Pluripotency of embryonic stem cells (ESCs) are maintained under specific culture conditions. The requirements for proliferation and undifferentiation of ESCs vary among different species. The feeder cells are considered as the important factor for providing both a suitable attachment substrate and critical soluble factors. Although equine ESCs were isolated and able to proliferate on mouse embryonic fibroblast (MEF) feeder cells, maintenance of undifferentiated state is still a problem. Because tissue engineering and regenerative medicine have been recently proposed for the treatment of musculoskeletal and ostheoarticular diseases in horses, the equine ESCs are of particular interest for the cell therapy. Due to the pluripotency of ESCs, equine ESCs are potentially able to repair tendon, ligament and bone damages. For clinical treatment, purified ESCs without contamination of mouse MFE cells are needed. In the present study, mouse embryonic fibroblast cells and horse embryo-derived cells were evaluated for equine ESC culture. Heterologous MEF and autologous horse embryo-derived cells were used as feeder cells for maintaining equine ESCs, respectively. We derived horse ESC from the ICM of day 7 horse blastocysts fertilized in vivo and maintain their pluripotencies in two different feeder conditions. In result, the primary horse ESCs were able to self-renew when they were cultured in a stem cell basic medium supplemented with hLIF on γ-irradiated MEFs for several passages. The putative horse ESCs showed that some cells in the colonies were positive for Oct4, SSEA-1, GCTM-2, TRA1-60 and TRA1-81. However, γ-irradiated autologous horse feeder cells could not support equine ESC to be in undifferentiated state, which is similar to embryonic fibroblast feeders for cow and sheep. This may suggest that the feeder cells from these species do not secrete the necessary factors to support self-renew. Furthermore, feeder free condition was assessed for maintaining culture of equine ESCs. However, we found MEF feeder layer is obligatory for the growth of horse ESCs. Based on our results, the new autologous feeder cell types and feeder free conditions are needed to investigate further to develop equine ESC culture and maintenance technology.

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