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: 8328969
• 负责人: SUSAN J. HENNING
• 金额: $ 33.3万
• 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/8328969
• 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; stem; 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）：本提案是根据肠干细胞（ISC）联盟的RFA提交的。正如《RFA》所指出的，尽管几十年前就已经知道小肠上皮中存在干细胞，但目前还没有公开的方法来分离这些细胞的纯制剂。2005年，本课题组(1)利用流式细胞术侧群（SP）分选首次分离出富含ISCs的部分，通过富集Musashi-1 mRNA（当时唯一已知的ISCs标记物）来判断。随后的微阵列、原位杂交和切除（2;3）研究加强了小鼠小肠SP细胞包含ISCs的证据。虽然我们从一开始就强调(1)SP不是纯的ISC制剂，但从正常小鼠或人小肠中分离ISC的更好方法尚未报道。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）的干细胞性的证据一直受到阻碍。最近关于ISC新标记的报道表明可能存在活跃和静止的亚群。然而，这些尚未被分离出来，对这两种细胞群和表达其他ISC标记的细胞在肠道发育过程中的行为知之甚少。为了解决这些重大差距，我们提出了一种联合肠道发育，干细胞分离，肠道生长手术模型，干细胞遗传学和关键肠道生长因子信号改变小鼠模型专家的合作方法。具体而言，我们提出以下目标：1。通过鉴定合适的膜抗原进行抗体分选，改进ISC的分离方法，并利用功能特性分离活性和静止的ISC；2. 使用三种新方法开发体内试验，以功能验证假定的ISC制剂的自我更新和多谱系分化；和3。研究ISC在肠道发育过程中的行为特征，并建立一个小肠发育组织库，用于其他联盟成员定位已知或新的ISC标记。我们认为，实现这些目标是可行的。