Isolation And Culture Of Goat Embryonic Stem Cells

Goat embryonic stem cells (ESCs) and embryonic germ cells (EGCs) lines, which may be perfect cells for making bioreactor, 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. The results obtained were as follows:1. A total of 46 Guanzhong dairy goats were superovulated with a result of total 307 embryos including 39 embryos of 4-cell to 16-cell, 128 morulae, 89 blastocysts and 51 hatched blastocysts. In the study, we obtain 6.67 embryos from per goat. With the culture system including serun, which is better than no-serum in sustain growth of goat ESCs. The period of goat ESCs maintained proliferative and undifferentiated state is longer in FBS medium than in knockout serum replacement (KSR), but the cells differentiated spontaneously in culture. The ESCs colonies were subcultured for up to 10 passages in this study, in which the colonies were cultured on the MEF feeders and the media of Knockout DMEM including 5%FBS, 15%KSR, 0.1mmol/Lβ-Me, 0.1mmol/L NEA, 2mmol/L glutamine, 50IU/mL penicillin, 50μg/mL streptomycin, 1000IU/mL LIF and 20ng/mL bFGF.2. The adherence and propagation rate of ICM and passage culture of ESCs were studied in the media of Knockout DMEM including 5% FBS, 15% KSR, 0.1mmol/Lβ-Me, 0.1mmol/L NEA, 2mmol/L glutamine, 50IU/mL penicillin, 50μg/mL streptomycin and 1000IU/mL LIF. We found that the adherence rate of ICM is higher in microdrop culture than 4-well tissue culture dish, both on a feeder layer of mitomycin-inactivated mouse embryo fibroblasts (MEF). Use goat MSCs as feeder cells, we also sustain the growth of goat ESCs, and there is no difference between MSCs and MEF. The media added both LIF and bFGF sustain longer period of life than only added LIF,difference is significant, but there is no difference between LIF+bFGF group and LIF+bFGF+SCF group, which indicate that SCF seemed no used in promoting the growth of goat ESCs.3. Experiments indicated that the condition medium from mouse ESCs or SD rat NSCs can promote adherence and growth of goat ICMs,both better than the media of Knockout DMEM, but difference is quiet when comparing the rate of adherence (P>0.05), however,the difference is significant when comparing the colony rate (P<0.05). This indicated that both condition medium from ESCs or NSCs have ability to sustain the passage culture of goat ESCs. The ESCs colonies were subcultured for up to 10 passages in this essay.4. 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 both ntESCs and pESCs to passages 2.5. Forty-eight goat fetuses were collected from the Xi'an Abattoir. The putative EG-like clumps were derived from 34 goat fetuses in this study. We found that the colony rate was a little higher in the group that goat PGCs were cultured with 15% KSR than other groups with FBS, but difference is quiet (P=0.0506, P>0.05). And the EG-like colonies were successfully maintained 12 passages with the medium of high DMEM including 15% KSR, 1000 IU/mL LIF and 20 ng/mL bFGF.6. In order to enhance the growth of goat EG cells, we transfer expression vector, named pEGFP-N1-hTERT and pEGFP-C1-mNanog, into goat EG cells by Lipofectamine2000. Fluorescent observation demonstrated that hTERT and mNanog was expressed and had strong growth promotion capacity in goat EG cells. The results suggest that hTERT and mNanog may be useful in long-term culture of goat ESCs and establishment of the cells lines. This will provide the means to the study on manipulating these cells for the purpose of basic and applied research.7. The morphology of ESCs derived from goat embryonic ICM or PGCs of fetus was observed. These cell colonies were AKP positive and immunocytochemically characterized with positive Oct-4 and SSEA-1 staining and EGCs-like colonies also characterized with positive Nanog, Oct-4, SSEA-1 and TERT by RT-PCR eassy. Under the condition of delaying passage or suspension culture, those cells could differentiate into embryoid, adipocyte, neuron-like and epithelium-like cells in vitro.