? BONE ORGAN SPECIFIC PROJECT The bone is a highly complex organ made of three tissue types with distinct functions, including cells from hematopoietic, mesenchymal and endothelial lineages. Confined within one organ and thoroughly mixed with each other, these three tissues are mutually dependent for their development and homeostasis. The spatial organization of the various cell types and their cross-talk in the bone is a major gap of our knowledge. The goal of the bone Organ Specific Project (B-OSP) is to address this knowledge gap and generate high quality, single- cell resolution, longitudinal imaging and multi-omics data of normal bones across the human lifespan. We propose the following three specific aims: 1) To refine protocols for biospecimen processing, multi-omics and imaging assays and define inter-individual variability using our existing banked bone samples. Using existing biobanked normal bones from the autopsy program, we will perform a series of pilot studies to refine our protocols for biospecimen collection and processing, as well as single-cell omics and imaging assays. 2) To procure, archive, and annotate high-quality normal bone samples across human life span. We have established a streamlined procurement and biorepository infrastructure to support our bone OSP. We will procure normal bone from the same donor of 6 age groups across the human life span. 3) To spatially profile bone specimens across the human life span using a set of robust and scalable imaging and single-cell omics assays. We will use micro computed tomography to image every bone we collect at both the macroscale and mesoscale. At the microscale, we will perform single-nucleus RNA sequencing (snRNA-Seq) and single-cell assay for transposase-accessible chromatin using sequencing (scATAC-Seq) of two strategic anatomical sites per bone to determine cell type composition, as well as transcriptional and epigenetic signatures. We will perform multiplexed error-robust fluorescence in situ hybridization (MERFISH, for transcriptomics) and Co- Detection by Indexing (CODEX, for proteomics) analyses.
