Mechanisms of pancreatic endocrine cell differentiation

胰腺内分泌细胞分化机制

• 批准号: 8584780
• 负责人: Maike Sander
• 金额: $ 37.59万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8584780
• 关键词: Accounting; Achievement; Adult; Affect; Beta Cell; Cell Cycle; Cell Differentiation process; Cell Lineage; Cell Proliferation; Cell Therapy; Cell division; Cells; Characteristics; Competence; Cues; Development; Diabetes Mellitus; Duct (organ) structure; Ductal; Ductal Epithelium; Embryo; Embryonic Development; Endocrine; Environment; Epithelium; Funding; Gene Expression; Gene Targeting; Genetic; Goals; Grant; Human; Image; Imaging technology; In Vitro; Injury; Insulin; Insulin-Dependent Diabetes Mellitus; Knowledge; Laboratories; Learning; Life; Lifting; Link; Literature; Modeling; Monitor; Mus; Non-Insulin-Dependent Diabetes Mellitus; Notch Signaling Pathway; Organ; Organ Culture Techniques; Pancreas; Pluripotent Stem Cells; Population; Production; Protocols documentation; Repression; Resolution; Role; Signal Transduction; Source; Stem cells; Sum; System; Testing; Time; Tissues; To specify; Work; base; cell type; human embryonic stem cell; improved; in vitro activity; in vivo; inhibitor/antagonist; loss of function; mouse model; notch protein; novel; progenitor; programs; public health relevance; research study; stem cell differentiation; transcription factor

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.

描述(由申请人提供)：这项建议的总体目标是确定胰腺内分泌细胞形成的基础机制，并应用这一知识指导人类胚胎干细胞(HESCs)产生功能强大的胰岛素分泌β细胞。在过去的资助期间，这项赠款下的工作已经确定，胰腺中的导管前体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Sox9是赋予导管前体细胞启动内分泌基因表达程序的能力所必需的。此外，我们已经证明Sox9的表达受控于成纤维细胞生长因子和Notch信号通路，这表明祖细胞需要暴露于成纤维细胞生长因子和Notch信号才能启动内分泌细胞分化。在预赛中 为了支持这些研究的继续，我们表明，在hESCs向胰腺内分泌β细胞分化的现行方案中，成纤维细胞生长因子和Notch信号受到异常调节。我们推测，异常的成纤维细胞生长因子和Notch信号环境是导致体外产生的β-样细胞功能障碍的原因。在这个持续的更新申请中，PI提出了结合小鼠遗传和基于hESC的方法来(a.)进一步确定成纤维细胞生长因子和Notch信号调节内分泌细胞发育的机制和(b.)应用这一知识在体外从人类胚胎干细胞中产生具有功能的内分泌细胞。为了更好地了解内分泌细胞分化所需的特定信号环境，目标1将研究成纤维细胞生长因子和Notch信号通路如何协调控制内分泌细胞的规格和分化。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，我们将使用在PI的实验室建立的一种新的实时成像技术来监控启动 实时单细胞分辨率的内分泌细胞分化。基于其他组织和器官的证据，这一目的下的实验将探索细胞分裂、Notch活性和细胞分化启动之间的可能联系。在目标3中，我们将应用从我们的小鼠遗传学实验中学到的悖论来引导hESCs走向β细胞谱系。在这一目标下的实验将直接测试成纤维细胞生长因子和Notch信号的变化如何影响体外培养的人胚胎干细胞产生的内分泌细胞的成熟度。来自PI实验室的初步证据表明，通过提供与正常发育期间的环境更接近的成纤维细胞生长因子和Notch信号环境，可以改善内分泌细胞的成熟度。综上所述，这些实验将有助于确定支持在体外从hESCs分化为功能性β细胞的培养条件。