A framework for defining mesenchymal cell types associated with murine periosteal and endosteal bone

Single-cell RNA sequencing has led to numerous novel designations for mesenchymal cell types associated with bone. Consequently, there are now multiple designations for what appear to be the same cell type. In addition, existing datasets contain relatively small numbers of mature osteoblasts and osteocytes and there has been no comparison of periosteal bone cells to those at the endosteum and trabecular bone. The main goals of this study were to increase the amount of single cell RNA sequence data for osteoblasts and osteocytes, to compare cells from the periosteum to those inside bone, and to clarify the major categories of cell types associated with murine bone. To do this, we created an atlas of murine bone-associated cells by harmonizing published datasets with in-house data from cells targeted by Osx1-Cre and Dmp1-Cre driver strains. Cells from periosteal bone were analyzed separately from those isolated from the endosteum and trabecular bone. Over 100,000 mesenchymal cells were mapped to reveal 11 major clusters designated fibro-1, fibro-2, chondrocytes, articular chondrocytes, tenocytes, adipo-CAR, osteo-CAR, pre-osteoblasts, osteoblasts, osteocytes, and osteo-X, the latter defined in part by Postn expression. Osteo-X, osteo-CAR, and pre-osteoblasts were closely associated with osteoblasts at the trabecular bone surface. Wnt16 was expressed in multiple cell types from the periosteum but not in any cells from endocortical or cancellous bone. Fibro-2 cells, which express markers of skeletal stem cells, localized to the periosteum but not trabecular bone in adult mice. Suppressing bone remodeling eliminated osteoblasts and altered gene expression in pre-osteoblasts but did not change the abundance or location of osteo-X or osteo-CAR cells. These results provide a framework for identifying bone cell types in murine single cell RNA sequencing datasets and suggest that osteoblast progenitors reside near the surface of remodeling bone. Author Summary The skeleton of vertebrate animals is produced by bone forming cells known as osteoblasts. New osteoblasts are continually produced throughout life to maintain the skeleton. However, the identity of the cells from which new osteoblasts are derived is not clear. Single cell RNA sequencing provides a gene expression profile of individual cells and allows them to be grouped into clusters of cells with similar profiles. In many cases, distinct clusters represent distinct cell types. Recent single cell RNA sequencing studies of mouse bones by several different laboratories have identified many novel cell types, some of which may be osteoblast progenitors. In many cases, different laboratories have created different names for what appear to be the same cell type. To help clarify this situation, we performed single cell RNA sequencing studies and combined our results with those from several published studies to create a harmonized map of the cell types associated with mouse bones. We also identified gene signatures for each cell type that can be used to improve the consistency of cell type designation in future single cell RNA sequencing studies of mouse bone.