Unraveling the cell networks in bone marrow through transcriptomic and epigenetic profiling

Project Summary Bone marrow is a highly complex and heterogeneous environment, composed of hematopoietic, mesenchymal, nervous and endothelial tissues. It is the site of a wide range of diseases including blood and immune disease (leukemia, myelodysplastic syndromes, aplastic anemia, myelofibrosis), skeletal disease (osteoporosis, osteopetrosis, Paget’s disease), vascular disease (avascular necrosis, ischemic and hypertension-related bone disease), and cancer metastasis (breast, prostate, thyroid). There is a great appreciation for the role of mesenchymal cells in orchestrating bone remodeling, angiogenesis and hematopoiesis – functions that are critical to maintaining health and that deteriorate with aging – but there is little knowledge on how this population is maintained or how it helps regulate the cellular diversity in bone marrow. Transcriptomic data has started to shed light on bone marrow cell regulation, but since bone marrow is primarily composed of hematopoietic cells, current data, particularly from humans, lack a holistic representation of all bone marrow cell types. Additionally, there is a lack of human data regarding how mesenchymal stem and progenitor cells are maintained, how they repopulate the bone marrow stroma and which mesenchymal cells are critical to each stage of hematopoiesis. This project seeks to achieve two interconnected goals: generate a complete single-cell transcriptomic and regulomic map of human bone marrow cells and experimentally establish the mesenchymal hierarchy in human bone. In order to accomplish this, we developed a protocol to isolate single-cell suspensions from human femoral head tissue, enrich for rare or difficult to isolate cell populations (i.e. mesenchymal cells, adipocytes, hematopoietic stem and progenitor cells) and isolate single-nuclei for simultaneous transcriptomic and epigenomic sequencing. This information will provide insight into how cell fates are regulated and what cell-cell interactions support cell fate decisions. Of particular interest is what epigenomic changes determine mesenchymal cell fate and what epigenomic states of hematopoietic cells make them more amenable cell regulation be one mesenchymal cell over another based on transcriptomic signatures. Transcriptomic and epigenomic studies will complement in vivo explant and in vitro differentiation studies to determine mesenchymal lineage differentiation patterns. By performing flow sorting for four progenitor cells, the studies will define the differentiation potential and position each cell on the step-wise differentiation pathway to osteogenesis and adipogenesis. This will provide insight into how mesenchymal cell fate is regulated and what cell types are required for hematopoietic recruitment and maintenance. By unraveling the complex bone marrow cellular signaling networks, the long-term goal is to develop novel therapies for osteoporosis and blood disease and provide the research community with a rich dataset to catalyze further discoveries.