Embryonic stem cells (ESCs) are originally derived from the ICM of blastocysts and characterized by their ability to self-renew and their pluripotencies. ESC lines have been well established in mice and humans. Only a few of reports were published on ESC isolations in animals, even less in horses. However, it is still important to isolate equine ESCs for animal biotechnology and therapeutic applications. In the present study, we tried to derive horse ESC lines from the ICM of day 7 horse blastocysts fertilized in vivo and maintain their pluripotencies in different conditions. In result, the primary horse ESCs were able to self-renew when they were cultured in KODMEM supplemented with 10% FBS, 0.1 mM Non-essential Amino Acids, 2 mM L-glutamine, 1% Insulin-Transferrin-Selenium, 100 μg/ml of streptomycin, 100 units/ml of penicillin and 0.1mM 2β-mercaptoethanol on γ-irradiated MEFs. The culture medium was changed everyday after first passage and the passage was performed by mechanical dissociation into cellular clumps through manual scraping with a needle every 6-8 days. After 15 passages, immunohistochemistry of the putative horse ESCs showed that some cells in the colonies were positive for Oct4, SSEA-1, GCTM-2, TRA-60 and TRA-81. These results indicated that those cell colonies were not completely purified embryonic stem cells, but some of them were still pluripotent. Moreover, to optimize the culture conditions, these putative horse ESCs were cultured in basic medium supplemented with leukemia inhibitory factor (hLIF) only, basic fibroblastic growth factor (bFGF) only, or bFGF plus hLIF with or without MEF feeder cells. Based on our results, the feeder cells are necessary to maintain the undifferentiated state for horse ESCs; ESC-like cell morphology of horse putative ESCs were well maintained in the basic medium supplemented with or without hLIF. This result suggested that hLIF was neither prerequisite nor negative for maintenance of horse ESCs; bFGF seemed to be negative for maintenance of horse ECSs. The combination of LIF and bFGF was unable to improve the culture condition. Thus, the new factors need to be investigated further to maintain horse ESCs in purified population.

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