Understanding the role of the stromal cell niche in intestinal stem cell aging

了解基质细胞生态位在肠道干细胞衰老中的作用

• 批准号: 10591876
• 负责人: Norihiro Goto
• 金额: $ 13.67万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10591876
• 关键词: Address; Affect; Aging; Back; Biology; Cell Aging; Cell Count; Cells; ChIP-seq; Clinic; Clinical; Coculture Techniques; Complex; Dietary Intervention; Engineering; Enzymes; Epigenetic Process; Fibroblasts; Fostering; Gene Deletion; Genes; Goals; Growth; Health; Homocysteine; Hydrolase; In Vitro; Injury; Intervention; Intestines; Knockout Mice; LGR5 gene; Ligands; Lymphatic Endothelial Cells; Measures; Mediating; Metabolism; Methionine; Methionine Metabolism Pathway; Mus; Natural regeneration; Organoids; Patients; Population; Regenerative capacity; Rejuvenation; Research; Role; Signal Transduction; Sirolimus; Source; Stromal Cells; Supporting Cell; System; Testing; Time; Training; Translating; Uncertainty; Visualization; age effect; age related; aged; cell type; conditional knockout; dietary; dietary restriction; experimental study; human old age (65+); improved; in vitro testing; in vivo; in vivo evaluation; insight; intestinal epithelium; mouse model; novel; pharmacologic; programs; promoter; repaired; single-cell RNA sequencing; skills; stem cell aging; stem cell biology; stem cell function; stem cell homeostasis; stem cell self renewal; stem cells; tool; transcriptome sequencing

Project Summary Aging compromises the numbers/function of mammalian Lgr5+ intestinal stem cell (ISCs), which depend on niche factors produced by neighboring cell types like stromal cells. Although the necessity of these niche factors has been tested in vitro, many uncertainties remain regarding their in vivo sources and the impact of aging on them. To address these questions, we have focused on RSPO3, the dominant R-spondin in the mammalian intestine and Lgr5 ligand that drives ISC self-renewal. Using novel Rspo3-GFP mice, we have discovered that RSPO3 is expressed by two distinct populations in the intestinal stroma: RSPO3+GREM1+ fibroblasts (RG fibroblasts) and lymphatic endothelial cells (LECs). We have established heterotypic co-culture systems of RSPO3+ stromal cells with intestinal epithelial organoids, and have found that RG fibroblasts, more than LECs, support organoid growth. Importantly, the numbers/function of RG fibroblasts decline significantly in old mice. By RNA-seq, we have discovered that S-adenosyl-L-homocysteine hydrolase (Ahcy), a rate-limiting enzyme in methionine metabolism that hydrolyzes S-adenosyl homocysteine (SAH), is the most downregulated gene in aged mouse RG fibroblasts compared to their young counterparts. Furthermore, pharmacological inhibition of Ahcy recapitulates the age-related decline in the ability of RG fibroblasts to support ISCs, whereas short-term methionine restriction reverses the age-related decline of RG fibroblasts. We hypothesize that Ahcy loss and methionine accumulation in RG fibroblasts account for some of the age-related deficits of old ISCs that can be reversed by short-term dietary methionine restriction. In this proposal, we will test the hypothesis that RG fibroblasts are the dominant niche cells that foster ISCs in vivo (Aim 1); that loss of Ahcy leads to the age-related decline of RG fibroblasts through accumulation of methionine cycle intermediate metabolites (Aim 2); and that short-term dietary methionine restriction rejuvenates aged mouse RG fibroblasts to support ISCs and ISC-mediated regeneration (Aim 3). Through these aims, we will provide novel insights into how age-related changes in the ISC stromal niche contribute to ISC aging and how we can reverse it through modulating methionine metabolism. Identification of a new dietary intervention that may augment intestinal regeneration in old age will have important clinical implications. My goal is to discover novel insights into how aging influences stem cells with the long-term goal of translating these findings back to the clinic for the improvement of patient health. Because little is known about the aging and metabolism of stromal niche cells in ISC biology, the novel tools that I develop and the skill sets I acquire to assess metabolism of aging stromal niche cells during the K99 training period will permit me to establish a successful and independent research program as I transition to independence.

项目摘要 衰老损害哺乳动物Lgr 5+肠干细胞（ISCs）的数量/功能， 由邻近的细胞类型如基质细胞产生的生态位因子。虽然这些利基的必要性 虽然已经在体外测试了这些因素，但关于它们的体内来源和 在他们身上衰老。为了解决这些问题，我们集中于RSPO 3，在细胞中占主导地位的R-spondin。 哺乳动物肠和Lgr 5配体驱动ISC自我更新。使用新型Rspo 3-GFP小鼠，我们 发现RSPO 3在肠基质中由两个不同的群体表达：RSPO 3 + GREM 1 + 成纤维细胞（RG成纤维细胞）和淋巴管内皮细胞（LEC）。我们建立了异型共培养 系统的RSPO 3+基质细胞与肠上皮类器官，并发现RG成纤维细胞，更多 支持类器官生长。重要的是，RG成纤维细胞的数量/功能下降， 在年老的老鼠身上通过RNA-seq，我们发现了S-腺苷-L-高半胱氨酸水解酶（Ahcy）， 甲硫氨酸代谢中水解S-腺苷高半胱氨酸（SAH）的限速酶，是最 在老年小鼠RG成纤维细胞相比，年轻的同行下调基因。此外，委员会认为， Ahcy的药理学抑制重现了RG成纤维细胞支持细胞增殖能力的年龄相关性下降。 ISCs，而短期蛋氨酸限制逆转RG成纤维细胞的年龄相关性下降。我们 假设RG成纤维细胞中Ahcy丢失和甲硫氨酸积累解释了一些与年龄相关的 缺乏旧的ISCs，可以通过短期饮食蛋氨酸限制逆转。在本提案中，我们将测试 假设RG成纤维细胞是体内培养ISCs的优势小生境细胞（Aim 1）; 通过甲硫氨酸循环中间产物的积累导致RG成纤维细胞的年龄相关性下降 代谢物（目标2）;短期饮食蛋氨酸限制使衰老小鼠RG成纤维细胞恢复活力 以支持ISC和ISC介导的再生（Aim 3）。 通过这些目标，我们将提供新的见解，如何年龄相关的变化，在ISC间质生态位 有助于ISC老化，以及我们如何通过调节蛋氨酸代谢来逆转它。鉴定 一种新的饮食干预可能会增加老年人的肠道再生， 影响我的目标是发现衰老如何影响干细胞的新见解，长期目标是 将这些发现转化回临床，以改善患者健康。因为我们对 ISC生物学中基质小生境细胞的衰老和代谢，我开发的新工具和技能， 在K99训练期间获得评估老化基质小生境细胞的代谢将允许我 建立一个成功的和独立的研究计划，因为我过渡到独立。