Architecture and communication controlling the efficient generation of beta cells

描述（由申请人提供）：我们将生成翻译过程中的基本信息？-细胞从祖细胞分化，针对一个主要突出的问题：什么刺激和培养条件，以产生成熟？-体外细胞。我们的小组只关注组织来源吗？-细胞祖细胞：胰腺中部器官发生的干域上皮。时空协调的信号网络和细胞分裂/迁移过程控制？-细胞出生/成熟的定义非常模糊。但是，产生成熟b细胞的细胞自主和非自主程序必须在确定的上皮结构的约束下工作，其紧密相连的形态发生程序产生大量出芽，成熟的胰岛。我们将进行高分辨率的结构和细胞生物学分析内分泌祖细胞的位置，细胞-细胞组织和信号，和他们的行为产生分化？-细胞的后代或在上皮内作为循环的内分泌偏向细胞。我们将：(1)定义上皮结构如何提供控制内分泌偏向祖细胞的位置、数量和行为的信号龛。(2)定义不对称分裂如何产生促内分泌前体和有内分泌倾向的后代。(3)表征促内分泌细胞的定向分层及其向？-细胞或其他内分泌类型。(4)定位以细胞引导因子为标志的中间体，确定细胞自主和细胞非自主效应。(5)使用突变分析和谱系追踪来详细描述上皮内组织和所涉及的信号传导过程。(6)在正常和受干扰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理解，以及对关键过渡细胞状态标记物的新见解。(7)将来自BCBC细胞表面标记项目的新单克隆抗体映射到该框架上，为分化hESC的祖-子状态的鉴定/分类提供新的工具。(8)直接测试从我们的研究中得到的功能信息，如？体外诱导人胚胎干细胞（hESC）分化的细胞形成/成熟刺激。这样一个时空解决的“正常分化框架”将揭示hESC分化协议的缺陷。体外hesc衍生？细胞未成熟，数量少，部分分化，胰腺原细胞簇产生功能？-小鼠细胞成熟的影响：我们假设体外缺陷反映了无法建立适当的沟通和祖细胞质量。我们的项目旨在整合和补充其他BCBC团队在hESC分化和内分泌祖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、寿命和强大的bcbc内相互作用方面的家谱，使我们对上皮内分泌祖细胞产生了明确的共同兴趣。新工具、试剂、数据集将与所有BCBC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.