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

项目摘要 衰老影响哺乳动物Lgr5+肠道干细胞(ISCs)的数量/功能，这依赖于 由邻近的细胞类型产生的生态位因素，如基质细胞。尽管这些利基市场的必要性 因子已经在体外进行了测试，关于它们在体内的来源和影响仍然存在许多不确定性 他们正在变老。为了解决这些问题，我们将重点放在了RSPO_3上，RSPO_3是 哺乳动物肠道和驱动ISC自我更新的Lgr5配体。使用新的Rspo3-GFP小鼠，我们已经 发现RSPO_3在肠道间质中由两个不同的群体表达：RSPO_3+GREM1+ 成纤维细胞(RG成纤维细胞)和淋巴管内皮细胞(LECs)。我们已经建立了异型共培养 RSPO_3+间质细胞与肠上皮类器官的系统，并发现RG成纤维细胞，更多 而不是LEC，支持有机物生长。重要的是，RG成纤维细胞的数量/功能下降 在老年小鼠身上有显著差异。通过rna-seq，我们发现了S-腺苷-L-同型半胱氨酸水解酶(Ahcy)， 蛋氨酸代谢中的限速酶，对S-腺苷同型半胱氨酸(SAH)的水解酶，是最 与年轻小鼠RG成纤维细胞相比，老年小鼠RG成纤维细胞中基因下调。此外， AHcy的药理抑制概括了RG成纤维细胞支持能力的年龄相关性下降 而短期限制蛋氨酸可逆转与年龄相关的RG成纤维细胞的衰退。我们 假设RG成纤维细胞的Ahcy丢失和蛋氨酸积累可以解释一些与年龄相关的 可通过短期饮食蛋氨酸限制逆转的陈旧ISCs缺陷。在这份提案中，我们将测试 假设RG成纤维细胞是体内培养ISCs的主要生态位细胞(目标1)；Ahcy的丢失 通过蛋氨酸循环中间产物的积累导致视网膜成纤维细胞的增龄性衰退 代谢产物(目标2)；短期饮食蛋氨酸限制使衰老的小鼠视网膜成纤维细胞恢复活力 支持ISCs和ISC介导的再生(目标3)。 通过这些目标，我们将对ISC基质生态位的年龄相关变化提供新的见解 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。身份识别 一种可能促进老年人肠道再生的新的饮食干预措施将具有重要的临床意义 这意味着什么。我的目标是发现关于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，以改善患者的健康。因为人们对此知之甚少 ISC生物学中基质龛细胞的衰老和新陈代谢，我开发的新工具和技能集 在K99训练期间，获得评估老化基质细胞新陈代谢的能力将使我能够 在我向独立过渡的过程中，建立一个成功和独立的研究项目。

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.