Embryonic stem cells (ESCs) are originally derived from the ICM of blastocysts and are characterized by their ability to self-renew and their pluripotencies. Only a few reports have been published on ESC isolations and line establishment in animals, even fewer 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 blastocysts fertilized in vivo and maintain their pluripotencies in different conditions. The primary horse ESCs were able to self-renew when they were cultured in basic medium on γ-irradiated MEFs. After 15 passages, immunohistochemistry of the putative horse ESCs showed that some cells in the colonies were positive for Oct-4, SSEA-1, GCTM-2, TRA-1-60 and TRA-1-81. Moreover, to optimize the culture conditions, these putative horse ESCs were cultured in basic medium supplemented with human leukemia inhibitory factor (hLIF) only, human basic fibroblastic growth factor (hbFGF) only, or hbFGF plus hLIF with or without heterologous (MEF) feeder cells. Based on our results, the heterologous feeder (MEF) cells are necessary to maintain the undifferentiated state for horse ESCs, and ESC-like cell morphology of horse 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 and the combination of hLIF and bFGF was unable to improve the culture condition.

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