Anatomic and functional characterization of the intestinal crypt-villus niche

肠隐窝绒毛生态位的解剖和功能特征

基本信息

批准号：
10237318
负责人：
Ramesh A Shivdasani
金额：
$ 41.97万
依托单位：
DANA-FARBER CANCER INST
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10237318
关键词：
3-Dimensional Address Adopted Anatomy Ascending colon Award Basement membrane Biological Assay Bone Morphogenetic Proteins CD34 gene CD81 gene CRISPR/Cas technology Cell Compartmentation Cell Differentiation process Cell physiology Cell surface Cells Cities Colectomy Collaborations Communities Complementary DNA Confocal Microscopy Cre driver Data Data Set Descending colon Development Disease Duodenum EGF gene Elements Engineering Epithelial Evaluation FOXL1 gene Fibroblasts Flow Cytometry Foundations Generations Genes Goals Heterogeneity Human In Situ In Vitro Intestines Investigation Label Laboratories Lamina Propria Maps Mesenchymal Mesenchyme Messenger RNA Michigan Molecular Molecular Analysis Molecular Profiling Morphology Mouse Strains Mus Myofibroblast National Institute of Allergy and Infectious Disease National Institute of Diabetes and Digestive and Kidney Diseases Nature Organoids Pharmaceutical Preparations Platelet-Derived Growth Factor alpha Receptor Population Protocols documentation RNA Reagent Reporter Reproducibility Research Research Personnel Resolution Seeds Short Bowel Syndrome Signal Transduction Small Intestines Smooth Muscle Actin Staining Method Source Standardization Stream Stromal Cells Structure Testing Therapeutic Tissues Validation Villus Work base cell type combat data sharing epithelial injury epithelium regeneration experience ileum in vivo intestinal crypt intestinal epithelium jejunum member molecular marker mouse model novel regional difference repaired response single-cell RNA sequencing stem stem cells

项目摘要

Project Description Intestinal epithelial integrity, turnover, and function require signals from the underlying mesenchyme. The new focus of the Intestinal Stem Cell Consortium (ISCC) of the NIDDK and NIAID is to investigate in depth the mesenchymal cells that provide crucial support, their organization within the tissue, and their molecular signatures, including essential secreted factors. The PI has been a member of the Consortium for the last 4 years and this application is made in response to RFA-DK-18-507, a Limited Competition to renew the ISCC Research Centers. Although recent progress in the field implicates various cell populations (CD34+, Foxl1+, PDGFRA+, Gli1+) as key niche elements, their precise identities, overlap, and contributions remain uncertain. By integrated consideration of high-resolution whole-mount confocal microscopy and ensemble and single-cell (sc) RNA-seq analysis of well-defined cell populations isolated by flow cytometry, we have identified three distinct mesenchymal cell types as leading candidates for essential niche functions. Over the last 2 years, our sharing of data on these telocytes and two distinct (anatomic and molecular) PDGFRAlo cell populations has stimulated one important stream of investigation in several ISCC laboratories. Building on this experience and community, and following productive completion of all Aims from the current award period, we propose three lines of investigation. Specific Aim 1 will generate detailed molecular, anatomic, and functional maps of mouse stromal cell populations, with three purposes: (a) to identify the sources of Wnt, BMP, EGF, and other key mesenchymal signals at high spatial resolution, (b) to establish robust and reproducible culture conditions for selected important mesenchymal cell types, and (c) to inform identification and validation of the corresponding functional human cell populations. These studies consider the whole small intestine as a unit, and Aim 2 will advance purpose (c) further by considering each gut region (duodenum, jejunum, ileum, and ascending and descending colon) separately. Both Aims will integrate high-resolution anatomic and molecular analyses to generate accurate 3D maps, superimposed with assessments of mesenchymal cell functions conducted in collaboration with other ISCC groups. Aim 3 addresses an urgent need in the field and the specific RFA call to generate new Cre-driver and fluorescent reporter mice. Based on extensive molecular profiling in Aims 1 and 2, we will identify novel cell type-specific loci and use CRISPR/Cas9 editing to insert reporter constructs in up to 3 such loci. Each Aim involves extensive collaboration with ISCC laboratories under the umbrella of a comprehensive Collaborative Management Plan. In addition, we will contribute and adopt standardized protocols and share data, protocols, reagents, and mouse strains with ISCC investigators.

项目描述肠上皮完整性、周转和功能需要来自底层间充质的信号。 NIDDK 和 NIAID 肠道干细胞联盟 (ISCC) 的新重点是研究深入提供重要支持的间充质细胞、它们在组织内的组织及其分子特征，包括必需的分泌因子。 PI已成为联盟成员过去 4 年，本申请是为了响应 RFA-DK-18-507 的要求而提出的，这是一项有限竞争更新 ISCC 研究中心。尽管该领域的最新进展涉及各种细胞群（CD34+、Foxl1+、PDGFRA+、Gli1+）作为关键利基元素，它们的精确身份、重叠和贡献仍然不确定。通过综合考虑高分辨率整体共聚焦显微镜和对通过流式细胞术分离的明确细胞群进行整体和单细胞 (sc) RNA-seq 分析，我们已经确定了三种不同的间充质细胞类型作为重要生态位的主要候选细胞功能。在过去的两年里，我们共享了这些特细胞和两种不同的（解剖学和分子）PDGFRAlo 细胞群刺激了多个 ISCC 中的一项重要研究实验室。建立在这种经验和社区的基础上，并在有效地完成所有目标之后从当前的奖励期内，我们提出了三方面的调查。具体目标 1 将生成小鼠基质细胞群的详细分子、解剖和功能图谱，具有三个目的： (a) 识别高空间 Wnt、BMP、EGF 和其他关键间质信号的来源决议，(b) 为选定的重要间充质细胞建立稳健且可重复的培养条件细胞类型，以及 (c) 为相应功能性人类细胞的识别和验证提供信息人口。这些研究将整个小肠视为一个单元，目标 2 将推进目的 (c) 进一步考虑每个肠道区域（十二指肠、空肠、回肠、升胃和降胃）冒号）分开。这两个目标都将整合高分辨率解剖和分子分析来生成准确的 3D 地图，叠加对间充质细胞功能的评估与其他 ISCC 团体的合作。目标 3 解决现场和特定 RFA 的迫切需求调用生成新的 Cre-driver 和荧光报告小鼠。基于广泛的分子分析目标 1 和 2，我们将鉴定新的细胞类型特异性位点并使用 CRISPR/Cas9 编辑插入报告基因构建最多 3 个这样的基因座。每个目标都涉及与 ISCC 实验室的广泛合作全面的协作管理计划的总括。此外，我们将贡献并采用标准化方案并与 ISCC 研究人员共享数据、方案、试剂和小鼠品系。