Cell Type Specific Effect Of Wnt Signaling Pathway On Reprogramming

Objective:1.Compare the reprogramming rate and efficiency between different somatic cell types,including mouse embryonic fibroblasts cells and hematopoietic stem/progenitor cells.2.Examine the impact of Wnt signaling pathway on different somatic cell types and their reprogramming process.3.Illustrate the function of Tcf/Lef1 family in different somatic cell types and analyze their DNA binding sites,thus to understand the molecular basis of cell type specific effect of Wnt pathway on reprogramming.Methods:1.We employ the secondary reprogramming system,in which the reprogramming factors has been inserted into the genome of donor cells and their expression is under the control of Doxycycline,thus the induced pluripotent stem cells could be derived without extra introducing of exogenous genes.We compared the reprogramming rate and efficiency starting with MEF and HSPC.2.Activate and inhibit Wnt pathway by Gsk3 inhibitor CHIR99021 and IWR1-endo respectively,to confirm the effect of Wnt signaling on reprogramming of different cell types by comparing the rate and efficiency.3.Examine the expression of ?-catenin and Tcf/Lef1 by real-time q PCR and Western Blot.Using Co-IP to test the interaction between ?-catenin and Tcf/Lef1.Explore the function of Tcf/Lef1 in MEF and HSPC through knockdown and overexpression.4.Analyze the DNA binding sites and downstream target of Tcf/Lef1 in MEF and HSPC by Ch IP-seq.Results:1.Activate Wnt pathway had a significant improvement on reprogramming efficiency in HSPC but not in MEF.2.The expression of ?-catenin was lower in HSPC compare to MEF,but increased greatly following the addition of CHIR99021.And the responses of Tcf/Lef1 to CHIR99021 were different between MEF and HSPC.3.Knockdown of Tcf3 inhibited the increase of differentiation-associated genes and maintained the pluripotent genes at a relative high level in ESC differentiation.Conclusion:1.Different cell types response to Wnt signaling specifically.2.The activation of Wnt increase the protein level of ?-catenin,then the different level of Tcf/Lef1 may be responsible for the different effect of Wnt signaling on reprogramming of MEF and HSPC.