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

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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.