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

上皮极性作为人类胰腺发育过程中多能内分泌祖细胞的命运决定因素

• 批准号: 382408533
• 负责人: Dr. Ulf Tiemann
• 金额: --
• 依托单位: University of Copenhagen
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/382408533?language=en
• 关键词: Epithelial; polarity; fate; determinant; multipotent

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.

发育生物学的一个基本问题是，在由许多不同细胞类型组成的复杂器官形成过程中，相同的多能祖细胞如何诱导不同的细胞命运。我们使用发育中的胰腺作为模型系统来研究这个核心的一般性问题，并且最近发现上皮细胞的顶基极化在指导胰腺细胞命运规范中起着关键作用。基于初步实验，我们假设上皮极性是胰腺多能内分泌祖细胞分化为产生胰高血糖素的α细胞或产生胰岛素的β细胞的决定性线索。为了直接验证这一假设，我们将使用CRISPR/Cas9技术将内分泌祖标记物NGN3和极性标记物的荧光报告基因引入同一人类胚胎干细胞（hESC）细胞系。这些双报告型hESCs将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颖谱系追踪方法，我们将确定祖细胞极化与最终α或β细胞命运之间的相关性。宿主组先前的研究表明，顶基极化通过抑制Notch信号通路影响内分泌承诺。我们将通过结合RNA干扰和蛋白质共免疫沉淀等实验方法，剖析多能祖细胞中极性复合物与Notch信号和细胞命运决定之间的分子机制。预期的结果不仅将揭示基本生物学过程的未知细节，特别是人类胰腺发育，而且还将有助于设计更精细的hESCs体外生成β细胞的方案，从而有助于开发尖端的糖尿病细胞疗法。