Sox9 positive periosteal cells in repair of adult long bone fractures. Mus musculus

The phases of fracture healing have been well characterized. However, the exact source and genetic profile of the skeletal progenitors that participate in bone repair is somewhat unclear. Sox9 expression in skeletal elements precedes bone and cartilage formation and a Sox9+ cell type is retained in the adult periosteum. We hypothesized that Sox9+ periosteal cells are multipotent skeletal progenitors normally participating in fracture repair. To test this hypothesis we used tamoxifen (TM)-mediated lineage tracing of Sox9+ cells in Sox9CreErt2:Td-Tomato mice. TM injection indelibly labels Sox9+ cells and all their descendants with the fluorescent reporter protein Td-Tomato. Intact mouse femora were harvested 2 weeks after TM injection and analyzed by histology and immunostaining and RNA sequencing, to evaluate the skeletal distribution and gene expression profile of Td-Tomato positive cells in the adult femur. To assess the role of these cells in fracture repair, mice underwent a closed mid-diaphyseal femoral fracture and their hind limbs were harvested at 3, 9 or 56 days post-fracture to assess the contribution of Sox9+ and Td-tomato+ cells in the fracture healing process. In the intact adult mouse femur, Td-Tomato-labelled cells were observed in articular and growth plate cartilage where Sox9 is known to be expressed, but also in the primary spongiosa, periosteum, and endosteum. RNA sequencing and subsequent analysis showed that Td-Tomato positive periosteal cells were enriched in Sox9 transcripts, and transcripts for various osteogenic and chondrocyte specific genes. In a femoral fracture model, we showed that pre-labeled Td-Tomato positive descendent cells were mobilized during the fracture repair process, expanding and migrating towards the fracture site 3 days post-fracture. Here, Td-Tomato positive cells differentiate into chondrocytes and osteoblasts in the soft and hard callus, respectively, 9 days post-fracture. By 2 months post-fracture, descendants of the original Sox9 labelled cell population were prevalent in the cortex and periosteum, and amongst the differentiated osteocyte population embedded within the cortical bone. Thus, a Sox9+ progenitor population resides in the adult periosteum. Fate tracing shows that these cells likely play a substantial role in repair of the fractured femur giving rise to chondrocytes, osteoblasts and mature cortical osteocytes. To our knowledge this is the first report of this Sox9+ cell population in the periosteum of the adult long bone. Taken together with developmental studies on skeletal formation, our data suggest that Sox9+ osteochondroprogenitors play a continuous role in skeletal formation throughout life. Overall design: Double heterozygous mice, carrying one allele of Sox9CreErt2 driver and one allele of Td-Tomato reporter, were generated by crossing heterozygous Sox9CreErt2 (Sox9tm1(cre/ERT2)Haak) with homozygous Td-Tomato (Gt(ROSA)26Sortm14(CAG-tdTomato)Hze) mice. The mice received 3 intraperitoneal injections of 100µL tamoxifen (TM) solution (20 mg/mL diluted in corn oil), at 48-hour intervals. Intact mouse femora were harvested 2 weeks after the last tamoxifen injection and analyzed with RNA sequencing to determine the gene expression profile of periosteal cells of the femur. Femoral diaphyses were digested in a liberase solution (100ug/mL in PBS) for 2 hours in a fast shaking rocker at 37 oC, 100 rpm to obtain diaphyseal periosteum cells. Digested cells were filtered and single cell suspensions were subjected to FACS sorting to collect Td-Tomato positive and negative cells. About 10,000 Td-Tomato positive cells and 10,000 Td-Tomato negative control cells were isolated after two collections from 18 femora (9 mice). Total RNA was extracted from each cell population separately with Qiagen RNAasy micro kit with DNaseI digestion according to the manufacturer’s instructions. Duplicated experimental RNA sequencing libraries were made with a SMARTer Universal Low Input RNA Kit and 75 nucleotide paired-end sequencing performed on an Illumina Nextseq 500 sequencer in USC’s Epigenomics Core facility. Sequence reads were mapped by the Star aligner and quantified within the Partek Genomics Suite software (http://www.partek.com) to identify differentially expressed genes. The resulting gene list was uploaded to DAVID GO analysis tool (https://david.ncifcrf.gov/) to extract the enriched gene ontology terms of TdTomato positive periosteal cells.