Screening Of Natural Compounds For Promoting Oct4 Expression And Maintaining Stem Cell Pluripotency

Stem cell is one of the hotspots in the field of biology and medicine research.With the deepening of stem cell research,application of stem cells in tissue engineering and organ reconstruction has gradually become possible.However,the number of stem cells in the body is extremely rare and very highly undirected.Therefore,how to maintain the pluripotency of stem cells in vitro is an urgent problem to be solved.Oct4 is one of the marker of stem cell pluripotency and plays an important role in keeping pluripotency and self-renewal of stem cells.Oct4 also plays an irreplaceable role in the process of inducing somatic cells to differentiate into pluripotent stem cells.In this paper,Oct4 promoter and Oct4 m RNA 3’UTR are used as targets to screen the natural small molecule compounds that can promote Oct4 expression,and its role in the maintenance of stem cell pluripotency and the underlying mechanisms were studied preliminaryly.1.Screening and Identification of Oct4 Expression PromotersFirstly,the Oct4 promoter-driven luciferase reporter vector or the Oct4 m RNA3’UTR luciferase reporter vector were transfected into cells,and then the cells were treated with a variety of natural small molecule compounds respectively to screen the activator of Oct4 expression by detecting the luciferase activities.The results showed that compounds T2-78,T2-79,T3-76,T3-77 can enhance the activity of Oct4 promoter,while T3-21,T3-76,T3-77,and T5-46 can increase the stability of Oct4 m RNA.Then,RT-q PCR and Western blot experiments were conducted and the results show that these 6 small molecules can promote the expression of Oct4 in both m RNA and protein levels.2.The role of Oct4 expression enhancer in maintaining the pluripotency of stem cells.To determine whether these six small molecule compounds could stimulate stem cell characteristics,we examined the effects of six small molecular compounds on the pluripotency and self-renewal of P19 cells.Western blot showed that only T2-78,T2-79,T3-77 could increased the expression of Oct4,but also increased the expression of Sox2 and Nanog.Soft agar assay results showed that T2-78,T2-79,T3-77 can promote the formation of P19 cell clones,and the clones can expressed alkaline phosphatase highly.These results further confirmed that T2-78,T2-79,T3-77 have the potential to maintain stem cell properties.Since stem cells can form teratomas,and differentiate to the three germ layers.Therefore,the P19 cells induced by T2-78,T2-79 or T3-77 were injected subcutaneously into the back of nude mice.Teratoma formation test results showed that T2-78,T2-79,T3-77 can promote the expression of endodermal(AFP),mesoderm(c Tn T),and ectodermal(β3-tublin)markers in teratomas when compare with the control group.These results indicated that T2-78,T2-79 and T3-77 can promote the pluripotency of P19 cells.3.The mechanisms of compounds up-regulation of Oct4 expressionTo demonstrate how these three small molecular compounds maintain cell pluripotency,we detected the effects of three compounds on the three common signal pathways that can sustain the pluripotency of murine stem cells in previous reports.The results showed that T2-78 can promote the phosphorylation of Jak1 and Stat3,activate the Jak/Stat3 signaling pathway.,while treatment the cells with the Jak/Stat3 signalling inhibitor Ruxolitinib inhibited the expression of the pluripotency factor Oct4.It is suggested that compound T2-78 can regulate the expression of Oct4 by activating the Jak1/Stat3 signaling pathway,thereby maintaining stem cell self-renewal and pluripotency.T2-79 and T3-77 can increase the level of phosphorylated GSK3β in the cytoplasm and promote the nuclear translocation ofβ-catenin,while the expression of Oct4 was decreases when the signaling pathway is blocked by Salinomycin which is a Wnt signaling pathway inhibitor.It is suggested that these two compounds maintain the self-renewal and pluripotency of stem cells by activating the canonical Wnt signaling pathway.At the same time,T2-79 and T3-77 can also promote the phosphorylation of PI3 K and AKT.When the signal is inhibited by PI3K/Akt signaling pathway inhibitor LY294002,the expression of intracellular Oct4 is decreased.It is suggested that T2-79 and T3-77 can also promote the expression of Oct4 by activating the PI3K/Akt signaling pathway,thereby maintaining the pluripotency of the cells.In summary,this article uses Oct4 as a target to screen out three natural small molecule compounds that can promote Oct4 expression and maintain stem cell self-renewal and pluripotency,and further clarified the mechanisms involved in these process.This study provided small molecule medium supplement that can maintain the cell pluripotency in vitro amplification of stem cells,and also provided candidate inducers for the preparation of induced pluripotent stem cells.