Abstract
BackgroundThe transcription factor STAT3 is a downstream target of the LIF signalling cascade. LIF signalling or activation is sufficient to maintain embryonic stem (ES) cells in an undifferentiated and pluripotent state. To further investigate the importance of STAT3 in the establishment of ES cells we have in a first step derived stable pluripotent embryonic stem cells from transgenic FVB mice expressing a conditional tamoxifen dependent STAT3-MER fusion protein. In a second step, STAT3-MER overexpressing cells were used to identify STAT3 pathway-related genes by expression profiling in order to identify new key-players involved in maintenance of pluripotency in ES cells.ResultsTransgenic STAT3-MER blastocysts yielded pluripotent germline-competent ES cells at a high frequency in the absence of LIF when established in tamoxifen-containing medium. Expression profiling of tamoxifen-induced transgenic FVB ES cell lines revealed a set of 26 genes that were markedly up- or down-regulated when compared with wild type cells. The expression of four of the up-regulated genes (Hexokinase II, Lefty2, Pramel7, PP1rs15B) was shown to be restricted to the inner cell mass (ICM) of the blastocysts. These differentially expressed genes represent potential candidates for the maintenance of pluripotency of ES cells. We finally overexpressed two candidate genes, Pem/Rhox5 and Pramel7, in ES cells and demonstrated that their overexpression is sufficient for the maintenance of expression of ES cell markers as well as of the typical morphology of pluripotent ES cells in absence of LIF.ConclusionOverexpression of STAT3-MER in the inner cell mass of blastocyst facilitates the establishment of ES cells and induces the upregulation of potential candidate genes involved in the maintenance of pluripotency. Two of them, Pem/Rhox5 and Pramel7, when overexpressed in ES cells are able to maintain the embryonic stem cells in a pluripotent state in a LIF independent manner as STAT3 or Nanog.
Highlights
The transcription factor signal transducer and activator of transcription 3 (STAT3) is a downstream target of the leukaemia inhibitory factor (LIF) signalling cascade
In summary the data presented here indicates that the overexpression of functional STAT3-MER in FVB/N blastocysts sustains and facilitates the establishment of germline competent embryonic stem (ES) cells in absence of LIF
Our findings open up the possibility of establishing germline competent ES cells from non-permissive mouse strains by manipulation of the STAT3 signal transduction pathway
Summary
LIF signalling or activation is sufficient to maintain embryonic stem (ES) cells in an undifferentiated and pluripotent state. Murine ES cells are maintained in a pluripotent state by co-culturing with mitotically-inactivated feeder cells, such as embryonic fibroblasts, and/or the addition of leukaemia inhibitory factor (LIF: [4,5]). These ES cells can be maintained indefinitely in the presence of LIF, and express markers of the undifferentiated and pluripotent state, including the POU-domain transcription factor OCT-3/4 (POU5F1), a factor that is essential for the development of the ICM (reviewed by [6]; [7]). The shared usage of signal transducers (i.e. gp130) in the multichain cytokine receptor complexes clearly explains the functional redundancies of these cytokines (reviewed by [9])
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