Anatomic and functional characterization of the intestinal crypt-villus niche

项目描述 肠上皮的完整性、更新和功能需要来自底层间充质的信号。 NIDDK和NIAID的肠道干细胞联盟（ISCC）的新重点是研究 深入提供关键支持的间充质细胞，它们在组织中的组织，以及它们的 分子特征，包括必需的分泌因子。PI一直是联盟的成员 在过去的4年中，本申请是为了响应RFA-DK-18-507，有限的竞争， 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+，Foxl 1+，PDGFRA+，Gli 1+）作为关键生态位元件，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装共焦显微镜和 通过流式细胞术分离的明确定义的细胞群的整体和单细胞（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必需小生境的主要候选者， 功能协调发展的在过去的2年里，我们分享了这些端细胞和两个不同的（解剖和 分子）PDGFRA 10细胞群刺激了几个ISCC中的一个重要研究流 laboratories.在这一经验和社区的基础上，在富有成效地完成所有目标之后， 从目前的奖励期开始，我们建议采取三种调查方式。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，有三个目的： (a)为了在高空间分辨率下识别Wnt、BMP、EGF和其他关键间充质信号的来源， 分辨率，（B）建立用于所选重要间充质干细胞的稳健且可再现的培养条件 细胞类型，以及（c）告知相应的功能性人细胞的鉴定和验证 人口。这些研究将整个小肠视为一个单元，Aim 2将推进目的 (c)进一步通过考虑每个肠道区域（十二指肠、空肠、回肠以及上行和下行 colon）分开。这两个目标将整合高分辨率解剖和分子分析， 精确的3D图，叠加在间充质细胞功能的评估中， 与其他ISCC团体合作。目标3涉及外地的迫切需要和外地住房准备金的具体情况 调用以产生新的Cre-driver和荧光报告小鼠。基于大量的分子分析， 目的1和2，我们将鉴定新的细胞类型特异性基因座，并使用CRISPR/Cas9编辑插入报告基因。 在多达3个这样的基因座中构建。每个目标都涉及与ISCC实验室的广泛合作 一个全面的合作管理计划的保护伞。此外，我们还将提供和采用 标准化的方案，并与ISCC研究人员共享数据、方案、试剂和小鼠品系。

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.