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.

项目概要 衰老会损害哺乳动物 Lgr5+ 肠干细胞 (ISC) 的数量/功能，这取决于 邻近细胞类型（如基质细胞）产生的利基因子。尽管这些利基市场的必要性 因素已经在体外进行了测试，但关于它们的体内来源和影响仍然存在许多不确定性 他们身上的衰老。为了解决这些问题，我们重点关注 RSPO3，它是细胞中占主导地位的 R-spondin。 哺乳动物肠道和驱动 ISC 自我更新的 Lgr5 配体。使用新型 Rspo3-GFP 小鼠，我们 发现 RSPO3 由肠基质中两个不同的群体表达：RSPO3+GREM1+ 成纤维细胞（RG 成纤维细胞）和淋巴内皮细胞（LEC）。我们建立了异型共培养 RSPO3+基质细胞与肠上皮类器官的系统，并发现RG成纤维细胞，更多 比 LEC 更支持类器官的生长。重要的是，RG 成纤维细胞的数量/功能下降 在老年小鼠中显着。通过RNA-seq，我们发现S-腺苷-L-同型半胱氨酸水解酶（Ahcy）， 蛋氨酸代谢中的一种限速酶，可水解 S-腺苷同型半胱氨酸 (SAH)，是最重要的酶 与年轻小鼠相比，老年小鼠 RG 成纤维细胞中的基因下调。此外， Ahcy 的药理学抑制概括了 RG 成纤维细胞支持能力与年龄相关的下降 ISCs，而短期蛋氨酸限制可逆转 RG 成纤维细胞与年龄相关的衰退。我们 假设 RG 成纤维细胞中 Ahcy 丢失和蛋氨酸积累是一些与年龄相关的原因 旧的ISCs的缺陷可以通过短期饮食蛋氨酸限制来逆转。在这个提案中，我们将测试 RG 成纤维细胞是体内培养 ISC 的主要生态位细胞的假设（目标 1）；失去阿希 通过蛋氨酸循环中间体的积累导致 RG 成纤维细胞与年龄相关的衰退 代谢物（目标 2）；短期饮食蛋氨酸限制可使衰老小鼠 RG 成纤维细胞恢复活力 支持 ISC 和 ISC 介导的再生（目标 3）。 通过这些目标，我们将提供关于 ISC 基质生态位与年龄相关的变化如何变化的新颖见解 有助于 ISC 衰老，以及我们如何通过调节蛋氨酸代谢来逆转它。鉴定 一种可能增强老年肠道再生的新饮食干预措施将具有重要的临床意义 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 将这些发现转化回诊所以改善患者的健康。因为人们知之甚少 ISC 生物学中基质微环境细胞的衰老和代谢，我开发的新工具和我的技能 在 K99 训练期间获得评估老化基质细胞的代谢将使我能够 当我过渡到独立时，建立一个成功且独立的研究计划。