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: 8113022
• 负责人: SUSAN J. HENNING
• 金额: $ 0.8万
• 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/8113022
• 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）：本提案是针对肠干细胞（ISC）联盟的RFA提交的。正如RFA中所指出的，尽管几十年来人们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。在2005年，我们的小组（1）使用流式细胞术侧群（SP）分选首次分离富集ISC的级分，如通过富集Musashi-1 mRNA（当时唯一已知的ISC标志物）所判断的。随后的微阵列、原位杂交和切除（2; 3）研究加强了来自小鼠小肠的SP细胞包括ISC的证据。虽然我们从一开始就强调了SP不是一种纯的ISCs，但从正常小鼠或人小肠分离ISCs的更好方法尚未报道。此外，迄今为止，用SP细胞（或来自小肠的任何其他分离的细胞部分）证明干细胞性的证据由于缺乏证明自我更新和多谱系分化的功能测定而受到阻碍。最近关于ISC新标记的报道表明可能存在活跃和静止亚群。然而，这些细胞尚未被分离，并且对这两个细胞群和表达其他ISC标记物的细胞在肠发育期间的行为知之甚少。为了解决这些重大差距，我们提出了一种合作方法，该方法将肠道发育，干细胞分离，肠道生长手术模型，干细胞遗传学和小鼠模型与关键肠道生长因子的改变信号传导相结合。具体而言，我们提出以下目标：1.通过鉴定合适的膜抗原用于基于抗体的分选来改进ISC的分离方法，并利用功能特性来分离活动和静止ISC; 2.开发体内试验，以使用三种新方法功能性地验证推定的ISC制剂的自我更新和多谱系分化;以及3.描述肠道发育过程中ISC的行为，并建立小肠发育组织库，以供其他联盟成员定位已知或新的ISC标记物。我们认为，实现这些目标是可行的。 公共卫生关系：了解肠道干细胞的生物学是发展再生医学方法来治疗肠道功能受损的各种疾病的核心。例如，扩大残余干细胞库将是预防或治疗肠衰竭和短肠综合征的理想方法。此外，在干细胞本身受损的情况下（例如在放射或化疗后），健康干细胞的移植可以提供一种新的有效疗法。