Vallier Ludovic, Reynolds Daniel, Pedersen Roger A
Department of Surgery, University of Cambridge, Cambridge CB2 2QQ, United Kingdom.
Dev Biol. 2004 Nov 15;275(2):403-21. doi: 10.1016/j.ydbio.2004.08.031.
Genetic studies in fish, amphibia, and mice have shown that deficiency of Nodal signaling blocks differentiation into mesoderm and endoderm. Thus, Nodal is considered as a major inducer of mesendoderm during gastrulation. On this basis, Nodal is a candidate for controlling differentiation of pluripotent human embryonic stem cells (hESCs) into tissue lineages with potential clinical value. We have investigated the effect of Nodal, both as a recombinant protein and as a constitutively expressed transgene, on differentiation of hESCs. When control hESCs were grown in chemically defined medium, their expression of markers of pluripotency progressively decreased, while expression of neuroectoderm markers was strongly upregulated, thus revealing a neuroectodermal default mechanism for differentiation in this system. hESCs cultured in recombinant Nodal, by contrast, showed prolonged expression of pluripotency marker genes and reduced induction of neuroectoderm markers. These Nodal effects were accentuated in hESCs expressing a Nodal transgene, with striking morphogenetic consequences. Nodal-expressing hESCs developing as embryoid bodies contained an outer layer of visceral endoderm-like cells surrounding an inner layer of epiblast-like cells, each layer having distinct gene expression patterns. Markers of neuroectoderm were not upregulated during development of Nodal-expressing embryoid bodies, nor was there induction of markers for definitive mesoderm or endoderm differentiation. Moreover, the inner layer expressed markers of pluripotency, characteristic of undifferentiated hESCs and of epiblast in mouse embryos. These results could be accounted for by an inhibitory effect of Nodal-induced visceral endoderm on pluripotent cell differentiation into mesoderm and endoderm, with a concomitant inhibition of neuroectoderm differentiation by Nodal itself. There could also be a direct effect of Nodal in the maintenance of pluripotency. In summary, analysis of the Nodal-expressing phenotype suggests a function for the transforming growth factor-beta (TGF-beta) growth factor superfamily in pluripotency and in early cell fate decisions leading to primary tissue layers during in vitro development of pluripotent human stem cells. The effects of Nodal on early differentiation illustrate how hESCs can augment mouse embryos as a model for analyzing mechanisms of early mammalian development.
对鱼类、两栖动物和小鼠的遗传学研究表明，Nodal信号通路的缺陷会阻碍中胚层和内胚层的分化。因此，Nodal被认为是原肠胚形成过程中中内胚层的主要诱导因子。基于此，Nodal是控制多能性人类胚胎干细胞（hESC）分化为具有潜在临床价值的组织谱系的一个候选因子。我们研究了Nodal作为重组蛋白和组成型表达转基因对hESC分化的影响。当对照hESC在化学成分确定的培养基中培养时，其多能性标志物的表达逐渐降低，而神经外胚层标志物的表达则强烈上调，从而揭示了该系统中神经外胚层默认的分化机制。相比之下，在重组Nodal中培养的hESC显示多能性标志物基因的表达延长，神经外胚层标志物的诱导减少。在表达Nodal转基因的hESC中，这些Nodal效应更加明显，具有显著的形态发生后果。作为胚状体发育的表达Nodal的hESC包含一层围绕着内层类上胚层细胞的内脏内胚层样细胞外层，每层细胞具有不同的基因表达模式。在表达Nodal的胚状体发育过程中，神经外胚层标志物没有上调，也没有诱导确定中胚层或内胚层分化的标志物。此外，内层表达多能性标志物，这是未分化hESC和小鼠胚胎上胚层的特征。这些结果可以通过Nodal诱导的内脏内胚层对多能细胞分化为中胚层和内胚层的抑制作用来解释，同时Nodal本身也抑制神经外胚层分化。Nodal在维持多能性方面也可能有直接作用。总之，对表达Nodal表型的分析表明，转化生长因子-β（TGF-β）生长因子超家族在多能性以及多能人类干细胞体外发育过程中导致初级组织层形成的早期细胞命运决定中发挥作用。Nodal对早期分化的影响说明了hESC如何作为分析早期哺乳动物发育机制的模型来补充小鼠胚胎。
