Project Summary – Overall Bone marrow produces blood cells whose functions range from oxygen delivery to anti-microbial defense to hemostasis, all originating from hematopoietic stem cells (HSC). To sustain and regulate this process, bone marrow stromal cells form multiple niche microenvironments, each tailored to the needs of a particular developing blood cell population. Using highly-multiplexed imaging technologies, our proposed Bone Marrow Tissue Mapping Center (TMC) aims to systematically and quantitatively dissect the cellular composition and spatial organization of human bone marrow microenvironments. The resulting detailed maps will serve as an open and global platform for understanding which cells and interactions are critical for each branch of hematopoietic maturation, and how these vary by anatomical site and across diverse patient demographics. The TMC will define cellular identities and cell states at the transcriptional, translational, and post- translational levels using Nanostring DSP, Multiplexed Ion Beam Imaging (MIBI), and MALDI-MSI, which generate quantitative spatial maps of RNA, protein, and N-glycans, respectively. Our cross-disciplinary team not only includes the inventors of MIBI and a pioneer of MALDI-MSI, but also experts in human HSCs and human hematopoiesis and a practicing hematopathologist with expertise in histopathologic bone marrow diagnosis. To overcome the unique challenges of working with hard, mineralized bone, we will leverage parallel, robust, clinically-validated bone marrow processing pipelines which maximize and standardize sample quality and compatibility with current and future technologies. Integrating seamlessly into standard clinical workflows, our pipelines enable convenient sharing of prospectively-collected materials with the Tissue Core. Samples will be collected from three different sources: (1) prospective, patient-matched multi-site collection from deceased donors to examine differences between anatomical sites, (2) prospective collection of femoral head from hip arthroplasty specimens for differences between age ranges, (3) iliac crest bone in the Stanford Pathology archive for differences between races and genders. These multiple collection strategies, multiple sites, and different investigational focuses complement prior HuBMAP projects. The Data Analysis Core team has pioneered multiple novel data processing pipelines, including pixel-based analyses, cell-based analyses including state-of- the-art cell segmentation and cell clustering and enumeration, and neighborhood analyses. These tools are broadly-applicable to all highly-multiplexed quantitative imaging technologies. Overall, our team and strategy are exceedingly well-suited for executing the vision of the proposed Bone Marrow TMC. The spatial structure of bone marrow reflects the evolutionary mechanisms that terraformed bone to create unique microenvironments meeting the nutritional needs of developing blood cells with divergent functions. The interdependence between bone marrow tissue structure and hematopoiesis is informative not just in blood cell maturation, but for understanding metabolism, aging, and development of cellular therapies.

Project Narrative – Overall Defining bone marrow cell identities and states using quantitative spatial maps of RNA, protein, and N- glycans, our proposed Bone Marrow Tissue Mapping Center will dissect the cellular composition and spatial structure of bone marrow niches to understand which cells and interactions are critical to each branch of hematopoietic maturation. The multi-arm sample collection strategy, leveraging of clinically-validated pre- analytical pipelines, assay technologies (Nanostring DSP, Multiplexed Ion Beam Imaging, and MALDI-MSI), computational tools (pixel-based analyses, cell-based analyses, neighborhood analyses), and biological principles are broadly applicable to and complement other HuBMAP projects. Interrogating the interdependence between bone marrow tissue structure and hematopoiesis is informative not just in blood cell maturation, but for understanding metabolism, aging, and development of cellular therapies.