Oeda Shiho, Hayashi Yohei, Chan Techuan, Takasato Minoru, Aihara Yuko, Okabayashi Koji, Ohnuma Kiyoshi, Asashima Makoto
Department of Life Sciences (Biology), Graduate School of Arts and Sciences, The University of Tokyo, Japan.
Int J Dev Biol. 2013;57(5):383-9. doi: 10.1387/ijdb.130058ma.
Renal lineages including kidney are derived from intermediate mesoderm, which are differentiated from a subset of caudal undifferentiated mesoderm. The inductive mechanisms of mammalian intermediate mesoderm and renal lineages are still poorly understood. Mouse embryonic stem cells (mESCs) can be a good in vitro model to reconstitute the developmental pathway of renal lineages and to analyze the mechanisms of the sequential differentiation. We examined the effects of Activin A and retinoic acid (RA) on the induction of intermediate mesoderm from mESCs under defined, serum-free, adherent, monolayer culture conditions. We measured the expression level of intermediate mesodermal marker genes and examined the developmental potential of the differentiated cells into kidney using an ex vivo transplantation assay. Adding Activin A followed by RA to mESC cultures induced the expression of marker genes and proteins for intermediate mesoderm, odd-skipped related 1 (Osr1) and Wilms Tumor 1 (Wt1). These differentiated cells integrated into laminin-positive tubular cells and Pax2-positive renal cells in cultured embryonic kidney explants. We demonstrated that intermediate mesodermal marker expression was also induced by RA receptor (RAR) agonist, but not by retinoid X receptor (RXR) agonists. Furthermore, the expression of these markers was decreased by RAR antagonists. We directed the differentiation of mESCs into intermediate mesoderm using Activin A and RA and revealed the role of RAR signaling in this differentiation. These methods and findings will improve our understanding of renal lineage development and could contribute to the regenerative medicine of kidney.
包括肾脏在内的肾谱系源自中间中胚层，中间中胚层由尾侧未分化中胚层的一个子集分化而来。哺乳动物中间中胚层和肾谱系的诱导机制仍知之甚少。小鼠胚胎干细胞（mESCs）可以作为一个很好的体外模型，用于重建肾谱系的发育途径并分析顺序分化的机制。我们在限定的、无血清、贴壁单层培养条件下，研究了激活素A和视黄酸（RA）对从mESCs诱导中间中胚层的影响。我们测量了中间中胚层标记基因的表达水平，并使用体外移植试验检测了分化细胞向肾脏的发育潜能。在mESC培养物中添加激活素A后再添加RA，可诱导中间中胚层、奇数跳过相关1（Osr1）和威尔姆斯肿瘤1（Wt1）的标记基因和蛋白质的表达。这些分化细胞整合到培养的胚胎肾外植体中的层粘连蛋白阳性管状细胞和Pax2阳性肾细胞中。我们证明，RA受体（RAR）激动剂也可诱导中间中胚层标记物的表达，但类视黄醇X受体（RXR）激动剂则不能。此外，RAR拮抗剂可降低这些标记物的表达。我们使用激活素A和RA将mESCs分化为中间中胚层，并揭示了RAR信号在这种分化中的作用。这些方法和发现将增进我们对肾谱系发育的理解，并可能有助于肾脏再生医学。
