Architecture and communication controlling the efficient generation of beta cells

描述(由申请者提供)：我们将生成关于祖细胞分化过程的翻译上的基本信息，针对一个主要的悬而未决的问题：在体外使用什么刺激和培养条件来产生成熟的？细胞。我们的团队将只专注于所有？细胞前体的组织来源：胰腺中部器官发生的主干区域上皮。控制细胞诞生/成熟的时空协调的信号网络和细胞分裂/迁移过程的定义非常模糊。但是，产生成熟b细胞的细胞自主和非自主程序必须在定义的上皮结构的约束下工作，其紧密联系的形态发生程序产生大量萌芽的、成熟的胰岛。我们将对内分泌前体细胞的位置、细胞-细胞组织和信号进行高分辨率的结构和细胞生物学分析，以及它们在产生分化的？细胞后代或作为内分泌偏向细胞在上皮内循环的行为。我们将：(1)定义上皮结构如何提供控制内分泌偏向前体细胞的位置、数量和行为的信号利基。(2)定义不对称分裂如何产生内分泌前体和内分泌受托后代。(3)表征前内分泌细胞的定向分层及其向β细胞或其他内分泌类型的进展。(4)定位以细胞导向因子为标志的中间产物，确定细胞自主和非自主效应。(5)使用突变分析和谱系追踪来详细说明所涉及的上皮内组织和信号传递过程。(6)在正常和不安的情况下对选定的细胞群体进行基因表达谱分析，以提供对所涉及的基因调控网络的前所未有的了解，以及对关键过渡细胞状态的标记的新见解。(7)将来自BCBC细胞表面标记项目的新的单抗映射到该框架上，为识别/分类区分hESC的祖代-子代状态提供新的工具。(8)直接检测本研究中发现的-细胞形成/成熟刺激对人胚胎干细胞(HESC)体外分化的功能信息。这样一个时空分辨的“正常分化框架”将揭示hESC分化方案中的不足。在体外，hESC来源的？-细胞不成熟且数量较少，而部分分化的前胰腺细胞团在小鼠成熟后产生功能性？-细胞：我们假设体外缺陷反映了无法建立适当的沟通和祖细胞特性。我们设计我们的项目是为了整合和补充其他BCBC团队在hESC分化和内分泌前体细胞鉴定方面的研究。该团队在谱系分析、细胞生物学和祖细胞控制、寿命和BCBC内强大的相互作用方面的血统导致了我们对上皮内分泌祖细胞的明确共同兴趣。新的工具、试剂和数据集将与BCBC的所有成员共享。

DESCRIPTION (provided by applicant): We will generate translationally essential information on the process of ?-cell differentiation from progenitor cells, targeting a major outstanding issue: what stimuli and culture conditions to employ to produce mature ?-cells in vitro. Our team will focus solely on the tissue source of all ?-cell progenitors: the trunk domain epithelium of mid-pancreas organogenesis. The spatiotemporally orchestrated signaling networks and cellular division/migration processes that control ?-cell birth/maturation are very poorly defined. But, cell-autonomous and non-autonomous programs that give rise to mature b cells must work within the constraints of a defined epithelial structure, with its tightly linked morphogenetic program that produces the large numbers of budded, mature islets. We will perform a high-resolution structural and cell biological analysis of endocrine progenitor locations, cell-cell organization and signaling, and their behavior in yielding differentiating ?-cell progeny or remaining as cycling endocrine-biased cells within the epithelium. We will: (1) Define how the epithelial structure provides signaling niches that control the location, number and behavior of endocrine-biased progenitors. (2) Define how asymmetric division yields pro-endocrine precursors and endocrine-committed progeny. (3) Characterize the directional delamination of pro-endocrine cells and their progression to ?-cell or other endocrine types. (4) Localize intermediates marked by cell-instructive factors, and determine cell-autonomous and cell-non-autonomous effects. (5) Use mutational analysis and lineage tracing to detail the intraepithelial organization and signaling processes involved. (6) Perform gene expression profiling of selected cell populations in normal and perturbed situations to provide an unprecedented understanding of the gene regulatory networks involved, plus new insights into markers of critical transitional cell states. (7) Map new monoclonal antibodies from the BCBC cell-surface-marker project onto this framework, providing new tools for identifying/sorting progenitor-progeny states in differentiating hESC. (8) Test directly the functional information emerging from our studies as ?-cell formation/maturation stimuli on human ES cell (hESC) differentiating in vitro. Such a spatiotemporally resolved 'normal differentiation framework' will shed light on deficiencies in hESC differentiation protocols. In vitro hESC-derived ?-cells are immature and low in number, while partly differentiated, propancreatic cell clusters produce functional ?-cells on maturation in mice: we hypothesize that the in vitro deficit reflects an inability to build proper communication and progenitor qualities. We designed our project to integrate with and complement studies of other BCBC teams on hESC differentiation and endocrine progenitor identification. The team's pedigree in lineage analysis, cell biology and progenitor control, longevity and strong intra-BCBC interactions, led to our sharply defined common interest in epithelial endocrine progenitors. Novel tools, reagents, data sets will be shared with all BCBC members.