| Embryonic stem cells are pluripotent cells derived from the inner cell mass of pre-implantation mouse embryos. They have extensive selfrenewal capacity and are competent to differentiate into any cell type of the body in vivo and in vitro. It is a tractable cellular system to investigate cellular and genetic programming of early development. In terms of clinical benefit, stem cells are generating many hopes for future regenerative medicine.Smooth muscle cells (SMC) of the vascular system form an intriguing population of cells that are relevant for maintaining vascular tone and function. Proper regulation of vascular smooth muscle cell (SMC) differentiation and growth is critical for vasculogenesis and the maintenance of homeostasis in the mature vessel wall. Abnormal growth and proliferation of vascular smooth muscle cells (SMCs) is a key feature of vascular diseases such as atherosclerosis, restenosis, and hypertension. An in vitro system that recapitulates human SMC differentiation would be invaluable for exploring the environmental cues and molecular mechanisms leading to the human diseases. Various types of cell lineages but not involving SMC have been derived from human embryonic stem cells through directed in vitro differentiation, however, the differentiation efficiency and the homogeneity of the differentiated cells remained a major issue for potential application of these cells in therapeutic medicine.In this study, we have established an innovative culture system for the efficient differentiation of smooth muscle cells from human embryonic stem (ES) cells by monolayer adherent culture without feeder cells, embryoid bodies or cell-sorting processes. The hESCs were cultivated in differentiation medium containing 10μM of RA, more than 93 percent of all the cells expressed SMα-actin and 64 percent of all the cells expressed SM-MHC after 15 days. Combining to detect some parameters, such as cell appearance, RT-PCR, immunofluorescence, western blot, responding to agonist, spontaneous contraction, it shows hESCs-derived cells has the typical characteristic of the smooth muscle cell. The advantage of this system includes simple procedure and high efficiency. This inducible system (single factor, the monolayer, high efficiency) in vitro provide a very good platform for studying the molecular mechanism controlling SMC differentiation. Furthermore, high pure hESCs-derived SMC would provide a good cell resource for cell therapy in the future.Through the RT-PCR analysis for expression of cyp26s, RARs and RXRs in induced process, study retinoic acid metabolism blocking agent's influence to this monolayer system and time-dependent effects of RA on the SMC Differentiation of hESCs in vitro, we found that the high efficiency of the SMC differentiation in this system appears to result from double effects of differentiation-inducing and apoptotic of high atRA on the starting hESCs. Because of RA's multi-biological effects, the ES cells started differentiation toward various cell lineages (include SMC lineage) under RA. however progenitor cells in early differentiation appeared obvious difference about the ability with RA inactivation due to their expression dissimilarity of the CYP26s. These differences urged parts of cells (SMC progenitor cells) can succeed in escaping the destiny of apoptosis because of the high expression of the CYP26s. In the and, it make the hES cells differentiate SMC efficiently.To drive differentiation of mouse embryonic stem (ES) cells, various culture protocols have been previously developed that most require the formation of embryoid bodies or co-culture, Considering that, in terms of cell to-cell interactions, one-dimensional culture introduces lower complexity to the system compared to three-dimensional EBs, we reasoned that differentiation protocols involving only monolayer culture must be developed in order to: (1) make large scale production of neurodifferentiated cells more effective and to (2) allow for analyzing the molecular mechanisms that drive early events of neural differentiation in vitro.A significant problem for monolayer adherent differentiation is how to gain pure ES cells. We established a method of separating murine embryonic fibroblast from embryonic stem cells. By comparing the different adhesion rate of MEF and ES cells to find out the opitimum time for MEF removing. The result shows that 1.5h and 0.5% gelatin concentration is the opitimum condition for MEF removing. Furthermore, the ES cells which have been purified have the same plating efficiency and the ablity of three germ layers differentiation as the unpurified ES cells. After differential adhesion there is strong ALP activity in ES cells. All the results show that the purified ES cells are still in the state of undifferentiation and maintain the pluripotent.On the basis of establishing the ESC differentiation system by monolayer adherent culture, we try to apply sodium butyrate to research neural differentiation. It make the period of the ESC direct differentiate into neuron shorter, the efficiency is higher, manipulation is more simple. Use HDAC inhibitors (NaB) to breaks the balance of histone acetylation, make chromatin loose, thus repress embryo stem cells to propagate, make it turn into differentiation appearance as soon as possible, induce medium N2B27 was adopts by monolayer adherent culture immediately. The results show that high proportion of neuron were obtained by this method, there are mostly nestin-positive neural progenitor cell at 4-6 days, and ESCs were differentiated into GFAP-positive gails and neuron that expresβ-tubulin III after two weeks. ESCs-derived neuron have the function that let out electric potential by LAPS chip examination.
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