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

• 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
• 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

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，周转率和功能需要基础间质的信号。 NIDDK和NIAID的肠道干细胞联盟（ISCC）的新焦点是在 深度为提供至关重要的支持的间充质细胞，组织内的组织及其 分子特征，包括基本的分泌因素。 PI是财团的成员 在过去的四年中 更新ISCC研究中心。尽管该领域的最新进展暗示了各种细胞种群 （CD34+，FOXL1+，PDGFRA+，GLI1+）作为关键的小众元素，其精确身份，重叠和贡献 保持不确定。通过整合考虑高分辨率全置共聚焦显微镜和 通过流式细胞仪分离的明确定义的细胞群体的集合和单细胞（SC）RNA-seq分析， 我们已经确定了三种不同的间充质细胞类型是必需利基的主要候选者 功能。在过去的两年中，我们共享这些链球菌的数据和两个不同的（解剖和解剖和 分子）PDGFRALO细胞种群刺激了几个ISCC的一项重要研究流 实验室。建立在这种经验和社区的基础上，并遵循所有目标的有效完成 从目前的奖励期开始，我们提出了三条调查。特定目标1将产生 小鼠基质细胞群的详细分子，解剖和功能图，具有三个目的： （a）识别高空间上的Wnt，BMP，EGF和其他钥匙间充质信号的来源 分辨率，（b）为选定的重要间充质建立鲁棒和可重复的培养条件 细胞类型，以及（c）为相应功能性人体细胞的识别和验证提供信息 人群。这些研究将整个小肠视为一个单位，AIM 2将促进目的 （c）进一步考虑每个肠道区域（DuodeNum，Jejunum，Ileum和上升和下降 分别结肠。两种目标都将整合高分辨率的解剖和分子分析以产生 准确的3D图，并叠加在对中充质细胞功能的评估中叠加 与其他ISCC小组的合作。 AIM 3解决了该领域的迫切需求和特定的RFA 致电以生成新的CRE驱动器和荧光记者小鼠。基于广泛的分子分析 AIM 1和2，我们将确定新颖的细胞类型特异性基因座，并使用CRISPR/CAS9编辑来插入记者 最多3个此类基因座的构造。每个目标都涉及与ISCC实验室的广泛合作 综合合作管理计划的伞。此外，我们将贡献并采用 标准化协议并与ISCC研究人员共享数据，协议，试剂和小鼠菌株。