Department of Developmental Biology, Stanford University School of Medicine, Stanford, Califorina, USA.
Stem Cells Dev. 2012 Aug 10;21(12):2273-87. doi: 10.1089/scd.2011.0416. Epub 2012 Feb 29.
Embryonic definitive endoderm (DE) generates the epithelial compartment of vital organs such as liver, pancreas, and intestine. However, purification of DE in mammals has not been achieved, limiting the molecular "definition" of endoderm, and hindering our understanding of DE development and attempts to produce endoderm from sources such as embryonic stem (ES) cells. Here, we describe purification of mouse DE using fluorescence-activated cell sorting (FACS) and mice harboring a transgene encoding enhanced green fluorescent protein (eGFP) inserted into the Sox17 locus, which is expressed in the embryonic endoderm. Comparison of patterns of signaling pathway activation in native mouse DE and endoderm-like cells generated from ES cells produced novel culture modifications that generated Sox17-eGFP⁺ progeny whose gene expression resembled DE more closely than achieved with standard methods. These studies also produced new FACS methods for purifying DE from nontransgenic mice and mouse ES cell cultures. Parallel studies of a new human SOX17-eGFP ES cell line allowed analysis of endoderm differentiation in vitro, leading to culture modifications that enhanced expression of an endoderm-like signature. This work should accelerate our understanding of mechanisms regulating DE development in mice and humans, and guide further use of ES cells for tissue replacement.
胚胎 definitive endoderm (DE) 生成肝脏、胰腺和肠道等重要器官的上皮细胞。然而，哺乳动物中 DE 的纯化尚未实现，限制了对内胚层的分子“定义”，并阻碍了我们对内胚层发育的理解和从胚胎干细胞 (ES) 等来源产生内胚层的尝试。在这里，我们描述了使用荧光激活细胞分选 (FACS) 纯化小鼠 DE 的方法，以及携带 Sox17 基因座插入增强型绿色荧光蛋白 (eGFP) 转基因的小鼠，该基因在胚胎内胚层中表达。对天然小鼠 DE 和从 ES 细胞生成的类内胚层细胞中信号通路激活模式的比较产生了新的培养改良方法，这些方法产生的 Sox17-eGFP ⁺ 后代的基因表达比标准方法更接近 DE。这些研究还为从非转基因小鼠和小鼠 ES 细胞培养物中纯化 DE 提供了新的 FACS 方法。对一种新型人类 SOX17-eGFP ES 细胞系的平行研究允许对体外内胚层分化进行分析，导致培养改良，增强了类内胚层特征的表达。这项工作应该加速我们对调控小鼠和人类 DE 发育的机制的理解，并指导进一步使用 ES 细胞进行组织替代。
