Csf1 from marrow adipogenic precursors is required for osteoclast formation and hematopoiesis in bone.

Colony-stimulating factor 1 (Csf1) is an essential growth factor for osteoclast progenitors and an important regulator for bone resorption. It remains elusive which mesenchymal cells synthesize Csf1 to stimulate osteoclastogenesis. We recently identified a novel mesenchymal cell population, marrow adipogenic lineage precursors (MALPs), in bone. Compared to other mesenchymal subpopulations, MALPs expressed Csf1 at a much higher level and this expression was further increased during aging. To investigate its role, we constructed MALP-deficient Csf1 CKO mice using AdipoqCre. These mice had increased femoral trabecular bone mass, but their cortical bone appeared normal. In comparison, depletion of Csf1 in the entire mesenchymal lineage using Prrx1Cre led to a more striking high bone mass phenotype, suggesting that additional mesenchymal subpopulations secrete Csf1. TRAP staining revealed diminished osteoclasts in the femoral secondary spongiosa region of Csf1 CKOAdipoq mice, but not at the chondral-osseous junction nor at the endosteal surface of cortical bone. Moreover, Csf1 CKOAdipoq mice were resistant to LPS-induced calvarial osteolysis. Bone marrow cellularity, hematopoietic progenitors, and macrophages were also reduced in these mice. Taken together, our studies demonstrate that MALPs synthesize Csf1 to control bone remodeling and hematopoiesis.

YAP and TAZ couple osteoblast precursor mobilization to angiogenesis and mechanoregulated bone development.

Endochondral ossification requires coordinated mobilization of osteoblast precursors with blood vessels. During adult bone homeostasis, vessel adjacent osteoblast precursors respond to and are maintained by mechanical stimuli; however, the mechanisms by which these cells mobilize and respond to mechanical cues during embryonic development are unknown. Previously, we found that deletion of the mechanoresponsive transcriptional regulators, YAP and TAZ, from Osterix-expressing osteoblast precursors and their progeny caused perinatal lethality. Here, we show that embryonic YAP/TAZ signaling couples vessel-associated osteoblast precursor mobilization to angiogenesis in developing long bones. Osterix-conditional YAP/TAZ deletion impaired endochondral ossification in the primary ossification center but not intramembranous osteogenesis in the bone collar. Single-cell RNA sequencing revealed YAP/TAZ regulation of the angiogenic chemokine, Cxcl12, which was expressed uniquely in vessel-associated osteoblast precursors. YAP/TAZ signaling spatially coupled osteoblast precursors to blood vessels and regulated vascular morphogenesis and vessel barrier function. Further, YAP/TAZ signaling regulated vascular loop morphogenesis at the chondro-osseous junction to control hypertrophic growth plate remodeling. In human cells, mesenchymal stromal cell co-culture promoted 3D vascular network formation, which was impaired by stromal cell YAP/TAZ depletion, but rescued by recombinant CXCL12 treatment. Lastly, YAP and TAZ mediated mechanotransduction for load-induced osteogenesis in embryonic bone.

Single cell transcriptomics identifies a unique adipose lineage cell population that regulates bone marrow environment.

Bone marrow mesenchymal lineage cells are a heterogeneous cell population involved in bone homeostasis and diseases such as osteoporosis. While it is long postulated that they originate from mesenchymal stem cells, the true identity of progenitors and their in vivo bifurcated differentiation routes into osteoblasts and adipocytes remain poorly understood. Here, by employing large scale single cell transcriptome analysis, we computationally defined mesenchymal progenitors at different stages and delineated their bi-lineage differentiation paths in young, adult and aging mice. One identified subpopulation is a unique cell type that expresses adipocyte markers but contains no lipid droplets. As non-proliferative precursors for adipocytes, they exist abundantly as pericytes and stromal cells that form a ubiquitous 3D network inside the marrow cavity. Functionally they play critical roles in maintaining marrow vasculature and suppressing bone formation. Therefore, we name them marrow adipogenic lineage precursors (MALPs) and conclude that they are a newly identified component of marrow adipose tissue.

Increasing radiation dose improves immunotherapy outcome and prolongation of tumor dormancy in a subgroup of mice treated for advanced intracerebral melanoma.

Previously, we developed a clinically relevant therapy model for advanced intracerebral B16 melanomas in syngeneic mice combining radiation and immunotherapies. Here, 7 days after B16-F10-luc2 melanoma cells were implanted intracerebrally (D7), syngeneic mice with bioluminescent tumors that had formed (1E10(5) to 7E10(6) photons per minute (>1E10(6), large; <1E10(6), small) were segregated into large-/small-balanced subgroups. Then, mice received either radiation therapy alone (RT) or radiation therapy plus immunotherapy (RT plus IT) (single injection of mAbPC61 to deplete regulatory T cells followed by multiple injections of irradiated granulocyte macrophage colony stimulating factor transfected B16-F10 cells) (RT plus IT). Radiation dose was varied (15, 18.75 or 22.5 Gy, given on D8), while immunotherapy was provided similarly to all mice. The data support the hypothesis that increasing radiation dose improves the outcome of immunotherapy in a subgroup of mice. The tumors that were greatly delayed in beginning their progressive growth were bioluminescent in vivo-some for many months, indicating prolonged tumor "dormancy," in some cases presaging long-term cures. Mice bearing such tumors had far more likely received radiation plus immunotherapy, rather than RT alone. Radiotherapy is a very important adjunct to immunotherapy; the greater the tumor debulking by RT, the greater should be the benefit to tumor immunotherapy.