Regulation Of Rat Embryonic Stem Cell Self-renewal By BFGF Via Phosphoinositide 3-kinase-dependent Signaling

Embryonic stem(ES)cells are isolated from the inner cell mass of a blastocyst.According to different extracellular signals,ES differentiate into cells of all three germ layers.Thus,ES cells have become a powerful tool in the field of regenerative medicine and human diseases modeling.So far,ES cells have been derived from several species(including mouse,rat,human,monkey and so on).However,only the ES cells from mouse and rat have been proved holding ability of contribution to chimera and germline transmission.Compared with the most commonly-used biological mouse,rats are more relevant to human and have been widely applied in experiments of physiological,behavioral science,pharmacology,toxicity.However,Rat embryonic stem cells can not be maintained in the commonly-used culture condition of mouse ES cells.So precise genetic modification in rat is hard to achieve and the application of rats is considerably limited.In recent years,the researchers have found that ES cells can be maintained in an undifferentiated state through regulation of a combination of signaling pathways.For example,mouse embryonic stem cells can be maintained self-renewal under the feeder–free culture conditions supplemented with leukemia inhibitory factor(LIF)and two small molecule inhibitors(PD0325901 and CHIR99021,2I).Moreover,the condition of LIF/2I can also maintain rat ES self-renewal with support of feeder cells.Feeder cells usually secrete LIF,basic Fibroblast Growth Factor(bFGF)and so on.LIF promote the self-renewal of mouse ES cells via activation of STAT3 signaling pathway.bFGF have proved to promote self-renewal of human embryonic stem cells.Human embryonic stem cells are considered in a primed state because human ES are more similar with cells derived from post-transplanted mouse epiblast.However,recent studies which trying to convert human ES cell to na?ve state show that bFGF signaling pathways also play an important role in regulation of na?ve state of human ES cells.Here,we try to culture rat embryonic stem cells in the serum and feeder free medium supplemented with LIF and 2i,and investigate the effect of b FGF on the self-renewal of rat embryonic stem cells and the underlying mechanism.The first part: bFGF regulate self-renewal of rat ES cellsRat ES cells were cultured in serum free medium supplemented with LIF,PD0325901,CHIR99021 and with or without bFGF.Small molecular chemical SU5402 and LY294002 were used to inhibit FGF receptor and PI3 K,respectively.AKP staining,ICC and RT-PCR were performed to identify the pluripotency of rat ES cells.Western Blot was used to analysis the phosphorylation level of AKT.The results show that,bFGF promoted self-renewal of rat ES cells under a feeder cell and serum free condition.Rat ES cells maintained with LIF 2I and bFGF expressed Oct-4 and Nanog and could differentiate to cells of ectoderm,mesoderm and endoderm.bFGF promoted the phosphorylation level of AKT and this effect could be inhibited by SU5402 or LY294002.Both SU5402 and LY294002 abolished the promoting effect of bFGF on the self-renewal of Rat ES cells.These results indicate that bFGF promote self-renewal of rat ES cells and this effect may mainly regulate through PI3K/AKT dependent signaling pathway.The second part: isolation and culture of rat ES cells under feeder free condition4.5 days of DA rat blastocyst were separated and then cultured in the feeder-free medium supplememted with LIF and 2i and with or without bFGF.The results showed that LIF+2i+bFGF can support the growing of cells from blastocyst and following passage culture.No evident cell colonies obtained in the medium without bFGF.These results showed that LIF+2i+bFGF can not only maintain self-renewal of ES cells lines,but also support primary culture of ES celllines from rat.And bFGF is indispensable for derivation of rat ES cells.Through our research,we draw the following conclusion: First,The culture condition of LIF+2i+bFGF can maintain rat embryonic stem cells self-renewal in the absence of feeder cells.Both of LIF and bFGF promote self-renewal of rat ES cells.Second,bFGF mainly mediate self-renewal of rat ES cells via PI3 K dependent signaling pathways.Inhibition of PI3 K signaling pathways which were activated by bFGF can cancel the positive influence of bFGF in rat ES cells self-renewal.