Studies On Transformation Of Human Embryonic Pluripotent Stem Cells Into Lslet-secreting Cells

human embryonic pluripotent stem cells is a kind of stem cells with multi-functional differentiation, which can differentiate into a variety of connective tissue and Ectodermal Organization. it can differentiate into dermis, fat, bone, cartilage, skeletalmuscle, tendon, ligament, bone marrow, islet cells and nerve under Different inducing factors. Recently, the main source of cell is still bone marrow, which has some defects, such as small number(with aging the number of stem cells will greatly decrease), poor Differentiation potential and strong immune prototype, so it will easily lead to post-transplant immune rejection, some physical factors such as weakness, disease and infection other can also restrict the application of autologous transplantation. While, fetal-derived stem cells have a much larger number than adult stem cells, which is more primitive and more Proliferation potential, and it also have relatively low Immunogenicity, so fetal-derived stem cells have a better prospect for clinical application.Currently, adherent screening is the main Method to separate and Screen mesenchymal stem cells, this method's name is Natural purification method, which uses strong Adherent ability and viability that embryonic stem cells contain, in the process of cell culture, it can eliminate the non-embryonic stem cells which have poor adhesion and low viability. This method applies to the isolation of the bone marrow-derived mesenchymal stem cells first, Campagonli and chunhua Zhao had proved that the selection method is also worked in isolated fetal liver-derived mesenchymal stem cells.This article provides an overview of human embryo-derived mesenchymal stem cells differentiation induced by experimental research At home and abroad, separation and purification of the source of abortion fetal liver fetal mesenchymal stem cells, and identify its biological characteristics preliminarily, then differentiate these cells into insulin-secreting cells through a variety of Inducible factors, identified the induced cells by various methods. In this paper,We use bFGF, GLP-1 and nicotinamide to induce human embryonic mesenchymal stem cells and differentiate it into insulin-secreting cells. We purify fetal liver-derived mesenchymal stem cells through the separation of the Abortion fetal liver, and cultured in the high glucose DMEM medium with 10% fetal calf serum, that is in a incubator with 37℃and 5%CO2. After subculturing 3 generations, By adding the above-mentioned three kinds of growth factors, We collected the cells after 25-35 days of culture. The observation of human fetal liver tissue-derived MSCs'morphology: the cells of the original generation grows slowly, while the sizes of the new cells are not uniform, showing polygonal. After 24 hours the cells will Adherent and the Cells'shape became round, 72 hours later, majority of the cells had cytoplasmic processes, the shape of the adherent cells became spindle-shaped or long spindle, the Growth of the cells were still very slow. 80% of the cells were fusion after 1 week's primary culture, The cells look like vortex or flame, showing epithelioid cells and spindle cells. At this point it can be subcultured, after subcultured the cells entered the logarithmic phase, In this period the cells grow rapidly and gradually purified. They have less and uniform cytoplasm and no particles. The shape of the cells are fusiform or rhomboid. Under the microscope, we can see that these cells has a high ratio of plasma/nuclear, and the nuclear are large. Some have dual-core. All of these indicate that the cells has a very strong capacity for splitting. After 7~10 days, cells can be passage once, the logarithmic phase cells were induced in P3 generation, after 7 days'induction, some cells became round gradually, and a small part of the cells gathered into a cell mass, a number of Oval cell clusters which can be seen after 15 days'induction. Continue to induced the cells , the number of cell clusters became more and the shape became gradually circular. Induced in high glucose conditions , the fetal liver-derived mesenchymal stem cells clustered together, Cluster around the cell mass cells positive brown granules can be seen by Immunocytochemical staining, but the middle part of the clusters of cells are stained negative. When adding nocotinamide in the cells, the clusters'diameter became large, positive cells began to increase in the clusters, dark brown positive granules can be shown in cytoplasm, and the cells around the positive cells decreased. Dithizone showed positive staining, Cells are stained brown to dark red, and non-inducing cells were not stained, indicating that the cells containing high concentrations of zinc. Compared with the DNA in non-inducing cells, the inducing cells after RT-PCR amplification can specific express the gene PDX-1, INS-1, Somatostatin which related to insulin-secreting cells. The result of a variety of detection methods indicates that human embryonic mesenchymal stem cells have been directed to differentiate into insulin-secreting cells.In summary, the experimental results show that: human embryonic mesenchymal stem cells can be induced into insulin-secreting cells by the above-mentioned agents in vitro, and the cells have the role of regulation of blood sugar. However, there is no conclusive evidence to prove that the cells can be used to treat diabetes. Because the real islet B cells are interact with the environment in body, and the cells induced by our experiment can be tested to demonstrate that the induced-cells only have some part characteristics which insulin-secreting cells have, so it need to further prove that it can be transplanted into diabetic animals so that blood sugar back to normal, finally, we can draw the conclusion that human fetal liver-derived mesenchymal stem cell can be induced into a real insulin-secreting cells.