Isolation,Culture And Identification Of Chicken Embryonic Stem Cells

Isolation of embryonic stem cells of mice was first put forth in 1981, since then research on stem cells has become one of the topic issue in biology and much more progress have been achieved. But it has also been proven that only the mice embryonic stem cells have pluripotency to date. The purpose of this study is to investigate the effect of cytokines and other factors on the culturing of chicken blastdermal cells (CB cells), then build a suitable culture system and finally obtain the chicken embryonic stem cells (CES cells).The CB cells derived from x stage blastdermal cells of chicken hare been cultured on mitotically inactive feeder layer cells for four generations. The culture medium is composed of DMEM (high glucose) supplemented with FBS x beta-mercaptoethanoK SCF- bFGF, LIF etc. These cells exhibit features similar to those of murine Es cells such as typical morphology .. strong AKP activity. They also present high capacities to differentiate in vitro into various cell types. Production of chimeras after injection of the cultivated cells reinforces the view that our culture system maintains in vitro some chicken putative ES cells.Among the factors that influence the CB cells, feeder layer cells are the crucial one. We use primary chicken embryonic fibroblast (PCEF) cells, primary mice embryonic fibroblast (PMEF) cells and SNL cells respectively to make feeder cells and then observe the growth behavior of the CB cells. The experiment result indicates that the most effective one for the growth of the CB cells is PMEF cells. In addition, cytokines have different impact on the CB cells. SCF, bFGF can activate the growth and propagation of cells but without LIF the CB cells will differentiate more readily. If the mount of LIF were enough (2000IU/ml) the CB cell would have a fine state even without the feeder layer cells. We also find that the mixture of these three cytokines has47the greatest positive effect on the CES cells culture especially for the subculture. Besides feeder cells and cytokines, there are many other factors that are influential on the CB cells such as cell-dispersed liquid, the density of FBS etc.After cultured 24h the CB cell-colonies composed of 10-20 cells appear on the feeder layer cells and these colonies become more obvious after another 24h. 3~4days later we can see some nest-like ^ hill-like or sunflower-like colonies. 5 or 6 days later the edge of these colonies is clearer but there are also some differentiated cells. Thus it is the time to reproduce.In conclusion the cultured CB cells have the character of AKP positive. They can differentiate to several kinds of cells under the condition of devoid of LIF and feeder layer cells. And now we have gained three chimeras. All of which indicate that the CB cells are like-CES cells. They can be cultured in vitro for a long term using PMEF cells as feeder cel^ DMEM (High glucose) as basic medium supplemented with FBS^ cytokine etc.