Single cell sequencing shows that Sox9-expressing cells populate most mesenchymal lineages postnatally in mouse bone

In growing bones of mice, multiple cell types contribute to the osteoblast lineage, including growth plate chondrocytes, perichondrial cells and CXCL12-abundant reticular (CAR) marrow stromal cells. Here we use single cell RNA sequencing and lineage tracing to show that all these osteoblast precursors, even postnatally, derive from Sox9-expressing progenitors. We also characterize a distinct group of chondrocytes between the perichondrium and the columns of growth plate chondrocytes that contribute to the osteoblast lineage. Overall design: Sox9-CreERT2;tdTomato transgenic mice received a single, 2mg dose of tamoxifen. At days 2, 7, 13 and 35 post-tamoxifen administration, Isolated hindlimbs were mechanically crushed into 2mL of Hank's Balanced Salt Solution (Sigma Aldrich, H6648). Supernatant was filtered through a 70µm filter (Corning, 352350) into a 50mL Falcon tube. The remaining tissue was further incubated with 2 Wunsch units of LiberaseTM (Sigma Aldrich, 5401127001) and 0.25% Trypsin-EDTA (Fisher Scientific, 15400054) at 37°C for 30 min with agitation. The supernatant was again filtered through a 70µm filter into a 50mL Falcon tube. Cells were then stained with CD45-APC (ThermoFisher, MCD4505), Ter119-APC (ThermoFisher, 17-5921-82) and DAPI (Invitrogen, D1306) for 30min on ice. Cells were washed with FACS Buffer (Phosphate Buffered Solution plus 2% fetal bovine serum). Flow cytometry and sorting was performed with a 4-laser BD LSR II and FACSDivia. TdTomato+/DAPI-/APC- cells were sorted into FACS Buffer.