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.


CONCLUSIONS
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.
Cinelli
Paolo
P
Institute of Laboratory Animal Science, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. paolo.cinelli@ltk.uzh.ch
Casanova
Elisa A
EA
Uhlig
Syndi
S
Lochmatter
Priska
P
Matsuda
Takahiko
T
Yokota
Takashi
T
Rülicke
Thomas
T
Ledermann
Birgit
B
Bürki
Kurt
K
D000818:Animals; D053624:Blastocyst Inner Cell Mass / Q000166:cytology / Q000378:metabolism; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D020869:Gene Expression Profiling; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D051379:Mice; D008822:Mice, Transgenic; D000071317:Nanog Homeobox Protein; D020411:Oligonucleotide Array Sequence Analysis; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D000071317:Nanog Homeobox Protein; D011960:Receptors, Estrogen; D050796:STAT3 Transcription Factor; D014157:Transcription Factors; C475540:Nanog protein, mouse; C494087:Stat3 protein, mouse; C498048:Rhox5 protein, mouse