Optimization of Buffalo (Bubalus Bubalis) Embryonic Stem Cell Culture System

Objective: in order to retain an undifferentiated pluripotent state, embryonic stem (es)cells have to be cultured on feeder cell layers. however, use of feeder layers limits stemcell research, since experimental data may result from a combined es cell and feeder cellresponse to various stimuli.materials and methods: in this experimental study, a buffalo es cell line was establishedfrom in vitro derived blastocysts and characterized by the alkaline phosphatase (ap) andimmunoflourescence staining of various pluripotency markers. we examined the effect ofvarious factors like fibroblast growth factor 2 (fgf-2), leukemia inhibitory factor (lif) andy-27632 to support the growth and maintenance of bubaline es cells on gelatin coateddishes, in order to establish feeder free culture systems. we also analyzed the effect offeeder-conditioned media on stem cell growth in gelatin based cultures both in the presenceas well as in the absence of the growth factors.results: the results showed that y-27632, in the presence of fgf-2 and lif, resultedin higher colony growth and increased expression of nanog gene. feeder-conditionedmedium resulted in a significant increase in growth of buffalo es cells on gelatin coatedplates, however, feeder layer based cultures produced better results than gelatin basedcultures. feeder layers from buffalo fetal fibroblast cells can support buffalo es cells formore than two years.conclusion: we developed a feeder free culture system that can maintain buffalo escells in the short term, as well as feeder layer based culture that can support the longterm maintenance of buffalo es cells.