Epithelial polarity as a fate determinant of multipotent endocrine progenitors during human pancreas development

发育生物学中一个基本的悬而未决的问题是，在由许多不同类型的细胞组成的复杂器官的形成过程中，如何从相同的多能祖细胞诱导出不同的细胞命运。我们使用发育中的胰腺作为一个模型系统来研究这一中心普遍问题，最近发现上皮细胞的顶底极化在指导胰腺细胞命运规范方面起着关键作用。在初步实验的基础上，我们假设上皮极性是胰腺内多能内分泌前体细胞分化为产生胰高血糖素的α细胞或产生胰岛素的β细胞的决定性信号。为了直接验证这一假设，我们将使用CRISPR/Cas9技术将内分泌前体标记NGN3和极性标记NGN3的荧光报告引入同一人类胚胎干细胞(HESC)系。这些双报告的hESCs将被引导在体外向胰腺内分泌细胞分化。使用一种基于共聚焦实时成像的新的谱系追踪方法，我们将确定祖细胞极化与最终的α或β细胞命运之间的相关性。宿主组以前的研究表明，心尖-基底极化通过抑制Notch信号通路影响内分泌承诺。我们将结合实验方法，如RNA干扰和蛋白质免疫共沉淀，剖析将极性复合体与Notch信号和细胞命运决定联系在一起的分子机制。预期的结果不仅将阐明基本生物学过程的未知细节，特别是人类胰腺发育的未知细节，还将有助于设计更精细的方案，从hESCs体外生成β细胞，从而为开发尖端的糖尿病细胞疗法做出贡献。

A fundamental unanswered question in developmental biology is how different cell fates are induced from the same multipotent progenitors during the formation of complex organs that are composed of many different cell types. We use the developing pancreas as a model system to study this central general question and has recently discovered that apical-basal polarization of epithelial cells plays a critical role for steering pancreatic cell fate specification. Based on preliminary experiments, we hypothesize that epithelial polarity is the decisive cue for the commitment of multipotent endocrine progenitors in the pancreas to differentiate to either glucagon-producing alpha cells or insulin-producing beta cells. To directly test this hypothesis, we will use CRISPR/Cas9 technology to introduce fluorescent reporters for the endocrine progenitor marker NGN3 and a polarity marker into the same human embryonic stem cell (hESC) line. These double-reporter hESCs will be directed to differentiate in vitro toward pancreatic endocrine cells. Using a novel lineage-tracing approach based on confocal live-imaging, we will determine the correlation between progenitor cell polarization and eventual alpha or beta cell fate. The host group’s previous investigations indicate that apical-basal polarization affects endocrine commitment by repression of the Notch signaling pathway. We will dissect the molecular mechanisms that link the polarity complex to Notch signaling and cell fate decisions in multipotent progenitors by a combination of experimental approaches such as RNA interference and protein co-immunoprecipitation. The expected results will not only shed light on unknown details of basic biological processes and of human pancreas development in particular, but will also help design more refined protocols for the in vitro generation of beta cells from hESCs, thus contributing to the development of cutting-edge cellular therapies for diabetes.