Architecture and communication controlling the efficient generation of beta cells

描述（由申请人提供）：我们将生成有关祖细胞分化为α细胞的过程的翻译重要信息，针对一个主要的悬而未决的问题：采用什么刺激和培养条件在体外产生成熟的α细胞。我们的团队将只关注所有 β 细胞祖细胞的组织来源：中胰腺器官发生的主干域上皮。控制β细胞出生/成熟的时空协调信号网络和细胞分裂/迁移过程的定义非常不明确。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限制内发挥作用，其紧密相连的形态发生程序会产生大量出芽的成熟胰岛。我们将对内分泌祖细胞的位置、细胞-细胞组织和信号传导，以及它们在产生分化的α细胞后代或在上皮内保留为循环内分泌偏向细胞的行为进行高分辨率的结构和细胞生物学分析。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、数量和行为的信号传导生态位。 (2) 定义不对称分裂如何产生促内分泌前体和内分泌定向后代。 (3) 表征前内分泌细胞的定向分层及其向α细胞或其他内分泌类型的进展。 (4)定位细胞指导因子标记的中间体，并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说明所涉及的上皮内组织和信号传导过程。 (6) 在正常和扰动情况下对选定的细胞群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，为识别/分类分化 hESC 的祖代-子代状态提供新工具。 (8) 直接测试我们研究中出现的功能信息，作为人 ES 细胞 (hESC) 体外分化的 β 细胞形成/成熟刺激。这种时空解析的“正常分化框架”将揭示 hESC 分化方案的缺陷。体外 hESC 衍生的 α 细胞不成熟且数量较少，而部分分化的胰岛细胞簇在小鼠成熟时产生功能性 α 细胞：我们假设体外缺陷反映了无法建立适当的通讯和祖细胞质量。我们设计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、长寿和强大的 BCBC 内相互作用方面的谱系，导致我们对上皮内分泌祖细胞有着明确的共同兴趣。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.