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 衰老损害了哺乳动物LGR5+肠干细胞（ISC）的数字/功能，这取决于 关于邻近细胞类型（如基质细胞）产生的细分因子。虽然这些利基市场的必要性 在体外测试了因素，关于其体内来源的许多不确定性以及 对他们老化。为了解决这些问题，我们专注于RSPO3， 驱动ISC自我更新的哺乳动物肠和LGR5配体。使用新颖的RSPO3-GFP小鼠，我们有 发现RSPO3在肠道基质中由两个不同的种群表示：RSPO3+GREM1+ 成纤维细胞（RG成纤维细胞）和淋巴内皮细胞（LEC）。我们已经建立了异型共培养 RSPO3+基质细胞的系统，带有肠上皮器官，发现RG成纤维细胞有更多 比LEC支持器官生长。重要的是，RG成纤维细胞的数量/功能下降 在老鼠中显着。通过RNA-Seq，我们发现S-腺苷-L-联合半胱氨酸水解酶（AHCY）， 水解S-腺苷同型半胱氨酸（SAH）的蛋氨酸代谢中的限速酶是最多的酶 与年轻的小鼠相比，老年小鼠RG成纤维细胞的基因下调。此外， Ahcy的药理学抑制概括了与年龄相关的RG成纤维细胞支持能力的下降 ISC，而短期甲硫氨酸限制逆转了与年龄相关的RG成纤维细胞下降。我们 假设Ahcy损失和蛋氨酸在RG成纤维细胞中的积累是与年龄有关的一些 旧ISC的缺陷可以通过短期饮食蛋氨酸限制来逆转。在此建议中，我们将测试 RG成纤维细胞是促进体内ISC的主要细胞的假设（AIM 1）；阿西的损失 通过蛋氨酸周期中间体的积累，导致RG成纤维细胞与年龄相关的下降 代谢物（目标2）；而短期饮食中的蛋氨酸限制使老化的小鼠RG成纤维细胞恢复活力 支持ISC和ISC介导的再生（AIM 3）。 通过这些目标，我们将提供有关年龄相关的ISC Stromal利基市场变化的新颖见解 有助于ISC衰老，以及如何通过调节蛋氨酸代谢来逆转它。识别 一种新的饮食干预措施，可能会增加老年肠再生的新饮食干预措施将具有重要的临床 含义。我的目标是发现衰老如何以长期目标影响干细胞的新颖见解 将这些发现转移回诊所以改善患者健康。因为对 ISC生物学中基质细胞的衰老和代谢，我开发的新工具以及技能集I 在K99训练期内获得评估衰老基质细胞的代谢将使我能够 在我过渡到独立性时，建立成功的独立研究计划。