Integration Analysis Of Single Cell RNA Sequencing From Human Primary Bone Marrow Mesenchymal Stem Cells And Osteoblasts

Osteoporosis is one of the most common systemic and metabolic bone diseases.It is characterized by decreased bone mass and changes in bone tissue structure,leading to decreased bone density and increased risk of fragility fractures.As the main organ supporting the body,bones maintain mechanical properties and calcium homeostasis in the body through continuous remodeling.The process of bone remodeling is mainly undertaken by bone-forming osteoblasts and bone-resorbed osteoclasts.Osteoblasts are derived from bone marrow mesenchymal stem cells(BMMSCs).In addition to osteoblasts,bone marrow mesenchymal stem cells can also differentiate into adipocytes and chondrocytes.Under normal physiological conditions,bones will remain in a balanced state of bone formation and bone resorption.When the body is in various pathophysiological conditions such as obesity,diabetes and aging,the bone remodeling process will be out of balance and lead to bone diseases.The current clinical drug treatment methods mainly use the principle of inhibiting the bone resorption of osteoclasts and reducing the degree of bone loss,but there are still certain limitations.In this study,we focused on bone-forming osteoblasts and bone marrow mesenchymal stem cells.We used single-cell transcriptome sequencing(scRNA-seq)technology and bioinformatics to integrate human primary bone marrow mesenchymal stem cells and osteoblasts,to identify the expression changes of key transcription factors during osteogenesis,and analyze the biological processes and signal pathways enriched in the osteogenic cells.The study verified the known such as BMMSCs clusters and osteoblast clusters BMMSC1,BMMSC2,Pre OB1,Immat OB and Mat OB,and defined two new osteoblast clusters,Pre OB2 and Pre OB3.Among them,the Pre OB2 cluster highly expressed the NR4A1 and NR4A2 genes,which could inhibit the formation of osteoclasts.The Pre OB3 cluster,which highly expressed ATF3,CCL2,CXCL2 and IRF1 genes,had the function of inhibiting osteoblast differentiation,maintaining cells in a poorly differentiated state and recruiting osteoclasts.Meanwhile,it was found that the cellular communication between BMMSCs and osteoblasts was widespread at the single-cell level,especially mature osteoblasts had a regulatory effect on all stages of the process of osteogenesis.In the protein-protein interaction network analysis,we found a sub-network of FZD1,WIF1 and SFRP4 interaction.FZD1 was highly expressed in BMMSCs cluster BMMSC1.WIF1 and SFRP4,which were highly expressed in cluster Mat OB,have been shown to inhibit osteoblast differentiation in previous studies.Combined with PPI analysis,we speculated that WIF1 and SFRP4 inhibited the binding of FZD1 and Wnt ligand in bone marrow mesenchymal stem cells,thereby further inhibiting the regulation of osteoblast differentiation.This study revealed the development trajectory of osteoblasts,and provided a more systematic and comprehensive understanding of the heterogeneity of skeletal cells and the interaction between BMMSCs and osteoblasts.