Discovery of a periosteal stem cell mediating intramembranous bone formation

Abstract

Bone is comprised of separate inner endosteal and outer periosteal compartments, each with distinct contributions to bone physiology and each maintaining separate pools of cells due to physical separation by the bone cortex. While the skeletal stem cell giving rise to endosteal osteoblasts has been extensively studied, the identification of a periosteal stem cell has been elusive 1 – 5 . Here, we identify a periosteal stem cell (PSC) present in the long bones and calvarium of mice that displays clonal multipotency, self-renewal and sits at the apex of a differentiation hierarchy. Single cell and bulk transcriptional profiling show that PSCs display distinct transcriptional signatures in comparison with both other skeletal stem cells and mature mesenchymal cells. While other skeletal stem cells form bone via an initial cartilage template using the endochondral pathway 4 , PSCs form bone via a direct intramembranous route, providing a cellular basis for the divergence between intramembranous versus endochondral developmental pathways. However there is plasticity in this division, as PSCs acquire endochondral bone formation capacity in response to injury. Genetic blockade of the ability of PSCs to give rise to bone-forming osteoblasts results in selective impairments in cortical bone architecture and defects in fracture healing. A cell analogous to PSCs is present in the human periosteum, raising the possibility that PSCs are attractive targets for drug and cellular therapy for skeletal disorders. Moreover, the identification of PSCs provides evidence that bone contains multiple pools of stem cells, each with distinct physiologic functions.

The Unmixing Problem: A Guide to Applying Single-Cell RNA Sequencing to Bone

“…In keeping with this, comparison of in vivo transcriptional profiles on fixed cells to cells undergoing a typical isolation protocol in muscle suggests that enzymatic digestion is the major step that can disrupt the in vivo transcriptional profile . This emphasizes the importance of minimizing the duration and harshness of enzymatic digestion, and we note that brief digestion protocols can provide robust yields of mesenchymal cells, particularly in younger mice . Alternatively, there are several approaches designed to circumvent isolation‐associated artifacts, including in vivo fixation before cell isolation, though fixation can negatively impact cell isolation efficiency and RNA quality .…”

“…Consideration of these points is needed to ensure that the population of interest will be sufficiently represented to allow for robust downstream analysis. Wherever feasible, performing scRNA‐seq on cells isolated directly from bone without intervening culture is advised, as certain stem cell and progenitor populations may be lost upon culture, even when “basal” culture conditions are utilized . An important decision when planning the cell isolation is whether cells will be subjected to FACS before single‐cell sequencing or if cells will be directly utilized after enzymatic digestion.…”

“…Endothelial cells can be depleted based on negative selection on the pan‐endothelial marker CD31 (gene symbol PECAM1). Notably, capture of some number of unwanted cell populations is inevitable even with FACS and these cells must be accounted for during analysis . Additionally, use of cell type–specific fluorescent reporters, such as a GFP variant driven by a reporter active in osteoblasts or cre‐based lineage tracing methods, can allow for positive selection of populations of interest.…”

“…Disadvantages of adding a pre‐sequencing FACS step include the additional time and experimental complexity added. Additionally, FACS itself can have a negative effect on cell viability, though optimization of nozzle sizes and flow rates can minimize these effects; a larger nozzle size with a slower flow rate generally offers a better outcome . Regardless of whether FACS is used, optimization of enzymatic digestion conditions for cell viability and yield is critical, as in our experience this represents the most common point of experimental failure in scRNA‐seq studies.…”

“…Moreover, unappreciated differences in the cellular composition of heterogeneous skeletal mesenchymal cultures are likely to be a major contributor to problems with experimental reproducibility among labs . Some of these issues potentially extend to in vivo studies using single markers to identify populations of interest, as many available markers capture not one but multiple cell populations . In addition to these methodological factors that confound progress in understanding the cellular basis of bone formation, mesenchymal biology has inherent features that make resolving discrete populations of mesenchymal cells and determining their hierarchy challenging.…”