reprogramming vectors, to provide a more suitable material for the research of stem cells.In this paper, ADSCs were isolated from subcutaneous adipose tissue located in inguinal of 15-day old SD rats, and cultured in DMEM complete medium. The biological characteristics and differentiation potential were analyzed by experimental techniques of cell biology and molecular biology, such as RT-PCR, immunocytochemical staining, specific stain and so on. What's more, we obtained the vectors:Lent-EF1-OCT3/4-IRES-EGFP,Lent-EF1-SOX2-IRES-EGFP, Lent-EF1-c-MYC-IRES-EGFP and Lent-EF1-KLF4-IRES-EGFP, which were gifted by Doctor XiaoLei, and we indentified the length of aim genes instructed to the vectors, and package them in 293T cell, what's more, we detected the infection ability of the four vectors, which establish a basis for the future study.1.The Isolation and Culture of Adipose-Derived Stromal Cells.Stromal vascular fraction was isolated from subcutaneous adipose tissue of 15-day old SD rats, then passaged and purified to obtain ADCs. The fresh isolated-cells contained some kind of cells, showed triangle, fusiform, globular and polygon, after passage, the other cells disappeared except for fusiform cells, and these cells formed fibroblast-like colonies.2.The Biological characteristics of ADSCsThe results of RT-PCR showed that ADSCs expressed many of transcriptional factors, such as Oct3/,Sox2,Nanog,cMyc, and so on, meanwhile, immunocytochemical staining proved that ADSCs expressed CD105,Oct3/4, Sox2, Nanog, cMyc, SSEA1 and telomerase, but not CD31 and CD34, and the expression levels of Oct3/4, Sox2, Nanog and SSEA1 were 2.0%,3.7%,2.4% and 2.6%, respectively, what's more, the staining for AP showed positive.3.ADSCs differentiated to adipocytesUnder induction of insulin, hydrocortisone and transferrin, ADSCs differentiated to adipocytes gradually which contained a lot of lipid droplets, the Oil red O staining showed positive, and the cells expressed PPARy, FAS and LPL, which increased along with the induction.4. ADSCs differentiated to osteoblastsUnder induction of LiCl, ADSCs changed to square like from fibroblast like gradually, and the activity of AP improved, what's more, the cells formed many calcified nodules, the alizarin bordeaux staining showed positive, and the OSX and OPN also showed positive.5.ADSCs differentiated to neural-like cellsUnder culture of ADSCs in medium without serum supplement but withβ-mercaptoethanol, the cells contracted and stretched synapsis and connected to form network. The immunocytochemical staining showed the expression of Nestin, Tau and NF.6. The construction and identification of vectors containing transcriptional factorsLentivirus vectors containing Oct3/4, Sox2, cMyc and K1f4 were identified by RT-PCR and enzyme digestion, then we packaged these four vectors in 293T cell line and detected the transfection efficiency, founding the efficiency was too low to used for the future study.In a word, we isolated ADSCs from adipose tissue of rat, and the results showed that ADSCs expressed the marker of mesenchyme stem cells CD105,but not CD34 and CD31, which were the marker of hemopoietic stem cell and epidermic cell, respectively, and expressed many of specific markers of ESCs. Under the condition we established, ADSCs could differentiate to adipocyte, osteoblast and neural-like cell. What's more, we obtained vectors for reprogramming somatic cells into pluripotent stem cells, while the efficiency of infection should be improved.