Isolation And Cultivation Of Mouse And Goat Embryonic Stem Cells

In the past few years, due to its totipotency, multipotency operability and plasticity, the embryonic stem cells(ESCs) have become one of the most popular research topics in the fields of medical and life science. Mouse ESCs have been widely used in those research fields like developmental biology, human disease and drug development. The establishing technique of ESCs series have provided reference standard and theoretical basis for the establishment of human and animals ESCs series. In this research,the mouse embryonic fibroblasts(MEFs) were made by 12.5 to 13.5 days mouse fetus, and we also compered the effects of glycerine and DMSO as cryoprotectant to MEF.Approaching the effects of different developmental stages of embryos, blood-serums, growth factors and the ways of passage to the ICR ESCs with the subjects of 3.5 days mouse embryos.The results are as follows:1. Using the DMEM solution with 10% glycerine as the common cryoprotectant is better than the DMEM solution with 10% DMSO when freezing the MEF, and the vitalities are 0.787 and 0.728.2. Most of the 3.5 days mouse embyos are blastulas and morulas. The blastulas'adherence and ICM growth rate(75.4%, 72.8%)are both significant lager than morulas(57.9%, 52.6%)(P<0.05) after seeding on the MEF feeder layers with the ESCs medium contained with 15% FBS. Further more the speed of adherence and multiplication of morula is slower than blastulas, so it's better to isolated culture ICM with blastula.3. The blastular aherence rate and ICM proliferation rate are both lager than the mediums contained with 15%KSR or 5%FBS ad 10%KSR(42.9%, 28.6%; 75%, 54.2%)when using the medium contained with 15% FBS(79.3%, 69%) by the subjects of ICR blastulas. That's to say it's better to use the mediums contained with 15%FBS to blastular aherence and ICM proliferation, while subcultring the mouse ESCs it is better to use the mediums contained with 5%FBS and 10%KSR and coloned to sixth passage.4. and coloned to sixth passage.5. The added factors can play a positive role on blastular adherence, ICM proliferation and passage. When comparing with the control group which had no fators, the culture with added 10ng/mL LIF and 10ng/mL SCF was better than the one added only one fator,and it coloned to sixth passage which was higher than the passage number of the culture with only additional one kind of fator(forth passage,second passage).6. The mouse ICM which were sprarated by the ESCs medium contianed with 15%FBS were subcultured in three kinds of passage ways with the ESCs culture contianed with 5%FBS ad 10%KSR. The diversity adherence way is the best way of passage, and it coloned to eighth passage, as well the enzyme digestion colonged to forth passage as the mechanical method ad enzyme digestion coloned to sixth passage.7. In this experiment, we got the ICR mouse ESCs which had typical ES colony morphology showed the high activity of alkaline phosphatase(AKP).8. In this research, we got the embryoid through the way of suspension culture the ICR mouse ESCs, and during the culture process in which the mouse ESCs spontaneously differentiated into fibroblast-like cells, epithelial-like cells, fat-like cells and nerve-like cells and so on, as well the emergence of vacuolization.At present, there has been some detailed report about the research regarding human and mouse ESCs series. However, the studies about animal ESCs are still inadequate with little report about animal ESCs series. Goat ESCs and embryonic germ cells(EGCs), which may be perfect cells for producing transgenic, coloned animal and making bioreactor, Nevertheless, these has not been any successful report in relation to any of the establishment of the serious. The aim of this experiment was to compare the effects of feeder layer and growth factors on isolation and culture the goat ESCs with the subjects of 30 to 50-day goat fetus and 6 to 8-day goat blastula.The results are as follows:1. The research compared the effects of two kinds of feeder layers (MEF and GEF) and two kinds of cultures which contained different factors on the goat ESCs with the subjects of the goat ICM which were separated by mechanical method.The result showed that the ICM proliferation rate was better by using the MEF feeder layer than GEF feeder layer. In addition, using the same MEF feeder layer, when added with LIF SCF and insulin, they all had positive impact to the goat ICM adherence, proliferation and passage. However, best effect to goat ESCs when LIF and SCF were both added into the meduim with MEF feeder layer and coloned to third passage.2. In the experiment, we got the goat ESCs which had typical ES colony morphology showed the high activity of alkaline phosphatase(AKP).3. During the experiment,the goat ESCs spontaneously differentiated into fibroblast-like cells, epithelial-like cells, fat-like cells and nerve-like cells and so on.