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

背景：骨髓（BM）生态位通过细胞间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）。间充质间质祖细胞（MSPCs）是骨髓生态位的重要组成部分，通过分泌干细胞因子1 （SCF1）、C-X-C基序趋化因子12 （CXCL12）和多营养因子来维持造血干细胞。除了支持造血功能外，mscs还能产生各种中胚层细胞群，包括成骨细胞和脂肪细胞。为了研究骨髓间质干细胞的分子异质性，我们之前对骨髓间质干细胞进行了单细胞RNA测序（scRNA-seq）。我们的分析揭示了两个重要的观察结果。首先，它表明骨髓间质干细胞有两个关键的转录不同的亚群：脂肪和骨引物。其次，脂肪引物MSPCs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 MSPCs表达Notch3受体水平上调，并且Notch信号通路富集。为了从功能上测试Notch信号在间充质祖细胞中的作用，我们通过删除Nicastrin （Ncstn）来破坏Notch信号，Ncstn在BM MSPCs （LeprCRE-NcstnFL/FL）成熟Notch蛋白的加工中起重要作用。我们发现Notch信号的缺失增强了MSPC的克隆潜能和分化。根据我们的初步数据，我们假设Notch信号通过抑制MSPC向成骨细胞和脂肪细胞的分化来维持骨髓内稳态。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MSPCs分子通路的变化。2)确定Notch信号在MSPCs中支持造血的作用。意义：MSPCs在半个世纪前被发现，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和支持造血的能力。MSPCs在体外维持和扩展是具有挑战性的，因为它们很快就会失去自我更新的能力。此外，BM MSPC支持hsc的能力在离体培养中迅速丧失。在体内控制bmmspc静止和分化的分子机制尚不清楚。在这里，我们建议将先进的基因组方法与BM MSPCs的体内遗传操作相结合，以确定驱动BM MSPCs维持和功能的分子机制。总的来说，这项工作将对(i)骨再生和（ii）造血干细胞研究具有重要意义。

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.