Intestinal Stem Cell Culture and Entero-endocrine Lineage Development

描述（由申请人提供）：尽管最近在肠道干细胞领域开展了大量工作，但研究人员尚未证明单个假定的干细胞可以在体外或体内环境中被分离、操纵、维持并随后分化为所有指定的谱系。该项目的主要目标是证明一种特定的假定干细胞可以产生具有多谱系潜力的长寿克隆。我们的中心假设是，分离和繁殖肠道干细胞/祖细胞的方法的改进将促进体外和体内沿着由独特标记定义的分泌谱系发育。我们的目标是：(A)改进技术和材料，以促进体外和体内细胞簇或单细胞的生长；(B)研究肠肌成纤维细胞对肠干细胞生态位的贡献；(C)在体内和体外模型中调节干细胞/祖细胞形成完全成熟的肠内分泌细胞的能力；(D)鉴定和表征小鼠从未确定祖细胞到成熟肠内分泌细胞的其他标记物。为了实现这些目标，我们提出了四个具体目标。具体目标1：开发克服肠道上皮细胞锚定依赖性的方法，促进这些细胞在体内和体外的存活、生长和分化。特异性目的2：在一种新型克隆细胞培养系统中，评估贲门周围肌成纤维细胞在调节干细胞增殖和肠内分泌细胞成熟中的作用。特异性目的3：在体内和体外模型中调节来自分离的非承诺和承诺内分泌祖细胞的肠内分泌细胞的成熟。特异性目标4：进一步开发和验证一组独特的谱系特异性标记，这些标记定义了从未确定的祖细胞到完全成熟的肠内分泌细胞的不同阶段。

DESCRIPTION (provided by applicant): Despite the recent flurry of work in the field of gut stem cells, investigators have yet to demonstrate that a single putative stem cell can be isolated, manipulated, maintained and subsequently differentiate into all specified lineages in either an in vitro or in vivo setting. This project's main goal is to demonstrate that a specific putative stem cell can give rise to long-lived clones that have multi-lineage potential. Our central hypothesis is that refinement of methods to isolate and propagate gut stem/progenitors cells will facilitate the in vitro and in vivo development along the secretory lineage as defined by unique markers. Our goals are to (A) refine the techniques and materials that will promote the growth of clusters or single cells in vitro and in vivo; (B) investigate the contribution of intestinal myofibroblasts to the intestinal stem cell niche; (C) modulate the ability of stem/progenitor cells to form fully mature enteroendocrine cells in both in vivo and in vitro models; and (D) identify and characterize other markers from uncommitted progenitors to mature enteroendocrine cells in mice. To address these goals we propose four specific aims. Specific Aim 1: To develop methods that overcome the anchorage dependency of gut epithelial cells, and promotes the survival, growth and differentiation of these cells in both in vivo and in vitro settings. Specific Aim 2: To assess the role of pericryptal myofibroblast in modulating proliferation of stem cells and the maturation of enteroendocrine cells in a novel clonogenic cell culture system. Specific Aim 3: To modulate the maturation of enteroendocrine cells from isolated uncommitted and committed endocrine progenitors in an in vivo and in vitro model. Specific Aim 4: Develop and verify further a group of unique lineage-specific markers that define distinct stages in the progression from uncommitted progenitor cells to fully mature enteroendocrine cells.