The Regulation Of Mouse Embryonic Stem Cells Differentiated To Hematopoietic Stem/Progenitor Cells Mediated By Wnt Signaling Pathway Activation

Objects:In this work, we aimed to obtain a stable and efficient feeding layer cell to culture mouse embryonic stem cells, identify the characteristics of E14TG2a mouse embryonic stem cells.The regulation of cell differentiation mediated by Wnt/β-catenin signal pathway activation from mouse embryonic stem cells to hematopoietic stem/progenitor cells was also investigated.Methods:The feeder layer cells were prepared with mouse embryonic fibroblast. Mitomycin C concentrations in culture medium were determined by MTT to establish a highly efficient and stable mouse embryonic stem cell feeding layer cultivation system. The AP staining, cell cycle detection and karyotype analysis were performed to identify the characteristics of the E14TG2a mouse embryonic stem cells. The β-catenin level in E14TG2a cells was tested by cell immunofluorescence and western blot after treated with exogenous protein wnt3a (100ng/ml) for 21 days, and the expression of downstream target gene of Wnt signal pathway was detected by qRT-PCR to investigate whether the pathway was activated. To explore the effect of Wnt/β-catenin signaling pathway activation, the hematopoietic stem cell surface markers CD34+/Sca-1+ were detected by flow cytometry (FCM), and the expression of hematopoietic associated genes were measured by qRT-PCR.Results:1. We successfully establish a stable and efficient feeding layer cell to culture mouse embryonic stem cells.2. The E14TG2a mouse stem cells maintain undifferentiated state and high cell proliferation activity; its chromosomes did not show abnormity including deletions, amplification or shift.3. The β-catenin level in E14TG2a mouse stem cells is up-regulated after treated with wnt3a (100ng/ml) for 21 days. The expression of Wnt signal pathway’s downstream target genes such as Pitx2, Frizzled, Sox17 and Oct4 showed significant increase.4. After activating the Wnt/β-catenin signal pathway we induce E14TG2a mouse stem cells to differentiate to hematopoietic stem cells. The number of CD34+/Sca-1+ cells is significantly higher than that of the control group. The expression of hematopoietic associated genes BMP4, FLK2 and CD34 increases while the hematopoietic differentiation inhibitor gene SmadS is suppressed.Conclusion:Our data suggest that the activation of Wnt/β-catenin signal pathway could promote the directional differentiation of E14TG2a mouse stem cells to hematopoietic progenitor cells by treated with exogenous wnt3a (100ng/ml) for 21 days.