Identification Of Human Primary Osteoblast Subtypes At Single Cell Resolution

Osteoporosis is a bone disease that seriously threatens human health.Its pathogenesis is mainly caused by the metabolic imbalance between bone formation and bone resorption during bone remodeling.Osteoblasts are multifunctional bone forming cells,which are mainly responsible for new bone formation during bone remodeling.Several potential osteoblast subtypes have been identified in previous studies in mice,but the identification of human primary osteoblast subtypes has not yet been explored.Therefore,this study uses single-cell RNA sequencing（sc RNA-seq）technology to explore the subtype characteristics of human primary osteoblasts at a single cell level.In this study,we collected a sample of the hip joint of a 31-year-old Chinese man,and used the fluorescence-activated cell sorting method to separate and enrich human primary osteoblasts.We first used sc RNA-seq technology for transcriptome sequencing,and used the sc Impute method to accurately and reliably impute the missing values of missing genes in the osteoblast sc RNA-seq dataset,thereby obtaining the transcription of7656 osteoblasts.In which,on average,each cell expresses 6659 genes,and it is found that compared with the un-imputed original data,the number of osteoblasts in the imputed data has increased by about 24%.Then,through data dimensionality reduction and visualization,and cell clustering analysis,we have identified six different cell subpopulations,including osteoblast progenitors,pre-osteoblasts,mature osteoblasts and three undetermined rare osteoblast subpopulations(undetermined osteoblasts 1(IGFBP2^high/LOXL1^high),undetermined osteoblasts 2(NR4A1^high/NR4A2^high),and undetermined osteoblasts 3(ATF3^high/NAMPT^high)).Combined with gene expression patterns and the reconstruction of cell developmental lineages,it is found that these six osteoblast subgroups are in one cell developmental lineage,and only mature osteoblast was a terminal differentiation cell subtype.Finally,based on functional enrichment analysis and differentially expressed genes analysis of each subgroup,it was found that in addition to bone formation,six osteoblast subsets were involved in maintaining hematopoietic niche homeostasis and regulating hematopoiesis by expressing CXCL12,GAS6 and ZFP36;undetermined osteoblasts 1 and3 express ATF3,CCL2,CXCL2,NAMPT,IGFBP2,HP and LBP at a high level,so these two subpopulations may have immunomodulatory function and participate in the development of osteoarthritis and other diseases;undetermined osteoblasts 2 and 3 are involved in regulating the differentiation of osteoclasts and adipocytes through NR4A1,NR4A2,fosl2,KLF4 and SOCS1.They may affect the occurrence of osteoporosis by regulating the balance and relative proportions of adipocytes and osteoclasts in vivo.In this study,we identified six human primary osteoblast subtypes and their developmental trajectories at the single cell level,revealed the different functions of osteoblast subsets,and provide new insights for bone physiology,and provide a crucial theoretical basis for the realization of"precision treatment"of osteoporosis.