Department of Surgery, University of British Columbia, Vancouver, Canada.
PLoS One. 2010 Nov 30;5(11):e14146. doi: 10.1371/journal.pone.0014146.
Pluripotent embryonic stem cells hold a great promise as an unlimited source of tissue for treatment of chronic diseases such as Type 1 diabetes. Herein, we describe a protocol using all-trans-retinoic acid, basic fibroblast growth factor and dibutyryl cAMP (DBcAMP) in the absence of embryoid body formation, for differentiation of murine embryonic stem cells into definitive endoderm that may serve as pancreatic precursors. The produced cells were analyzed by quantitative PCR, immunohistochemistry and static insulin release assay for markers of trilaminar embryo, and pancreas. Differentiated cells displayed increased Sox17 and Foxa2 expression consistent with definitive endoderm production. There was minimal production of Sox7, an extraembryonic endoderm marker, and Oct4, a marker of pluripotency. There was minimal mesoderm or neuroectoderm formation based on expression levels of the markers brachyury and Sox1, respectively. Various assays revealed that the cell clusters generated by this protocol express markers of the pancreatic lineage including insulin I, insulin II, C-peptide, PDX-1, carboxypeptidase E, pan-cytokeratin, amylase, glucagon, PAX6, Ngn3 and Nkx6.1. This protocol using all-trans-retinoic acid, DBcAMP, in the absence of embryoid bodies, generated cells that have features of definitive endoderm that may serve as pancreatic endocrine precursors.
多能胚胎干细胞作为治疗 1 型糖尿病等慢性疾病的组织的无限来源，具有巨大的应用潜力。在此，我们描述了一种在不形成类胚体的情况下使用全反式视黄酸、碱性成纤维细胞生长因子和二丁酰环腺苷酸（DBcAMP）将小鼠胚胎干细胞分化为具有潜在胰腺前体细胞功能的三胚层胚胎内胚层的方案。通过定量 PCR、免疫组织化学和静态胰岛素释放试验对产生的细胞进行分析，以鉴定三胚层胚胎和胰腺的标志物。分化细胞 Sox17 和 Foxa2 的表达增加，表明其为内胚层的产生。 Sox7（胚胎外胚层标志物）和 Oct4（多能性标志物）的产生量很少。根据 brachyury 和 Sox1 标志物的表达水平，分别检测到极少的中胚层或神经外胚层形成。各种检测方法表明，该方案生成的细胞簇表达胰岛谱系标志物，包括胰岛素 I、胰岛素 II、C 肽、PDX-1、羧肽酶 E、泛细胞角蛋白、淀粉酶、胰高血糖素、PAX6、Ngn3 和 Nkx6.1。该方案使用全反式视黄酸和 DBcAMP，不形成类胚体，生成的细胞具有内胚层的特征，可能作为胰腺内分泌前体细胞。
