Collaborative Approaches to the Study of Intestinal Epithelial Stem Cells

肠上皮干细胞研究的合作方法

• 批准号: 7935374
• 负责人: SUSAN J. HENNING
• 金额: $ 31.08万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7935374
• 关键词: Achievement; Address; Antibodies; Antigens; Behavior; Biological Assay; Biology; Cell Fraction; Cell Separation; Cell membrane; Cells; Development; Disease; Doxorubicin; Epithelial; Excision; Failure; Flow Cytometry; Genetic; Goals; Growth; Growth Factor; Histocompatibility Testing; Human; In Situ Hybridization; Intestines; Label; LacZ Genes; Magnetism; Membrane; Messenger RNA; Methods; Modeling; Mus; Population; Preparation; Principal Investigator; Property; Publishing; Radiation; Regenerative Medicine; Reporting; Residual state; Short Bowel Syndrome; Side; Signal Transduction; Small Intestines; Sorting - Cell Movement; Stem cells; Surgical Models; Thymidine; Time; Tissue Banking; Tissue Banks; Transplantation; analog; base; blind; cell behavior; cell preparation; chemotherapy; effective therapy; improved; in vivo; intestinal epithelium; member; mouse model; novel; novel marker; novel strategies; postnatal; prevent; public health relevance; response; scaffold; self-renewal; stemness; subcutaneous

DESCRIPTION (provided by applicant): This proposal is submitted in response to an RFA for an intestinal stem cell (ISC) Consortium. As noted in the RFA, although the existence of stem cells in the small intestinal epithelium has been known for decades, there are currently no published methods to isolate pure preparations of these cells. In 2005 our group (1) used flow cytometric side population (SP) sorting to isolate for the first time a fraction enriched in ISCs as judged by enrichment for Musashi-1 mRNA (at that time the only known marker of ISCs). Subsequent microarray, in situ hybridization and resection (2; 3) studies have strengthened the evidence that SP cells from mouse small intestine include the ISCs. Although we empahsized from the outset (1) that the SP is not a pure ISC prepartion, better approaches to isolate ISCs from normal mouse or human small intestine have not yet been reported. In addition, to date proof of stemness with SP cells (or any other isolated cell fraction from the small intestine) has been hampered by lack of functional assays to demonstrate self-renewal and multi-lineage differentiation. Recent reports on new markers for ISC suggest that there may be active and quiescent subpopulations. However these have not yet been isolated and little is known about the the behavior of these two cell populations and cells expressing other ISC markers during intestinal development. To address these significant gaps we propose a collaborative approach which combines experts in intestinal development, stem cell isolation, surgical models of intestinal growth, genetics of stem cells and mouse models with altered signaling of key intestinal growth factors. Specifically, we propose the following aims: 1. To improve isolation methods for ISC by the identification of suitable membrane antigens for antibody-based sorting and to use functional properties to separate active and quiescent ISC; 2. To develop in vivo assays to functionally validate self-renewal and multi-lineage differentiation of putative ISC preparations using three novel approaches; and 3. To characterize ISC behavior during intestinal development and to create a developmental tissue bank of small intestine for localization of known or novel ISC markers by other Consortium members. We believe that achievement of these goals is feasible. PUBLIC HEALTH RELEVANCE: Understanding the biology of intestinal stem cells is central to the development of regenerative medicine approaches to various disorders in which intestinal function is compromised. For example expanding the pool of residual stem cells would be an ideal way to prevent or treat intestinal failure and short gut syndrome. Moreover, in situations where the stem cells themselves are damaged (e.g. after radiation or chemotherapy), transplantation of healthy stem cells may afford a novel and effective therapy.

描述（由申请人提供）：本提案是为了响应肠道干细胞 (ISC) 联盟的 RFA 而提交的。正如 RFA 中所指出的，尽管小肠上皮中干细胞的存在已为人所知数十年，但目前还没有公开的方法来分离这些细胞的纯制剂。 2005 年，我们的小组 (1) 使用流式细胞术侧面群体 (SP) 分选首次分离出富含 ISC 的组分，通过 Musashi-1 mRNA（当时唯一已知的 ISC 标记）的富集来判断。随后的微阵列、原位杂交和切除 (2; 3) 研究强化了来自小鼠小肠的 SP 细胞包含 ISC 的证据。尽管我们从一开始就强调 (1) SP 不是纯 ISC 制剂，但尚未报道从正常小鼠或人小肠中分离 ISC 的更好方法。此外，迄今为止，由于缺乏证明自我更新和多谱系分化的功能测定，SP细胞（或任何其他从小肠分离的细胞部分）的干性证据受到阻碍。最近关于 ISC 新标记的报告表明可能存在活跃和静止的亚群。然而，这些细胞尚未被分离出来，并且对于这两种细胞群和表达其他 ISC 标记物在肠道发育过程中的细胞的行为知之甚少。为了解决这些重大差距，我们提出了一种合作方法，该方法结合了肠道发育、干细胞分离、肠道生长手术模型、干细胞遗传学和改变关键肠道生长因子信号传导的小鼠模型方面的专家。具体来说，我们提出以下目标： 1. 通过鉴定适合基于抗体的分选的膜抗原来改进 ISC 的分离方法，并利用功能特性来分离活性和静态 ISC； 2. 使用三种新方法开发体内测定法，以功能验证假定的 ISC 制剂的自我更新和多谱系分化； 3. 表征肠道发育过程中的 ISC 行为，并创建小肠发育组织库，以便其他联盟成员定位已知或新颖的 ISC 标记。我们相信实现这些目标是可行的。 公共健康相关性：了解肠道干细胞的生物学特性对于开发针对肠道功能受损的各种疾病的再生医学方法至关重要。例如，扩大残留干细胞池将是预防或治疗肠道衰竭和短肠综合症的理想方法。此外，在干细胞本身受损的情况下（例如放疗或化疗后），健康干细胞的移植可能提供一种新颖且有效的治疗方法。