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？-细胞分化从祖细胞，针对一个主要的悬而未决的问题：什么刺激和培养条件，采用生产成熟？体外细胞我们的团队将专注于所有的组织来源？-细胞祖细胞：胰腺中部器官发生的主干域上皮细胞。时空协调的信号网络和细胞分裂/迁移过程控制？细胞出生/成熟的定义非常差。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，其紧密相连的形态发生程序产生大量出芽的成熟胰岛。我们将进行高分辨率的结构和细胞生物学分析的内分泌祖细胞的位置，细胞-细胞组织和信号，以及他们的行为产生分化？细胞后代或保持为上皮内的循环内分泌偏向细胞。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、数量和行为的信号微环境。(2)定义不对称分裂如何产生前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？细胞或其他内分泌类型。(4)定位由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描述上皮内组织和所涉及的信号传导过程。(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志物的新见解。(7)将BCBC细胞表面标记项目的新单克隆抗体映射到该框架上，为在分化hESC中识别/分选祖细胞-子代状态提供新的工具。(8)直接测试从我们的研究中出现的功能信息，细胞形成/成熟刺激对人ES细胞（hESC）体外分化的影响。这种时空分辨的“正常分化框架”将揭示hESC分化方案中的缺陷。体外hESC衍生的？-细胞是不成熟的，数量少，而部分分化，前胰腺细胞簇产生功能？细胞在小鼠中的成熟：我们假设体外缺陷反映了不能建立适当的交流和祖细胞品质。我们设计了我们的项目，以整合和补充其他BCBC团队对hESC分化和内分泌祖细胞鉴定的研究。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，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.