Department of Histology and Embryology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan.
Biochem Biophys Res Commun. 2010 Mar 19;393(4):877-82. doi: 10.1016/j.bbrc.2010.02.111. Epub 2010 Feb 19.
Embryonic stem cells (ESCs) are potentially powerful tools for regenerative medicine and establishment of disease models. The recent progress in ESC technologies is noteworthy, but ESC differentiation into renal lineages is relatively less established. The present study aims to differentiate mouse ESCs (mESCs) into a renal progenitor pool, the intermediate mesoderm (IM), without addition of exogenous cytokines and embryoid formation. First, we treated mESCs with a combination of small molecules (Janus-associated tyrosine kinase inhibitor 1, LY294002, and CCG1423) and differentiated them into BMP7-positive cells, BMP7 being the presumed inducing factor for IM. When these cells were cultured with adding retinoic acid, expression of odd-skipped related 1 (Osr1), which is essential to IM differentiation, was enhanced. To simplify the differentiation protocol, the abovementioned four small molecules (including retinoic acid) were combined and added to the culture. Under this condition, more than one-half of the cells were positive for Osr1, and at the same time, Pax2 (another IM marker) was detected by real-time PCR. Expressions of ectodermal marker and endodermal marker were not enhanced, while mesodermal marker changed. Moreover, expression of genes indispensable to kidney development, i.e., Lim1 and WT1, was detected by RT-PCR. These results indicate the establishment of a specific, effective method for differentiation of the ESC monolayer into IM using a combination of small molecules, resulting in an attractive cell source that could be experimentally differentiated to understand nephrogenic mechanisms and cell-to-cell interactions in embryogenesis.
胚胎干细胞（ESCs）是再生医学和疾病模型建立的潜在有力工具。最近 ESCs 技术的进展值得注意，但 ESC 向肾系分化的研究相对较少。本研究旨在不添加外源性细胞因子和类胚体形成的情况下，将小鼠胚胎干细胞（mESCs）分化为肾前体细胞池——中胚层（IM）。首先，我们用一组小分子（Janus 相关酪氨酸激酶抑制剂 1、LY294002 和 CCG1423）处理 mESCs，使它们分化为 BMP7 阳性细胞，BMP7 被认为是诱导 IM 的因子。当这些细胞用维甲酸培养时，对 IM 分化至关重要的 odd-skipped related 1（Osr1）的表达增强。为了简化分化方案，将上述四种小分子（包括维甲酸）组合并添加到培养物中。在这种条件下，超过一半的细胞呈 Osr1 阳性，同时通过实时 PCR 检测到 Pax2（另一个 IM 标志物）。外胚层标志物和内胚层标志物的表达没有增强，而中胚层标志物发生变化。此外，通过 RT-PCR 检测到肾脏发育所必需的基因，即 Lim1 和 WT1 的表达。这些结果表明，使用小分子组合建立了一种将 ESC 单层分化为 IM 的特异、有效的方法，为实验分化提供了有吸引力的细胞来源，以了解胚胎发生中肾发生机制和细胞间相互作用。
