Embryonic stem cells (ESCs) are originally derived from the ICM of blastocysts and characterized by their ability to self-renew and their pluripotencies. Although attempt to isolate equine ESCs has been done, proper growth factor(s) for their maintenance have not been still established. Different growth factors or even the same factors play different roles in ESC culture in different species. Our aim was to determine the influence of hLIF and bFGF on the horse embryonic stem cell culture. The putative eESC cell lines were derived by repeated passage of ICM cells recovered mechanically from day-7 blastocysts. The growth capabilities of the eESCs were evaluated when the putative eESCs were cultured in the basic medium supplemented with human leukemia inhibitory factor (hLIF) only, basic fibroblastic growth factor (bFGF) only, or bFGF plus hLIF with or without MEF feeder cells. 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 every day 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. Based on our results, the feeder cells are necessary to maintain the undifferentiated state for horse ESCs. Moreover, ESC-like cell morphology of putative eESCs could be maintained in the basic medium supplemented with or without hLIF. The results suggest that hLIF is not prerequisite for maintenance of eESCs; bFGF seem to be negative for maintenance of horse ECSs. The combination of LIF and bFGF is unable to support eESCs. Thus, the new factors need to be investigated further to maintain eESCs in purified population, and horse LIF-receptor need to be identified that whether they are compatible with human LIF to find out right signal transduction pathways in eESCs.

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