Analysis for mechanisms of self-renewal and differentiation signal transduction in human embryonic stem cells.

在LIF存在下，ES细胞的多能表型得以维持。LIF与由LIF受体β和gp 130组成的细胞表面受体复合物结合，通过该复合物激活包括MAP激酶和STAT 3在内的多种信号分子。我们报道了gp 130的胞内结构域在ES细胞的自我更新中起重要作用。在本研究中，我们研究了信号通路，通过它gp！30有助于ES细胞的自我更新。负责STAT 3激活的gp 130胞质结构域的突变分析是ES细胞自我更新所必需的，而SHP 2和MAP激酶激活所需的突变分析是不必要的。接下来，我们构建了由STAT 3的整个区域和雌激素受体的配体结合结构域组成的融合蛋白。该融合蛋白（STAT 3ER）在合成配体4-羟基他莫昔芬（4 HT）的存在下二聚化并活化。当稳定表达STAT 3ER的ES细胞在没有LIF和饲养细胞的4 HT存在下培养时，它们保持形态学未分化状态并表达未分化状态特异性标志物（SSEA-1和碱性磷酸酶）。此外，由STAT 3ER和4 HT维持的ES细胞在注射到囊胚中时有助于嵌合小鼠的产生。这些结果表明，STAT 3活化足以维持ES细胞的多能性。Oct-3/4转录因子在胚胎干细胞中表达，并具有胚胎干细胞特异性。已知它对于内细胞团的形成和维持ES细胞的未分化状态是必需的。LIF或STAT 3信号可能通过与Oct-3/4协同作用，通过尚未鉴定的因子抑制ES细胞的分化。

The pluripotent phenotype of ES cells is maintained in the presence of LIF. LIF binds to a cell surface receptor complex composed of LIF receptor β and gp130, through which several signaling molecules including MAP kinase and STAT3 are activated. We reported that the intracellular domain of gp130 plays an important role in self-renewal of ES cells. In the present study, we examined the signaling pathway through which gp!30 contributes to the self-renewal of ES cells. Mutational analysis of the cytoplasmic domain of gp130 responsible for STAT3 activation is necessary for self-renewal of ES cells, while that required for SHP2 and MAP kinase activation was dispensable. Next, we have constructed a fusion protein composed of the entire region of STAT3 and the ligand binding domain of estrogen receptor. This fusion protein (STAT3ER) was dimerized and activated in the presence of a synthetic ligand 4-hydroxytamoxifen (4HT). When ES cells stably expressing STAT3ER were cultured in the presence of 4HT without LIF and feeder cells, they maintained a morphologically undifferentiated state and expressed undifferentiated state-specific markers (SSEA-1 and alkaline phosphatase). Moreover, ES cells maintained by STAT3ER and 4HT contributed to chimeric mice production when they were injected into blastocysts. These results indicate that STAT3 activation is sufficient to maintain the pluripotency of ES cells. Oct-3/4 transcription factor is expressed in ES cells and ES cells specifically. It is known to be essential for the formation of inner cell mass and for the maintenance of undifferentiated state of ES cells. It is likely that LIF or STAT3 signals inhibit differentiation of ES cells through yet unidentified factor by cooperating with Oct-3/4.