Mechanisms of pancreatic endocrine cell differentiation

描述（由申请人提供）：本提案的总体目标是确定胰腺内分泌细胞形成的机制，并应用这些知识指导人胚胎干细胞（hESC）产生功能性胰岛素分泌β细胞。在过去的资助期间，这项资助下的工作已经确定胰腺中的导管祖细胞是胚胎发育期间内分泌细胞的主要来源。PI实验室的机制研究进一步表明，转录因子Sox 9是必要的赋予能力后，导管祖细胞启动内分泌基因表达程序。此外，我们已经证明，Sox 9的表达是在Fgf和Notch信号通路的控制下，这表明祖细胞需要暴露于Fgf和Notch信号，以启动内分泌细胞分化。初步 为了支持这些研究的继续，我们展示了FGF和Notch信号传导在目前的hESC向胰腺内分泌β细胞的分化方案中被异常调节。我们假设异常的FGF和Notch信号环境是体外产生的β样细胞功能障碍的原因。在该持续更新申请中，PI提出了小鼠遗传和基于hESC的方法的组合，以（a.）进一步定义FGF和Notch信号传导协调内分泌细胞发育的机制和（B.）应用这些知识在体外从hESC产生功能性内分泌细胞。为了更好地了解内分泌细胞分化所需的特定信号环境，目的1将研究FGF和Notch信号通路如何协调控制内分泌细胞的特化和分化。为了帮助这些实验，PI的实验室开发了独特的遗传小鼠模型。在目标2中，我们将采用PI实验室建立的新型实时成像技术来监测启动 在真实的时间内，内分泌细胞分化的单细胞分辨率。基于其他组织和器官中的证据，该目标下的实验将探索细胞分裂、Notch活性和细胞分化起始之间的可能联系。在目标3中，我们将应用从我们的小鼠遗传实验中学到的范式来指导hESC向β细胞谱系发展。在此目的下的实验将直接测试FGF和Notch信号传导的变化如何影响体外由hESC产生的内分泌细胞的成熟。来自PI实验室的初步证据表明，内分泌细胞成熟可以通过提供更接近正常发育期间环境的FGF和Notch信号环境来改善。总之，这些实验将有助于鉴定支持体外从hESC分化功能性β细胞的培养条件。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to define the mechanisms that underlie the formation of endocrine cells in the pancreas and to apply this knowledge to instruct human embryonic stem cells (hESCs) to produce functional insulin-secreting beta cells. During the past funding period, work under this grant has determined that ductal progenitors in the pancreas are the major source of endocrine cells during embryonic development. Mechanistic studies in the PI's laboratory have further shown that the transcription factor Sox9 is necessary to bestow competence upon ductal progenitors to initiate endocrine gene expression programs. Moreover, we have demonstrated that Sox9 expression is under control of the Fgf and Notch signaling pathways, suggesting that progenitors need to be exposed to Fgf and Notch signals for endocrine cell differentiation to be initiated. In preliminary studies presented to support a continuation of these studies, we show that Fgf and Notch signaling are aberrantly regulated in current differentiation protocols of hESCs towards pancreatic endocrine beta cells. We hypothesize that the aberrant Fgf and Notch signaling environment accounts for the malfunction of beta- like cells produced in vitro. In this continuing renewal application, the PI proposes a combination of mouse genetic and hESC-based approaches to (a.) further define the mechanisms by which Fgf and Notch signaling orchestrate endocrine cell development and (b.) apply this knowledge to generate functional endocrine cells from hESCs in vitro. To better understand the specific signaling environment necessary for endocrine cell differentiation, Aim 1 will investigate how the Fgf and Notch signaling pathways coordinately control the specification and differentiation of endocrine cells. To aid these experiments, the PI's laboratory has developed unique genetic mouse models. In Aim 2, we will employ a novel live imaging technology established in the PI's laboratory to monitor the initiation of endocrine cell differentiation at single cell resolution in real time. Based on evidence in othe tissues and organs, experiments under this aim will explore a possible connection between cell division, Notch activity, and the initiation of cell differentiation. In Aim 3, we will apply paradgms learned from our mouse genetic experiments to direct hESCs towards the beta cell lineage. Experiments under this Aim will directly test how changes in Fgf and Notch signaling affect the maturity of endocrine cells produced from hESCs in vitro. Preliminary evidence from the PI's laboratory suggests that endocrine cell maturity can be improved by providing a Fgf and Notch signaling environment that more closely resembles the environment during normal development. Together, these experiments will aid the identification of culture conditions that support the differentiation of functional beta cells from hESCs in vitro.