Cinelli Paolo, Casanova Elisa A, Uhlig Syndi, Lochmatter Priska, Matsuda Takahiko, Yokota Takashi, Rülicke Thomas, Ledermann Birgit, Bürki Kurt
Institute of Laboratory Animal Science, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
BMC Dev Biol. 2008 May 23;8:57. doi: 10.1186/1471-213X-8-57.
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.
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.
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.
转录因子信号转导与转录激活因子3（STAT3）是白血病抑制因子（LIF）信号级联反应的下游靶点。LIF信号传导或激活足以使胚胎干细胞维持未分化和多能状态。为了进一步研究STAT3在胚胎干细胞建立过程中的重要性，我们首先从表达条件性他莫昔芬依赖性STAT3-MER融合蛋白的转基因FVB小鼠中获得了稳定的多能胚胎干细胞。第二步，利用过表达STAT3-MER的细胞通过表达谱分析来鉴定与STAT3途径相关的基因，以确定参与维持胚胎干细胞多能性的新关键因子。
当在含他莫昔芬的培养基中培养时，转基因STAT3-MER囊胚在无LIF的情况下能高频产生具有生殖系能力的多能胚胎干细胞。对他莫昔芬诱导的转基因FVB胚胎干细胞系进行表达谱分析，发现一组26个基因与野生型细胞相比有明显的上调或下调。上调基因中的四个（己糖激酶II、Lefty2、Pramel7、PP1rs15B）的表达显示仅限于囊胚的内细胞团（ICM）。这些差异表达的基因是维持胚胎干细胞多能性的潜在候选因子。我们最终在胚胎干细胞中过表达了两个候选基因，Pem/Rhox5和Pramel7，并证明它们的过表达足以在无LIF的情况下维持胚胎干细胞标志物的表达以及多能胚胎干细胞的典型形态。
在囊胚内细胞团中过表达STAT3-MER有助于胚胎干细胞的建立，并诱导参与维持多能性的潜在候选基因上调。其中两个基因，Pem/Rhox5和Pramel7，在胚胎干细胞中过表达时能够像STAT3或Nanog一样以不依赖LIF的方式维持胚胎干细胞处于多能状态。
