Delineating the Role of Notch Signaling in Maintaining Bone Marrow Mesenchymal Progenitors

背景技术背景：骨髓（BM）龛通过细胞-细胞相互作用和可溶性因子的分泌维持所有造血祖细胞，包括造血干细胞（HSC）。间充质基质祖细胞（MSPC）是骨髓小生境的重要组成部分，通过分泌干细胞因子1（SCF 1）、C-X-C基序趋化因子12（CXCL 12）和多效生长因子维持HSC。除了它们在支持造血中的作用之外，MSCPs还产生各种中胚层细胞群，包括成骨细胞和脂肪细胞。为了研究BM MSPC的分子异质性，我们先前对BM MSPC进行了单细胞RNA测序（scRNA-seq）。我们的分析揭示了两个重要的观察结果。首先，它表明存在两个关键的转录上不同的BM MSPC子集：脂肪启动的和骨启动的。其次，脂肪致敏的MSPC表达高水平的关键促造血因子，包括IL-7、IL-15、IL-34和M-CSF。此外，我们发现BM MSPC表达上调水平的Notch 3受体，并富集Notch信号通路。为了在功能上测试Notch信号传导在间充质祖细胞中的作用，我们通过缺失在BM MSPC（LeprCRE-NcstnFL/FL）中加工成熟Notch蛋白中起重要作用的Nicastrin（Ncstrin）来破坏Notch信号传导。我们发现Notch信号传导的损失增强了MSPC的克隆形成潜力和分化。基于我们的初步数据，我们假设Notch信号通过抑制MSPC分化为成骨细胞和脂肪细胞来维持BM稳态。我们将在以下目标中检验这一假设。具体目的：1）评估Notch信号丢失后MSPC中分子通路的变化。 2)确定MSPC中Notch信号传导在支持造血中的作用。重要性：半个世纪前发现的MSPCs具有巨大的生物工程潜力，因为它们具有再生受损和患病骨骼并支持造血的能力。MSPC在体外维持和扩增具有挑战性，因为它们很快失去自我更新的能力。此外，BM MSPC支持HSC的能力在离体培养后迅速丧失。体内控制BM MSPC静止和分化的分子机制还不清楚。在这里，我们建议配对先进的基因组方法与遗传操作的BM MSPCs在体内，以确定分子机制驱动BM MSPCs的维护和功能。总的来说，这项工作将对（i）骨再生和（ii）造血干细胞研究产生重要影响。

BACKGROUND: The bone marrow (BM) niche maintains all hematopoietic progenitor cells, including hematopoietic stem cells (HSCs), through cell-cell interactions and the secretion of soluble factors. Mesenchymal stromal progenitor cells (MSPCs) comprise a significant component of the BM niche, maintaining HSCs through the secretion of stem cell factor 1 (SCF1), C-X-C motif chemokine 12 (CXCL12) and pleiotrophin. In addition to their role in supporting hematopoiesis, MSCPs give rise to various mesodermal cell populations, including osteoblasts and adipocytes. To investigate the molecular heterogeneity of BM MSPCs, we previously performed single-cell RNA sequencing (scRNA-seq) on BM MSPCs. Our analysis revealed tow important observations. First, it showed that there are two key transcriptionally distinct subsets of BM MSPCs: adipo- and osteo-primed. Second, adipo-primed MSPCs expressed high levels of key pro-hematopoietic factors, including IL-7, IL-15, IL-34, and M-CSF. Furthermore, we found that BM MSPCs expressed upregulated levels of the Notch3 receptor and are enriched for the Notch signaling pathway. To functionally test the role of Notch signaling in mesenchymal progenitors, we disrupted Notch signaling by deleting Nicastrin (Ncstn) that plays a significant role in processing mature Notch protein in the BM MSPCs (LeprCRE-NcstnFL/FL). We found that the resulting loss of Notch signaling enhances MSPC clonogenic potential and differentiation. Based on our preliminary data, we hypothesize that Notch signaling maintains BM homeostasis by suppressing MSPC differentiation into osteoblasts and adipocytes. We will test this hypothesis in the following aims. SPECIFIC AIMS: 1) To evaluate molecular pathways changes in MSPCs following Notch signaling loss. 2) To determine the role of Notch signaling in MSPCs in supporting hematopoiesis. SIGNIFICANCE: Identified half a century ago, MSPCs hold enormous bioengineering potential, as they possess the ability to both regenerate damaged and diseased bone and support hematopoiesis. MSPCs are challenging to maintain and expand in vitro, as they quickly lose their ability to self-renew. Furthermore, BM MSPC's ability to support HSCs is quickly lost upon ex vivo culture. The molecular mechanisms controlling BM MSPC quiescence and differentiation in vivo are not well understood. Here, we propose to pair advanced genomic approaches with genetic manipulations of BM MSPCs in vivo to identify molecular mechanisms driving BM MSPCs maintenance and function. Collectively, this work will have important implications for (i) bone regeneration and (ii) hematopoietic stem cell research.