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Brief introduction to the predominant signaling pathways in human embryonic stem cell (hESC) pluripotency: TGF-β, FGF and Wnt. Pluripotency at the molecular level. At the molecular level, the predominant signaling pathways involved in human ESC pluripotency and self-renewal, are TGF-β, which signals through SMAD2 and 3 and FGFR, which activates the MAPK and Akt pathways. The Wnt pathway also promotes pluripotency through activation of β-catenin. Signaling through these pathways results in the expression and activation of three key transcription factors Oct-4, Sox2 and Nanog. These transcription factors activate gene expression of ESC-specific genes, regulate their own expression and also serve as hESC markers. Other markers use to identify human ESCs are SSEA3 and 4 and TRA-1-60 and TRA-1-81. Pluripotency and self renewal are inhibited by the BNP4 pathway which uses SMAD1, 5 and 8 to block transcription factor expression. The pathway shown is for human cells, pluripotency in mice involved slightly different upstream signalling primarily the LIF leukemia inhibitory factor and STAT3 pathway, but results in expression of the same key transcription factors. Confocal immunofluorescent analysis showing expression and nuclear localization of the transcription factors Oct-4, Sox2 and Nanog in NTERA2 cells. Oct-4, Sox2 and Nanog antibodies have been labeled green. Actin filaments have been labeled red with Dy-554 Phalloidin. NTERA2 to is a human embryonic carcinoma cell line that expresses the same markers as ESCs. Note the presence of green signal in these cells. Hela cells are a non-pluripotent epithelial cell line used as a negative control and do not express these stem cell markers.