Establishment of an exogenous LIF-free culture system for mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs) have played a key role in the newly emerging fields of stem cell research. The traditional derivation and culture of mESCs have been based on the use of mouse embryonic fibroblasts (MEFs) treated with exogenous leukemia inhibitory factor (LIF). However, the rapid senescence of MEFs, coupled with the high cost of LIF, has significantly hampered the widespread use of mESCs in stem cell research. Thus, we present a novel exogenous LIF-free culture system for general mESCs applications, comprising fibroblast-like cells derived from the rabbit spleen (RSFs). We demonstrated that mESCs cultured on RSFs (mESCs-RSFs) maintained all mESC features after prolonged LIF-free culture, including alkaline phosphatase, cell surface markers (SSEA-1), molecular markers (OCT-4, NANOG, TERT, REX-1), karyotype, and pluripotency. The high expression level of both LIF and WNT3A in the RSFs may account for their ability to maintain mESCs without exogenous LIF. Moreover, this exogenous LIF-free culture system was verified to be of microbiological quality through analysis with electron transmission microscopy.