Raz R, Lee C K, Cannizzaro L A, d'Eustachio P, Levy D E
Department of Pathology, New York University School of Medicine, New York, NY 10016, USA.
Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2846-51. doi: 10.1073/pnas.96.6.2846.
Propagation of mouse embryonic stem (ES) cells in vitro requires exogenous leukemia inhibitory factor (LIF) or related cytokines. Potential downstream effectors of the LIF signal in ES cells include kinases of the Src, Jak, and mitogen-activated protein families and the signal transducer and transcriptional activator STAT3. Activation of nuclear STAT3 and the ability of ES cells to grow as undifferentiated clones were monitored during LIF withdrawal. A correlation was found between levels of STAT3 activity and maintenance of an undifferentiated phenotype at clonal density. In contrast, variation in STAT3 activity did not affect cell proliferation. The requirement for STAT3 was analyzed by targeted mutagenesis in ES cell lines exhibiting different degrees of LIF dependency. An insertional mutation was devised that abrogated Stat3 gene expression but could be reversed by Cre recombination-mediated excision. ES cells heterozygous for the Stat3 mutation could be isolated only from E14 cells, the line least dependent on LIF for self-renewal. Targeted clones isolated from other ES cell lines were invariably trisomic for chromosome 11, which carries the Stat3 locus, and retained normal levels of activated STAT3. Cre-regulated reduction of Stat3 gene copy number in targeted, euploid E14 clones resulted in dose-dependent losses of STAT3 activity and the efficiency of self-renewal without commensurate changes in cell cycle progression. These results demonstrate an essential role for a critical amount of STAT3 in the maintenance of an undifferentiated ES cell phenotype.
小鼠胚胎干细胞（ES细胞）在体外的增殖需要外源性白血病抑制因子（LIF）或相关细胞因子。ES细胞中LIF信号的潜在下游效应器包括Src、Jak和丝裂原活化蛋白家族的激酶以及信号转导子和转录激活子STAT3。在撤除LIF期间，监测了核STAT3的激活以及ES细胞作为未分化克隆生长的能力。发现在克隆密度下，STAT3活性水平与未分化表型的维持之间存在相关性。相反，STAT3活性的变化不影响细胞增殖。通过对表现出不同程度LIF依赖性的ES细胞系进行定向诱变，分析了对STAT3的需求。设计了一种插入突变，该突变消除了Stat3基因表达，但可通过Cre重组介导的切除作用使其逆转。Stat3突变杂合的ES细胞只能从E14细胞中分离出来，E14细胞系是对自我更新最不依赖LIF的细胞系。从其他ES细胞系分离的定向克隆对于携带Stat3基因座的11号染色体总是三体的，并保留了正常水平的活化STAT3。在定向的整倍体E14克隆中，Cre调节的Stat3基因拷贝数减少导致STAT3活性和自我更新效率呈剂量依赖性丧失，而细胞周期进程没有相应变化。这些结果表明，适量的STAT3在维持未分化ES细胞表型中起着至关重要的作用。
