Adult mesenchymal progenitor cells have enormous potential for use in regenerative medicine. However, the true identity of the progenitors in vivo and their progeny has not been precisely defined. We hypothesize that cells expressing a smooth muscle α-actin promoter (αSMA) directed Cre transgene represent mesenchymal progenitors of adult bone tissue. By combining complementary colors in combination with transgenes activating at mature stages of the lineage we characterized the phenotype and confirmed the ability of isolated αSMA+ cells to progress from a progenitor to fully mature state. In vivo lineage tracing experiments using a new bone formation model confirmed the osteogenic phenotype of αSMA+ cells. In vitro analysis of the in vivo labeled SMA9+ cells supported their differentiation potential into mesenchymal lineages. Utilizing a fracture-healing model, αSMA+ cells served as a pool of fibrocartilage and skeletal progenitors. Confirmation of the transition of αSMA+ progenitor cells to mature osteoblasts during fracture healing was assessed by activation of bone specific Col2.3emd transgene. Our findings provide a novel in vivo identification of defined population of mesenchymal progenitor cells with active role in bone remodeling and regeneration.

Fracture healing is a regenerative process that involves coordinated responses of many cell types, but characterization of the roles of specific cell populations in this process has been limited. We have identified alpha smooth muscle actin (αSMA) as a marker of a population of mesenchymal progenitor cells in the periosteum that contributes to osteochondral elements during fracture healing. Using a lineage tracing approach, we labeled αSMA-expressing cells, and characterized changes in the periosteal population during the early stages of fracture healing by histology, flow cytometry, and gene expression profiling. In response to fracture, the αSMA-labeled population expanded and began to differentiate toward the osteogenic and chondrogenic lineages. The frequency of mesenchymal progenitor cell markers such as Sca1 and PDGFRα increased after fracture. By 6 days after fracture, genes involved in matrix production and remodeling were elevated. In contrast, genes associated with muscle contraction and Notch signaling were downregulated after fracture. We confirmed that activating Notch signaling in αSMA-labeled cells inhibited differentiation into osteogenic and adipogenic lineages in vitro and ectopic bone formation in vivo. By characterizing changes in a selected αSMA-labeled progenitor cell population during fracture callus formation, we have shown that modulation of Notch signaling may determine osteogenic potential of αSMA-expressing progenitor cells during bone healing.

Unlike during embryogenesis, the identity of tissue resident progenitor cells that contribute to postnatal tendon growth and natural healing is poorly characterized. Therefore, we utilized 1) an inducible Cre driven by alpha smooth muscle actin (SMACreERT2), that identifies mesenchymal progenitors, 2) a constitutively active Cre driven by growth and differentiation factor 5 (GDF5Cre), a critical regulator of joint condensation, in combination with 3) an Ai9 Cre reporter to permanently label SMA9 and GDF5-9 populations and their progeny. In growing mice, SMA9+ cells were found in peritendinous structures and scleraxis-positive (ScxGFP+) cells within the tendon midsubstance and myotendinous junction. The progenitors within the tendon midsubstance were transiently labeled as they displayed a 4-fold expansion from day 2 to day 21 but reduced to baseline levels by day 70. SMA9+ cells were not found within tendon entheses or ligaments in the knee, suggesting a different origin. In contrast to the SMA9 population, GDF5-9+ cells extended from the bone through the enthesis and into a portion of the tendon midsubstance. GDF5-9+ cells were also found throughout the length of the ligaments, indicating a significant variation in the progenitors that contribute to tendons and ligaments. Following tendon injury, SMA9+ paratenon cells were the main contributors to the healing response. SMA9+ cells extended over the defect space at 1 week and differentiated into ScxGFP+ cells at 2 weeks, which coincided with increased collagen signal in the paratenon bridge. Thus, SMA9-labeled cells represent a unique progenitor source that contributes to the tendon midsubstance, paratenon, and myotendinous junction during growth and natural healing, while GDF5 progenitors contribute to tendon enthesis and ligament development. Understanding the mechanisms that regulate the expansion and differentiation of these progenitors may prove crucial to improving future repair strategies.

• Acute myeloid leukemia decreases osteoblast numbers in humans and mice.• Reinstatement of osteoblast number and function in leukemic mice by a pharmacologic approach reduces tumor burden in all sites and prolongs survival.The bone marrow niche is thought to act as a permissive microenvironment required for emergence or progression of hematologic cancers. We hypothesized that osteoblasts, components of the niche involved in hematopoietic stem cell (HSC) function, influence the fate of leukemic blasts. We show that osteoblast numbers decrease by 55% in myelodysplasia and acute myeloid leukemia patients. Further, genetic depletion of osteoblasts in mouse models of acute leukemia increased circulating blasts and tumor engraftment in the marrow and spleen leading to higher tumor burden and shorter survival. Myelopoiesis increased and was coupled with a reduction in B lymphopoiesis and compromised erythropoiesis, suggesting that hematopoietic lineage/progression was altered. Treatment of mice with acute myeloid or lymphoblastic leukemia with a pharmacologic inhibitor of the synthesis of duodenal serotonin, a hormone suppressing osteoblast numbers, inhibited loss of osteoblasts. Maintenance of the osteoblast pool restored normal marrow function, reduced tumor burden, and prolonged survival. Leukemia prevention was attributable to maintenance of osteoblast numbers because inhibition of serotonin receptors alone in leukemic blasts did not affect leukemia progression. These results suggest that osteoblasts play a fundamental role in propagating leukemia in the marrow and may be a therapeutic target to induce hostility of the niche to leukemia blasts. (Blood. 2014;124(18):2834-2846

The in vivo origin of bone-producing osteoblasts is not fully defined. Skeletal stem cells, a population of mesenchymal stem cells resident in the bone marrow compartment, are thought to act as osteoprogenitors during growth and adulthood. Quiescent bone lining cells (BLCs) have been suggested as a population capable of activation into mature osteoblasts. These cells were defined by location and their morphology and studies addressing their significance have been hampered by their inaccessibility, and lack of markers that would allow for their identification and tracing. Using lineage tracing models, we have observed labeled osteoblasts at time points extending beyond the reported lifespan for this cell type, suggesting continuous reactivation of BLCs. BLCs also make a major contribution to bone formation after osteoblast ablation, which includes the ability to proliferate. In contrast, mesenchymal progenitors labeled by Gremlin1 or alpha smooth muscle actin do not contribute to bone formation in this setting. BLC activation is inhibited by glucocorticoids, which represent a well-established cause of osteoporosis. BLCs express cell surface markers characteristic of mesenchymal stem/progenitors that are largely absent in osteoblasts including Sca1 and Leptin Receptor. BLCs also show different gene expression profiles to osteoblasts, including elevated expression of Mmp13, and osteoclast regulators RANKL and macrophage colony stimulating factor, and retain osteogenic potential upon transplantation. Our findings provide evidence that bone lining cells represent a major source of osteoblasts during adulthood.