Isolation And Culture Of Embryonic Stem Cells Derived From Goat

Goat embryonic stem cells (ESCs) and embryonic germ cells (EGCs), which may be perfect cells for coloned and transgenic animal, making bioreactor and Tissue engineering, have not yet been established. The aim of the present study was to isolate and culture ESCs and EGCs from goat embryos and fetuses of different development stage, and to compare some factors influencing the efficiency of establishing those stem cell lines by adopting the methods of mouse and human. And some detections were also done to the cells obtained. The results obtained were as follows:1. 4 Guanzhong dairy goats were superovulated with a result of total 42 embryos including 3 embryos of 4-cell to 16-cell, 5 morulae, 28 blastocysts and 6 hatched blastocysts. In the study, we obtain 10.05 embryos from per goat. We used whole embryo cultured,enzymatic digestion and immunosurgery methods separately to see the influence to the subculture by different stage of embryos in the primary cultured. And we found that the whole embryo method was best for the embryos hatching the dish, and the blastocyst was best for deriving ESCs, and the cells could be subcultured to 18 passage.2. We compared mechanical cutting and enzymatic digestion in the subculturing, and found that the first method was good for the proliferation, the second one was good for restraining the differentiation. In this experiment, we used mechanical cutting before 6 passage, enzymatic digestion after 6 passage, and obtained the 18 passage ESCs.3. The goat ESCs is derived from cloned and parthenogenetic embryos, which adherence rate is very low and the embryonic cells proliferate slowly. Compare the results of isolation and culture from different development stage embryos and found that early cloned or parthenogenetic morula is better than blastula for isolate the goat ESCs, which rate of adherence is higher. In this essay, we sustain ntESCs and pESCs separatly to passages 4 and passage 2.4. The immunochemistry and RT-PCR detection showed that the cells express SSEA-1, SSEA-3, Nanog, Oct4, TERT and CD117. They could also be induced to cardiomyocytes in vitro, which demonstrated that the cells we obtained have the characteristics of ESCs.5. 30 goat fetuses were collected from the Xi'an Abattoir, and the EGCs were derived from the germ ridge. We found that the colony rate was a little higher in the group that goat PGCs were cultured with 15% KSR than the other groups with 15% FBS, but the difference is quiet (P>0.05). The mechanical cutting method sustained the cells to passage 10, and the enzymatic digestion sustained the cells to passage 5, the first method was better for subculture, the differerce was obvious (P<0.05).6. The goat EGCs were positive for AKP, Oct4 and SSEA-1. They also express Nanog, Oct4 and TERT in the RT-PCR detection. Under the condition of delaying passage or suspension subculture, those cells could differentiate into embryoid, adipocyte, neuron-like and epithelium-like cells in vitro, which showed that the EGCs we obtained have the characteristics of ESCs.