Dvorak Petr, Dvorakova Dana, Koskova Stanislava, Vodinska Martina, Najvirtova Miroslava, Krekac Daniel, Hampl Ales
Laboratory of Molecular Embryology, Mendel University, Brno Zemedelska 1, 613 00 Brno, Czech Republic.
Stem Cells. 2005 Sep;23(8):1200-11. doi: 10.1634/stemcells.2004-0303. Epub 2005 Jun 13.
Although the detection of several components of the fibroblast growth factor (FGF) signaling pathway in human embryonic stem cells (hESCs) has been reported, the functionality of that pathway and effects on cell fate decisions are yet to be established. In this study we characterized expression of FGF-2, the prototypic member of the FGF family, and its receptors (FGFRs) in undifferentiated and differentiating hESCs; subsequently, we analyzed the effects of FGF-2 on hESCs, acting as both exogenous and endogenous factors. We have determined that undifferentiated hESCs are abundant in several molecular-mass isoforms of FGF-2 and that expression pattern of these isoforms remains unchanged under conditions that induce hESC differentiation. Significantly, FGF-2 is released by hESCs into the medium, suggesting an autocrine activity. Expression of FGFRs in undifferentiated hESCs follows a specific pattern, with FGFR1 being the most abundant species and other receptors showing lower expression in the following order: FGFR1 --> FGFR3 --> FGFR4 --> FGFR2. Initiation of differentiation is accompanied by profound changes in FGFR expression, particularly the upregulation of FGFR1. When hESCs are exposed to exogenous FGF-2, extracellular signal-regulated kinases are phosphorylated and thereby activated. However, the presence or absence of exogenous FGF-2 does not significantly affect the proliferation of hESCs. Instead, increased concentration of exogenous FGF-2 leads to reduced outgrowth of hESC colonies with time in culture. Finally, the inhibitor of FGFRs, SU5402, was used to ascertain whether FGF-2 that is released by hESCs exerts its activities via autocrine pathways. Strikingly, the resultant inhibition of FGFR suppresses activation of downstream protein kinases and causes rapid cell differentiation, suggesting an involvement of autocrine FGF signals in the maintenance of proliferating hESCs in the undifferentiated state. In conclusion from our data, we propose that this endogenous FGF signaling pathway can be implicated in self-renewal or differentiation of hESCs.
尽管已有报道称在人类胚胎干细胞（hESCs）中检测到了成纤维细胞生长因子（FGF）信号通路的多个组分，但该通路的功能以及对细胞命运决定的影响尚未明确。在本研究中，我们对未分化和分化中的hESCs中FGF家族的原型成员FGF-2及其受体（FGFRs）的表达进行了表征；随后，我们分析了FGF-2作为外源性和内源性因子对hESCs的影响。我们确定未分化的hESCs中存在多种分子量异构体的FGF-2，并且在诱导hESC分化的条件下，这些异构体的表达模式保持不变。值得注意的是，hESCs将FGF-2释放到培养基中，表明存在自分泌活性。未分化hESCs中FGFRs的表达遵循特定模式，其中FGFR1最为丰富，其他受体表达较低，顺序如下：FGFR1 --> FGFR3 --> FGFR4 --> FGFR2。分化的启动伴随着FGFR表达的深刻变化，特别是FGFR1的上调。当hESCs暴露于外源性FGF-2时，细胞外信号调节激酶被磷酸化并因此被激活。然而，外源性FGF-2的存在与否对hESCs的增殖没有显著影响。相反，随着培养时间的延长，外源性FGF-2浓度的增加导致hESC集落的生长减少。最后，使用FGFRs抑制剂SU5402来确定hESCs释放的FGF-2是否通过自分泌途径发挥其活性。令人惊讶的是，由此产生的FGFR抑制作用抑制了下游蛋白激酶的激活并导致细胞快速分化，表明自分泌FGF信号参与维持未分化状态下增殖的hESCs。根据我们的数据得出结论，我们认为这种内源性FGF信号通路可能与hESCs的自我更新或分化有关。
