Single-cell Transcriptomic And Functional Analysis Of Adult Skeletal Stem And Progenitor Cells

Skeletal stem and progenitor cells(SSPCs)play fundemental roles in skeletal development,maintenance and repair.Leptin receptor-positive(Lep R~+)bone marrow stromal cells(BMSCs)are enriched of adult bone marrow SSPCs,which can self-renew and differentiate into osteoblasts and adipocytes under steady state,and give rise to chondrocytes during skeletal repair after injuries.Lep R~+BMSCs mainly localized around bone marrow sinusoids and highly express hematopoietic stem cell(HSC)maintenance factors,thereby acting as an important cellular component of the hematopoietic mivroenvironment.Given that Lep R~+BMSCs play dual roles in skeletal and hematopoietic regulation,dissecting their cellular heterogeneity and functional divergence are of great significance for elucidating mechanisms underlying maintenance and regeneration of the skeletal and hematopoietic systems.In this study,we first combined genetic lineage tracing and single-cell RNA-sequencing(sc RNA-seq)to perform integrated analysis of long bone Prrx1-Cre~+and Lepr-Cre~+cells in adult mice,and found that Lepr-Cre~+cells include the majority of adult SSPCs.Next,we performed sc RNA-seq in long bone Lepr-Cre~+cells under homeostatic and stress conditions(aging,rosiglitazone-diet feeding,sublethal irradiation and femur fracture).After integrated analysis,17,224 single cells were clustered into 4 lineages,including adipogeneic,osteogenic,periosteal and cycling cells.The adipogeneic lineage cells are mainly composed of BMSC subsets that highly express HSC maintenance factors such as Scf and Cxcl12.Interestingly,we found a novel Notch3~+subset closely associated with the bone marrow vasculatures,which is quiescent and expresses the highest level of HSC maintenance factors.This suggests that the interaction between vascular endothelial cells and BMSCs is critical for maintaining the hematopoietic microenvironment.Pseudotime analysis within the osteogenic lineage cells revealed a clear differentiation trajectory from osteogenic progenitors to precursors and mature osteoblasts.By gene regulatory network analysis,we found a series of transcriptiona factors that could promote fracture healing,and validated that Npdc1 and Hoxb2 significantly promote osteogenic and chondrogenic differentiation.Although Lepr-Cre~+periosteal lineange cells are rare under homeostasis,they are quickly expanded after sublethal irradiation and bone fracture.We identified the previously reported Ctsk~+periosteal stem cells(PSCs)within this lineage,and discovered a Sca-1~+subsest that exhibits high clonogenic and adipogenic capacity but limited osteo-chondrogenic capacity,raising the possibility that it could participte in the repair of periosteum after injury.To explore novel periosteal/perichondrial SSPCs in an unbiased way,we performed sc RNA-seq of Prrx1-Cre~+cells in the periosteum/perichondrium of adult long bones,and divided the single cells into 2 categories:1)Stromal cell clusters that highly express Pdgfrαand Procr;2)Differentiated cell clusters that highly express Alpl,Acan or Acta2.Since Procr has been previously shown to label multiple tissue-specific stem and progenitor cells,we hypothesized that it might also label novel SSPCs.In vivo genetic lineage tracing study revealed that Procr labels periosteal stromal cells in adult long bones,which could play an important role in bone development and remodeling.In addition,Procr labels superficial cells in the articular cartilage and meniscus,which can give rise to chondrocytes during skeletal development.We excluded the contribution of Procr~+endothelial and hematopoietic cells to articular chondrocytes by dual recombinase lineage tracing.In conclusion,this study dissected the heterogeneity and lineage hierarchy of bone marrow SSPCs under homeostatic and stress conditions,and uncovered the functional divergence of different cell clusters in terms of HSC maintenance and skeletal regulation,which shed new light on the stem cell regulation of skeletal and hematopoietic systems and their cross-talk mechanisms.We also dissected the heterogeneity of periosteal SSPCs and discovered a novel Procr~+articular chondrocyte progenitor population,which provide new theory and strategy for regenerating articular cartilage and meniscus after injuries.