Mechanisms of pancreatic endocrine cell differentiation

胰腺内分泌细胞分化机制

• 批准号: 9095302
• 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 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9095302
• 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; Genetic study; Goals; Grant; Health; 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; Testing; Time; Tissues; To specify; Work; base; cell type; genetic approach; human embryonic stem cell; imaging genetics; imaging system; improved; in vitro activity; in vivo; inhibitor/antagonist; loss of function; mouse model; notch protein; novel; progenitor; programs; research study; stem cell differentiation; three dimensional cell culture; 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）：该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类胚胎干细胞（hESC）产生功能性胰岛素分泌β细胞。在过去的资助期间，该赠款下的工作已确定胰腺中的导管祖细胞是胚胎发育过程中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 对于赋予导管祖细胞启动内分泌基因表达程序的能力是必需的。此外，我们已经证明Sox9的表达受到Fgf和Notch信号通路的控制，这表明祖细胞需要暴露于Fgf和Notch信号才能启动内分泌细胞分化。在初步 为了支持这些研究的继续进行，我们表明，在当前 hESC 向胰腺内分泌 β 细胞的分化方案中，Fgf 和 Notch 信号传导受到异常调节。我们假设异常的 Fgf 和 Notch 信号传导环境是体外产生的 β 样细胞功能障碍的原因。在这个持续更新的应用中，PI 提出了小鼠遗传和基于 hESC 的方法的结合，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，以及 (b.) 应用这些知识来生成功能性内分泌细胞来自体外 hESC 的细胞。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 Fgf 和 Notch 信号通路如何协调控制内分泌细胞的规范和分化。为了帮助这些实验，PI 的实验室开发了独特的基因小鼠模型。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 以单细胞分辨率实时进行内分泌细胞分化。基于其他组织和器官的证据，该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可能的联系。在目标 3 中，我们将应用从小鼠遗传实验中学到的范式来将 hESC 引导至 β 细胞谱系。该目标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 hESC 产生的内分泌细胞的成熟度。 PI 实验室的初步证据表明，通过提供更接近正常发育过程中环境的 Fgf 和 Notch 信号传导环境，可以提高内分泌细胞的成熟度。总之，这些实验将有助于确定支持在体外从 hESC 分化出功能性 β 细胞的培养条件。