Directional Development And Differentiation Of Mouse Embryonic Stem Cells In Vitro And Visualized Through Atomic Force Microscope (AFM)

Embryonic stem (ES) cells have the character of high plasticity, apt proliferation and division and lower antigenic specificity, which can bring great expect to the cell replacement therapy in the treatment of desperate diseases. In order to investigate the membrane morphologic change and its corresponding biologic signification during the differentiation of ES cells under the control of cell growth factors in vitro, the ES cell line mouse BALB/c was cultured and differentiated into Pancreatic Islet-Like Cell Clusters (PICC). Observed the cell membrane morphologic change in different period of such process by atomic force microscope (AFM), this study has explored the application of AFM in the field of cell biology.In this study, the ES cell line mouse BALB/c was first cultured into embryonic bodies(Ebs) and differentiated spontaneously for four days. Then they were switched to gelatin-coated dishes in order to make the embryonic bodies attach and spread on the tissue culture plates. During this period, a series of cell growth factor such as bFGF, IGF-1 and Nicotinamide were added into medium in specific time in order to make the ES cells differentiated into Pancreatic Islet-Like Cell Clusters (PICC) directly. The ES cells were detected by AFM in every particular period during this process for the first time in the world. As a novel finding, ES cells division can be divided into two modes by ultrastructure (symmetrical division and non-symmetrical division), and the change of cell membrane morphology from EBs to clusters. When added the growth factor (bFGF), we could observe that the endoblast cells had proliferated obviously and the differentiation of ectoderm cells were changed in a order by the control of bFGF. Scanned the ectoderm cells by AFM, the plenty of cells, we found, were similar to the nerve fibre (including dendrite cells and axon cells). From then on, these cells were almost connected into a net and interweaved disorderly. We also found that some cells' shape were close to a circle, which might be the neuron cells that these cells might become the communication center. When adding the factor (IGF-1, Nicotinamide), we observed that the ectoderm cells were decreasing gradually by the time consuming we cultured, and the endoblast cells differentiated into PICC, however, we could not find the different change of theirIImembrane. Combining the results of ICC, we found that the number of B cells was enormous and there were lots of fibre tissues on the positive cells membrane, which we concluded that the fibre might be regulate the release of hormone. We proved the theory, first time in the world by ultrastructure, that the never and the pancreas stem cell was the same fountain, which supplied the credible evidence for cell replacement therapy. Furthermore, by adding the different growth factors, we also could induce the ES cell line mouse BALB/c into the liver cell and get the higher differentiation ratio(32%) in the end.By the use of AFM, we can observe and investigate our study objects totally different from the point of view of traditional biology and medicine, visualize the change of developmental process of the stem cell and obtain message not only special but also important. With the help of AFM, we can discover and study biologic phenomena and theories that can't be or still not be detected by other technologies yet. It extends the instrument to research the stem cell and be propitious to the development of cell biologic theory and technology.