Takenaga Masanori, Fukumoto Miki, Hori Yuichi
Department of Gastroenterological Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
J Cell Sci. 2007 Jun 15;120(Pt 12):2078-90. doi: 10.1242/jcs.004127. Epub 2007 May 29.
Nodal signaling induces the formation of the endoderm and mesoderm during gastrulation. Nodal expression persists until the definitive endoderm progenitor has completely formed, and disappears thereafter. A tightly regulated Nodal expression system is essential for the differentiation of embryonic stem (ES) cells into distinct tissue lineages. On this basis, we established an ES cell differentiation system with the tetracycline-regulated expression of Nodal. The upregulated Nodal signaling pathway and its downstream transcriptional targets induced the specification of ES cells into definitive endoderm and mesoderm derivatives, and the subsequent downregulation of Nodal signaling promoted further maturation of the gut tube both in vitro and in vivo. Sustained expression of the Nodal gene inhibited the maturation of the definitive endoderm owing to persistent Oct3 and/or Oct4 expression and teratoma formation. Furthermore, quantitative single cell analysis by flow cytometry using CXCR4, VEGFR2 and PDGFR-alpha indicated that this protocol for definitive endoderm and mesoderm differentiation is superior to any other available protocol. Our findings also indicated that the Nodal or Nodal-related molecules secreted from Nodal-expressing ES cells could cause genetically unmanipulated ES cells to induce the expression of the Nodal signaling pathway and its downstream targets, which consequently leads to the differentiation of the ES cells into definitive endoderm and mesoderm. Our differentiation system, using tightly regulated Nodal expression, enabled us to investigate the mechanism of ES cell differentiation into definitive endoderm or mesoderm derivatives. Our findings also demonstrate that Nodal-expressing ES cells might be a source of highly active proteins that could be used for developing endoderm or mesoderm tissues in regenerative medicine.
在原肠胚形成过程中，Nodal信号诱导内胚层和中胚层的形成。Nodal表达一直持续到确定的内胚层祖细胞完全形成，此后消失。一个严格调控的Nodal表达系统对于胚胎干细胞（ES细胞）分化为不同的组织谱系至关重要。在此基础上，我们建立了一个通过四环素调控Nodal表达的ES细胞分化系统。上调的Nodal信号通路及其下游转录靶点诱导ES细胞分化为确定的内胚层和中胚层衍生物，随后Nodal信号的下调促进了肠管在体外和体内的进一步成熟。Nodal基因的持续表达由于Oct3和/或Oct4的持续表达以及畸胎瘤的形成而抑制了确定内胚层的成熟。此外，使用CXCR4、VEGFR2和PDGFR-α通过流式细胞术进行的定量单细胞分析表明，这种用于确定内胚层和中胚层分化的方案优于任何其他可用方案。我们的研究结果还表明，从表达Nodal的ES细胞分泌的Nodal或Nodal相关分子可导致未经过基因操作的ES细胞诱导Nodal信号通路及其下游靶点的表达，从而导致ES细胞分化为确定的内胚层和中胚层。我们利用严格调控的Nodal表达的分化系统，使我们能够研究ES细胞分化为确定的内胚层或中胚层衍生物的机制。我们的研究结果还表明，表达Nodal的ES细胞可能是高活性蛋白质的来源，可用于再生医学中内胚层或中胚层组织的开发。
