NOTCH2 Promotes Osteoclast Maturation and Metabolism and Modulates the Transcriptome Profile During Osteoclastogenesis

Bone marrow derived macrophages (BMM) from Notch2tm1.1Ecan, harboring a NOTCH2 gain-of-function mutation, and control mice were cultured with macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL). Bulk RNA-Seq revealed enhanced cell metabolism, aerobic respiration, and mitochondrial function, all associated with osteoclastogenesis, in Notch2tm1.1Ecan cells. Single cell RNA-Seq data of BMMs treated with M-CSF or M-CSF and RANKL for 3 days identified 11 well-defined cellular clusters. There was an increased number of cells expressing gene markers associated with the osteoclast and with related clusters in Notch2tm1.1Ecan than in control BMMs. Overall design: To obtain BMMs, the bone marrow from 7 to 8-week-old experimental and control sex-matched littermate mice was removed by flushing. Cells were cultured in a-minimum essential medium (a-MEM) in the presence of 10% fetal bovine serum and M-CSF at 30 ng/ml on uncoated plastic petri dishes, grown, collected and seeded on tissue culture plates in the presence of M-CSF and RANKL. For bulk RNA-Seq, cultures were carried out until multinucleated tartrate resistant acid phosphotase (TRAP)-positive cells were formed. For scRNA-Seq, BMMS were seeded on culture plates coated with Cellmatrix Type I-A Collagen, grown in a-MEM in the presence of either M-CSF at 30 ng/ml alone or M-CSF and RANK-L at 10 ng/ml for 3 days. The collagen gel was digested and cells collected and processed for scRNA-Seq.