Mesenchymal regulation of fetal intestinal development and adult regeneration

胎儿肠道发育和成人再生的间质调节

• 批准号: 10040470
• 负责人: Neil McCarthy
• 金额: $ 13.22万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-17 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10040470
• 关键词: Ablation; Address; Adult; Agonist; Anatomy; Biological Assay; Biology; Birth; Bone Morphogenetic Proteins; CD81 gene; Cancer Etiology; Cell Culture Techniques; Cells; Cessation of life; Coculture Techniques; Colorectal Cancer; Constitution; Data; Development; Disease; Elements; Embryo; Environment; Epithelial; Epithelium; Equilibrium; Exhibits; Fetal Development; Fingers; Future; Genetic; Genetic Transcription; Growth; Growth Factor; Homeostasis; In Vitro; Inflammatory Bowel Diseases; Injury; Intestines; Knock-out; Ligands; Malignant Neoplasms; Mesenchymal; Mesenchyme; Methods; Microscopy; Molecular; Molecular Profiling; Mus; Natural regeneration; Organ; Organoids; PDGFRA gene; Pathway interactions; Population; Process; Proliferating; Protein Inhibition; Recombinants; Regenerative response; Regulation; Resolution; Rest; Role; Signal Transduction; Signaling Protein; Small Intestines; Stem Cell Development; Structure; Testing; Tissues; Tube; United States; Villous; Villus; base; cell injury; cell regeneration; cell type; crypt cell; experimental study; fetal; improved; in vivo; insight; intestinal crypt; intestinal epithelium; intestinal homeostasis; novel; progenitor; protein function; regenerative; repaired; response; restoration; single-cell RNA sequencing; stem cell homeostasis; stem cell niche; stem cell proliferation; stem cells; transcriptome sequencing

Project Summary Inflammatory bowel disorders represent insufficient epithelial repair and colorectal cancer (CRC), the second leading cause of cancer deaths in the United States, reflects disruption of intestinal stem cell (ISCs) homeostasis. ISCs depend in part on underlying mesenchymal signaling gradients that balance proliferation versus differentiation capacity of crypt cells, including ISCs. Moreover, ISCs regenerate efficiently after ablation, by de-differentiation of crypt cells. It is unknown which signals or mesenchymal cells promote this regenerative process. My preliminary studies uncover three distinct mesenchymal cell types, two of which orchestrate BMP signaling gradients. High PDGFRA- expressing cells, which correspond to recently described telocytes, lie closest to the epithelium, are most abundant at the villus base, and express high levels of BMP ligands. Low PDGFRA-expressing cells contain two subpopulations: a CD81+ fraction found exclusively below crypts and expressing high levels of the BMP antagonist Gremlin1, and a CD81- fraction (CP cells) which resides above and around crypts but lacks Grem1. In co-cultures of isolated crypt epithelium with each of these strictly purified cell types, CD81+PDGFRAlo cells functionally replace all growth factor-supplemented media, while CP cells do so minimally and telocytes do not. Furthermore, ablation of Grem1+ cells in vivo results in loss of ISCs, hence I call these cells trophocytes. How each of these distinct mesenchymal cell populations regulates fetal ISC plasticity during development and ISC regeneration during loss, is unknown. I hypothesize that telocyte, CP, and trophocyte functions evolve during development, leading to genesis of a stable crypt- villus niche and that some of the same populations promote crypt regeneration after ISC ablation. In Aim 1, I will investigate the molecular profiles and functions of telocytes, CP cells, and trophocytes during mouse fetal ISC development. I will determine when these cells arise during development, investigate their full transcriptional profiles, and use an established crypt co-culture method to define their supportive roles. In Aim 2, I will test how these mesenchymal cells respond to ISC loss and promote crypt regeneration. Using in vivo assays of ISC regeneration, combined with bulk and single-cell RNAseq and in vivo genetic knockouts, I will test which cells promote ISC regeneration and specifically if this occurs through supraphysiologic BMP inhibition. Collectively, these studies will provide fundamental insights into the development and regulation of ISCs in fetal development and regeneration, with broad implications for intestinal biology and disease.

项目摘要 炎症性肠病表现为上皮修复不足和结直肠癌(CRC)， 美国癌症死亡的第二大原因，反映了肠道干细胞的破坏 (ISCS)动态平衡。ISCs在一定程度上依赖于平衡的潜在间充质信号梯度 包括ISCs在内的隐窝细胞的增殖和分化能力。此外，ISCs可以再生 在消融后，通过去除隐窝细胞的分化而有效。尚不清楚是哪种信号或间充质 细胞促进这一再生过程。我的初步研究发现了三种不同的间充质细胞 类型，其中两种协调BMP信号梯度。高表达PDGFRA的细胞 与最近描述的端细胞相对应，端细胞位于最接近上皮的位置，在绒毛最丰富 碱基，并表达高水平的BMP配体。低表达PDGFRA的细胞含有两个 亚群：仅在隐窝下方发现的CD81+组分，表达高水平的BMP 拮抗剂Gremlin1和CD81组分(CP细胞)，它位于隐窝上方和周围，但缺乏 格雷姆1。在分离的隐窝上皮与这些严格纯化的细胞类型中的每一种共同培养时， CD81+PDGFRAlo细胞在功能上取代了所有添加生长因子的培养液，而CP细胞则是这样做的 最低限度，而端粒细胞则不是。此外，体内Grem1+细胞的消融会导致ISCs的丧失， 因此，我称这些细胞为滋养细胞。这些不同的间充质细胞群如何调控 胎儿ISC在发育过程中的可塑性以及ISC在丢失过程中的再生，目前尚不清楚。我假设 端粒细胞、CP和滋养细胞的功能在发育过程中进化，导致了稳定的隐窝的形成。 绒毛生态位和一些相同的种群在ISC消融后促进了隐窝的再生。在……里面 目的1、研究端粒细胞、CP细胞和端粒细胞的分子特征和功能。 小鼠胚胎间质干细胞发育过程中的滋养细胞。我将确定这些细胞何时在 开发，研究它们的完整转录图谱，并使用已建立的隐窝共培养 方法来定义他们的支持角色。在目标2中，我将测试这些间充质细胞如何响应 减少ISC损失，促进隐窝再生。使用体内ISC再生试验，联合 通过批量和单细胞RNAseq以及体内的基因敲除，我将测试哪些细胞促进ISC 再生，特别是如果这是通过超生理抑制BMP发生的话。总而言之，这些 研究将为胎儿ISCs的发育和调控提供基本的见解 发育和再生，对肠道生物学和疾病具有广泛的影响。