Effect Of Wnt3a On Pluripotent Maintenance Of Mouse Embryonic Stem Cells And Reprogramming Effeciency Of Induced Pluripotent Stem Cells

Wnt signaling pathway plays significant roles in embryonic differentiation and maintaining pluripotency of stem cells and so on. This signaling pathway initiating complicated series of intracellular signal mediation by extracellular proteins and cell surface receptor binding, consequently regulating the expressions of relative genes. However, the epigenetic modifications in maintaining and inducing the pluripotency of stem cells of Wnt signaling have not known yet.This study is aimed at using mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) as experimental objectives, utilizing the function of secreting Wnt3a proteins to extracellular space of L-Wnt3a cells, preparing L-Wnt3a conditioned medium or take L-Wnt3a cells as feeder layers to culture ESCs or induce iPSCs, in order to discuss the role of pluripotency maintenance of ESCs and inducing reprogramming iPSCs of Wnt signaling pathway. The results show that take L-Wnt3a cells as feeder layers of ESCs can maintain their pluripotent state well, this ESCs express alkaline phosphatase and other pluripotent relative genes and they can differentiate into embryoid bodies and teratomas. Then the expressions of epigenetic modification and Wnt3a signaling relative genes shows that the expression of histone H3K36 transmethylase kdm2b upregulated and H3K9me transmethylase downregulated. In addition, the expression of DNA hydroxymethylase tet1 downregulated, and there’s no significant difference in the expressions of chd1 and parp1. The expression of Wnt3a signaling regulating gene cd31 and Wnt signaling inhibiting gene dkkl were upregulated, the expression of stat3 downregulated and there’s no significant difference in birc5.Utilizing L-Wnt3a conditioned medium can maintain the clone form of ESCs in feeder-free conditions, and ESCs clones cultured in the common ESCs mediums with no Wnt3a factors differentiated quickly. The expression of pluripotent genes sox2, oct4, nanog, and epigenetic modification genes parp1, kdm2b of ESCs cultured in L-Wnt3a conditioned medium were all upregulated compared with ESCs cultured in ESCs medium. While there’s no significant difference of Wnt signaling pathyway between these two types of cells, compared with severe differentiated ESCs cultures in MEF medium, the expression levels of cd31 and birc5 were relatively high. While there’s no significant difference of Wnt signaling pathyway between these two types of cells, compared with severe differentiated ESCs cultures in MEF medium, the expression levels of cd31 and birc5 were relatively high. Moreover, ESCs cultured in L-Wnt3a conditioned medium can maintain the pluripotency well, and can differentiate into embryoid bodies and express higher marker genes of three germ layers in vitro, and can differentiate into teratomas in vivo.This study shows that, Wnt3a signaling pathway can promote the maintaining of pluripotent and upregulating the epigenetic modification genes of ESCs. Moreover, this signaling promote the inducing of mouse iPSCs significantly, and upregulating the expression of pluripotent related and epigenetic modification related genes. This study proved that, Wnt3a signaling pathway not only have signaling regulating function to pluripotent genes, but also have regulating functions in pluripotent related epigenetic gene expressions. This study lay a foundation of clarifying that how Wnt3a signaling pathway manipulating pluripotent, especially achieving pluripotent during inducing.