Expression profiling in transgenic FVB/N embryonic stem cells overexpressing STAT3

BACKGROUND: The 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. RESULTS: Transgenic 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. CONCLUSION: Overexpression 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

Expression profiling in transgenic FVB/N embryonic stem cells overexpressing STAT3-3

Formed after cultivation of the cells for 8 days without adding LIF to the medium. E14 ES cells differentiated under these conditions and lost the expression of OCT-3/4 and SSEA-1 and alkaline phosphatase. Both Pem/Rhox5 and Pramel7 overexpressing cells maintained the expression of the pluripotency markers. Nanog overexpressing cells, used as a control for the experiment, as expected maintained their pluripotency. Scale bar in both large and small panels: 100 μm.<p><b>Copyright information:</b></p><p>Taken from "Expression profiling in transgenic FVB/N embryonic stem cells overexpressing STAT3"</p><p>http://www.biomedcentral.com/1471-213X/8/57</p><p>BMC Developmental Biology 2008;8():57-57.</p><p>Published online 23 May 2008</p><p>PMCID:PMC2409313.</p><p></p

Expression profiling in transgenic FVB/N embryonic stem cells overexpressing STAT3-1

-1 (A) and alkaline phosphatase (B). The expression of all markers was restricted to ES cells. Mouse fibroblast used as feeder cells are negative for OCT-3/4, SSEA-1 and alkaline phosphatase. Scale bar in both large and small panels: 100 μm.<p><b>Copyright information:</b></p><p>Taken from "Expression profiling in transgenic FVB/N embryonic stem cells overexpressing STAT3"</p><p>http://www.biomedcentral.com/1471-213X/8/57</p><p>BMC Developmental Biology 2008;8():57-57.</p><p>Published online 23 May 2008</p><p>PMCID:PMC2409313.</p><p></p

Expression profiling in transgenic FVB/N embryonic stem cells overexpressing STAT3-0

TAT3. After 24 hours of LIF and OHT deprivation cells were cultivated till their homogenization in presence of either LIF or OHT. During LIF or OHT deprivation no changes in the protein expression levels could be detected, but after 24 hrs the Tyr705 residue of both transgenic and WT STAT3 was completely dephosphorylated. 10 minutes after addition of LIF the tyr705 residue of both STAT3 was phosphorylated whereas after addition of OHT complete phosphorylation was obtained only after 6 hrs. Dephosphorylation of Tyr705 was analyzed by eliminating LIF or OHT from respectively WT or 743 cells. Kinetics for the dephosphorylation were slower then for the phosphorylation, only after 48 hrs dephosphorylation of WT Tyr705 was complete whereas complete dephosphorylation of STAT3-MER occurred only after 72 hrs. Nanog and STAT3 expression levels were tested by RTQ-PCR. Values are normalized with β-actin and the values indicate fold changes compared to the WT. Both WT and 743 ES cell lines expressed Nanog and the transgenic 743 ES cells have an increased Nanog expression compared to WT. After injection of 743 in C57BL/6 host blastocysts a 50–60% chimera was generated. Littermates from the crossing of the chimera with a WT FVB/N female generated white littermates, 50% of which were hemizygous for the transgene, indicating germline competence of the 743 cell line.<p><b>Copyright information:</b></p><p>Taken from "Expression profiling in transgenic FVB/N embryonic stem cells overexpressing STAT3"</p><p>http://www.biomedcentral.com/1471-213X/8/57</p><p>BMC Developmental Biology 2008;8():57-57.</p><p>Published online 23 May 2008</p><p>PMCID:PMC2409313.</p><p></p

Expression profiling in transgenic FVB/N embryonic stem cells overexpressing STAT3-2

the morula and of the blastocyst. Expression of Hexokinase II, Lefty2 and PP1s15B was restricted to the cells of the inner part of the morula and to the ICM in the blastocyst. Sense probes in the same concentration of the antisense probes were used as negative controls for the hybridization. In situ hybridization with a Pramel7 antisense riboprobe of preimplantation embryos (a: two-cell embryo, b: four-cell embryo, c: eight-cell embryo; d, e: compacted morula, f, g: blastocyst, h: negative control with sense riboprobe). Magnification 40×.<p><b>Copyright information:</b></p><p>Taken from "Expression profiling in transgenic FVB/N embryonic stem cells overexpressing STAT3"</p><p>http://www.biomedcentral.com/1471-213X/8/57</p><p>BMC Developmental Biology 2008;8():57-57.</p><p>Published online 23 May 2008</p><p>PMCID:PMC2409313.</p><p></p