Abstract
Embryonic stem cells (ES cells) can be maintained its undifferentiated state with feeder cells or LIF, which can activate Jak/Stat3 pathway. Recently, it has been reported a new culture condition comprising serum-free medium with ERK and GSK3β inhibitors (2i) could drive ES cells into a state of pluripotency more like inner cell mass (ICM) in mouse blastocysts called ground state. However, although 2i could sustain ES cells self-renewal, LIF is routinely added. The roles of Stat3 activation are still unclear now. Here we investigated whether Jak/Stat3 might also contribute to the induction of ground state pluripotency. We introduced a lentiviral construct with 7-repeat Stat3-binding sequence to drive Renilla luciferase into ES cells, which can be used as a reporter to detect Stat3 activation by noninvasive bioluminescence imaging. Using this ES cells, we investigated the role of Stat3 activation in ground state maintenance. The results showed that Stat3 could be activated by 2i. Stattic, a chemical inhibitor of Stat3 phosphorylation, could effectively inhibit Stat3 activation in ES cells. When Stat3 activation was suppressed, ground state related genes were down regulated, and ES cells could not be maintained the ground state pluripotency even in 2i medium. All of these results indicate Stat3 activation is required in ground state maintenance.
Highlights
Mouse embryonic stem cells (ES cells) are derived from inner cell mass (ICM) of blastocyst embryos, which possess the ability of self-renewal and differentiation into any cell type of the three-germ layers [1]
To evaluate the role of signal transducer and activator of transcription 3 (Stat3) activation in regulating ES cells pluripotency, we used a phoshphor-Stat3 (p-Stat3) reporter gene to monitor the activation of Stat3
The reporter comprised a 7-repeat of Stat3-recognition sites and a small TA promoter, the design of tandem repetition could improve the expression of reporter gene significantly and Renilla luciferase could be used for quantitative bioluminescence imaging technique in vitro
Summary
Mouse embryonic stem cells (ES cells) are derived from inner cell mass (ICM) of blastocyst embryos, which possess the ability of self-renewal and differentiation into any cell type of the three-germ layers [1]. The optimal medium contents for the activation of signaling pathways could ES cells maintain the pluripotency in vitro. The role of leukemia inhibitory factor (LIF) in maintaining the pluripotency and self-renewal of mouse ES cells has been reported [5], and the biochemical function of LIF demonstrated a requirement for activation of the transcriptional factor signal transducer and activator of transcription 3 (Stat3) [6]. LIF binds to the cell surface LIF receptor (LIFR), causing the heterodimer formation of LIFR and the signal transducer glycoprotein 130 (gp130), which activates gp130-associated kinase (Jak) and phosphorylates the tyrosine residues in the gp130 cytoplasmic domain. The Stat proteins form dimmers enter the cell nucleus to bind response elements and regulate the expression of the target genes [7, 8]
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