Study Of Embryonic Stem Cells Differentiation Into Neural Cells In Vitro

The typical method of ES cells differentiation into neural cells in vitro includes embryoid bodies (EBs) formation and subsequent differentiation into neural cells under all-trans retinoic acid or serum-free conditions. Because EBs contain many different kinds of cells, and its differentiation procedure is too long, so the method without this stage was well developed in recent years. This study avoided formation of EBs and enabled differentiating ES cells into neural cell directly.This study focused mainly on the preparation of conditioned media (CM) from mouse astrocytes, rat astrocytes and liver mesenchymal cells, and investigated the effect of soluble factors in CM and sodium butyrate on ES cells differentiation into neural cells. The main contents are as follows:1. Isolation and culture of astrocytes.Astrocytes were obtained from ICR mouse, CDl mouse and SD rat.They were new bom within Id. Morphological observation showed that forms of astrocytes derived from different animals were very different. Furthermore, different culture conditions also induced different cell forms.2. Isolation and culture of fetal liver mesenchymal cells.Fetal liver mesenchymal cells were obtained from CDl mouse embryo of 14~16dpc.3. Preparation of CM and induced differention of ES cells into neural cells.This is the first report about the effects of soluble factors from astrocytes, fetal liver mesenchymal cells and astrocytoma lines U251 on the ES cells differentiation in vitro into neural stem cells. The efficiency of CM from different tissues to induce ES cells differentiation into neural cells were studied. It was found that the effect of astrocytes conditioned medium (ACM) from SD rat was most obvious, 50% ES cells being changed into epithelia-like cells. And with the increased inducing time, the ratio of those cells also increased, and neuron-like process bearings appeared on most of them. Such obvious effects were observed neither in ACM from ICR mouse or CDl mouse,nor in fetal liver mesenchymal cells CM from CDl mouse. The latter did not have the inducing effect as obvious as ACM from SD rat, but to compared with the control, they had smoother cell margin and the periphery ones of the aggregated cells had more neuron-like process bearings. 4. Sodium butyrate induced differentiation of ES cells into neural cellsThis is the first report on effects of sodium butyrate on ES cells differentiation into neural cells in vitro. After treating the ES cells of various density with different sodium butyrate concentration and time, we drew a primary conclusion mat dissociated ES cells at a density of 5X 104cells/ocm2"in sodium butyrate of 2.5 X 10~3 mol/L concentration for 3d appeared to be the best condition. In such condition, more than 50 % ES cells survived and 90 % of them were nestin positive cells. At the same time, fetal liver mesenchymal cells CM from CDl mouse could administer to increase the livabiliry of the differentiated cells.We sought to develop a simple system that would allow direct observation, analysis, and manipulation of the process of neural specification without the confounding influences of cell aggregation, coculture, cytokine or cell selection. We have established a new and better method for generating neural stem cells, by culturing dissociated ES cells in ACM from SD rat and sodium butyrate.The present method has many advantages over the previous ones besides the high efficiency . Firstly, using the soluble factors in ACM is more convenient than using membrane-bound factors such as SDIA, as ACM is readily available, easy to obtain and ready to use. Secondly, we found that differentiation of neural stem cell occured very rapidly, so this result showed that nestin positive cells (a marker of neural stem cells) can be generated from undifferentiated mouse ES cells within 24 hours, which is shorter than any of the previous methods.