Spatial transcriptomic interrogation of the murine bone marrow signaling landscape

Self-renewal and differentiation of skeletal stem and progenitor cells(SSPCs)are tightly regulated processes,with SSPC dysregulation leading to progressive bone disease.While the application of single-cell RNA sequencing(scRNAseq)to the bone field has led to major advancements in our understanding of SSPC heterogeneity,stem cells are tightly regulated by their neighboring cells which comprise the bone marrow niche.However,unbiased interrogation of these cells at the transcriptional level within their native niche environment has been challenging.Here,we combined spatial transcriptomics and scRNAseq using a predictive modeling pipeline derived from multiple deconvolution packages in adult mouse femurs to provide an endogenous,in vivo context of SSPCs within the niche.This combined approach localized SSPC subtypes to specific regions of the bone and identified cellular components and signaling networks utilized within the niche.Furthermore,the use of spatial transcriptomics allowed us to identify spatially restricted activation of metabolic and major morphogenetic signaling gradients derived from the vasculature and bone surfaces that establish microdomains within the marrow cavity.Overall,we demonstrate,for the first time,the feasibility of applying spatial transcriptomics to fully mineralized tissue and present a combined spatial and single-cell transcriptomic approach to define the cellular components of the stem cell niche,identify cell-cell communication,and ultimately gain a comprehensive understanding of local and global SSPC regulatory networks within calcified tissue.

Neuron-to-vessel signaling is a required feature of aberrant stem cell commitment after soft tissue trauma

The functional interdependence of nerves and blood vessels is a well-established concept during tissue morphogenesis, yet the role of neurovascular coupling in proper and aberrant tissue repair is an emerging field of interest. Here, we sought to define the regulatory relationship of peripheral nerves on vasculature in a severe extremity trauma model in mice, which results in aberrant cell fate and heterotopic ossification(HO). First, a high spatial degree of neurovascular congruency was observed to exist within extremity injury associated heterotopic ossification. Vascular and perivascular cells demonstrate characteristic responses to injury,as assessed by single cell RNA sequencing. This vascular response to injury was blunted in neurectomized mice, including a decrease in endothelial proliferation and type H vessel formation, and a downregulation of key transcriptional networks associated with angiogenesis. Independent mechanisms to chemically or genetically inhibit axonal ingrowth led to similar deficits in HO site angiogenesis, a reduction in type H vessels, and heterotopic bone formation. Finally, a combination of single cell transcriptomic approaches within the dorsal root ganglia identified key neural-derived angiogenic paracrine factors that may mediate neuron-to-vascular signaling in HO. These data provide further understanding of nerve-to-vessel crosstalk in traumatized soft tissues, which may reflect a key determinant of mesenchymal progenitor cell fate after injury.

PDGFRαreporter activity identifies periosteal progenitor cells critical for bone formation and fracture repair

The outer coverings of the skeleton,which is also known as the periosteum,are arranged in concentric layers and act as a reservoir for tissue-specific bone progenitors.The cellular heterogeneity within this tissue depot is being increasingly recognized.Here,inducible PDGFRαreporter animals were found to mark a population of cells within the periosteum that act as a stem cell reservoir for periosteal appositional bone formation and fracture repair.During these processes,PDGFRαreporter^(+)progenitors give rise to Nestin+periosteal cells before becoming osteoblasts and osteocytes.The diphtheria toxin-mediated ablation of PDGFRαreporter^(+)cells led to deficits in cortical bone formation during homeostasis and a diminutive hard callus during fracture repair.After ossicle transplantation,both mouse PDGFRαreporter^(+)periosteal cells and human Pdgfrα+periosteal progenitors expand,ossify,and recruit marrow to a greater extent than their counterpart periosteal cells,whereas PDGFRαreporter^(−)periosteal cells exhibit a predisposition to chondrogenesis in vitro.Total RNA sequencing identified enrichment of the secreted factors Fermt3 and Ptpn6 within PDGFRαreporter^(+)periosteal cells,which partly underlie the osteoblastogenic features of this cell population.

Comparison of skeletal and soft tissue pericytes identifies CXCR4+ bone forming mural cells in human tissues

Human osteogenic progenitors are not precisely defined,being primarily studied as heterogeneous multipotent cell populations and termed mesenchymal stem cells(MSCs).Notably,select human pericytes can develop into bone-forming osteoblasts.Here,we sought to define the differentiation potential of CD146 f human pericytes from skeletal and soft tissue sources,with the underlying goal of defining cell surface markers that typify an osteoblastogenic pericyte.CD146+CD31~CD45_pericytes were derived by fluorescence-activated cell sorting from human periosteum,adipose,or dermal tissue.Periosteal CD146+CD31—CD45 cells retained canonical features of pericytes/MSC.Periosteal pericytes demonstrated a striking tendency to undergo osteoblastogenesis in vitro and skeletogenesis in vivo,while soft tissue pericytes did not readily.Transcriptome analysis revealed higher CXCR4 signaling among periosteal pericytes in comparison to their soft tissue counterparts,and CXCR4 chemical inhibition abrogated ectopic ossification by periosteal pericytes.Conversely,enrichment of CXCR4+pericytes or stromal cells identified an osteoblastic/non-adipocytic precursor cell.In sum,human skeletal and soft tissue pericytes differ in their basal abilities to form bone.Diversity exists in soft tissue pericytes,however,and CXCR4+pericytes represent an osteoblastogenic,non-adipocytic cell precursor.Indeed,enrichment for CXCR4-expressing stromal cells is a potential new tactic for skeletal tissue engineering.