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The Role of Pou5f1 Gene on Differentiation into Germ Layers

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Pluripotent stem cells (PSCs) can be differentiated into all three germ layers in vitro and in vivo. Embryonic stem cells (ESCs) can be derived from the inner cell mass of blastocyst in vitro, and the epiblast derived pluripotent stem cells (EpiSCs) can be established from epiblast of post-implantation embryos. The pluripotent ESCs and EpiSCs express Oct4 (Pou5f1) gene. Oct4 gene is essential for maintenance of pluripotent state and levels of Oct4 regulate differentiation of pluripotent stem cells. The Oct4 gene contains two cis-regulatory elements, the distal enhancer (DE) and proximal enhancer (PE), which differentially control Oct4 expression in a cell-type-specific and stage-specific manner. We generated double transgenic mice carrying both Oct4-Δ PE-GFP and Oct4-ΔDE-tdTomato (RFP), enabling us to simultaneously monitor the activity of DE and PE. Oct4 expression is stage-specifically regulated by DE and PE during embryonic and germ cell development. We could classify pure populations of naive (GFP+RFP−) and primed (GFP−RFP+) PSCs by using double transgenic systems. Next, we established ZHBTc4 ESCs (Oct4 tetracycline conditional knockout ESCs) - derived epiblast stem cells like cells (ZHBTc4-EpiLCs) to determine function of Oct4 in differentiation into germ layers. ZHBTc4-EpiLCs could not be formed colonies in doxycycline (dox) contained EpiSC medium, indicated that Oct4 gene is essential for maintenance of primed PSCs. ZHBTc4-EpiLCs were differentiated into neuroectodermal or mesoendodermal cells in differentiation medium with or without dox. The ZHBTc4-EpiLCs could be efficiently differentiated into neuroectodermal lineage in neuroectodermal differentiation medium with dox, whereas mesoendodermal related genes were down-regulated in mesoendodermal differentiation medium with dox. This results showed that Oct4 gene regulates maintenance and differentiation potential in primed pluripotent stem cells.

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  • Hyun Woo CHoi

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