Sclerostin Antibody Administration Increases the Numbers of Sox9creER+ Skeletal Precursors and Their Progeny.

Blocking the Wnt inhibitor, sclerostin, increases the rate of bone formation in rodents and in humans. On a cellular level, the antibody against sclerostin acts by increasing osteoblast numbers partly by activating the quiescent bone-lining cells in vivo. No evidence currently exists, to determine whether blocking sclerostin affects early cells of the osteoblast lineage. Here we use a lineage-tracing strategy that uses a tamoxifen-dependent cre recombinase, driven by the Sox9 promoter to mark early cells of the osteoblast lineage. We show that, when adult mice are treated with the rat-13C7, an antibody that blocks sclerostin action in rodents, it increases the numbers of osteoblast precursors and their differentiation into mature osteoblasts in vivo. We also show that rat-13C7 administration suppresses adipogenesis by suppressing the differentiation of Sox9creER+ skeletal precursors into bone marrow adipocytes in vivo. Using floxed alleles of the CTNNB1 gene encoding ß-catenin, we show that these precursor cells express the canonical Wnt signaling mediator, ß-catenin, and that the actions of the rat-13C7 antibody to increase the number of early precursors is dependent on direct stimulation of Wnt signaling. The increase in osteoblast precursors and their progeny after the administration of the antibody leads to a robust suppression of apoptosis without affecting the rate of their proliferation. Thus, neutralizing the Wnt-inhibitor sclerostin increases the numbers of early cells of the osteoblast lineage osteoblasts and suppresses their differentiation into adipocytes in vivo. © 2021 American Society for Bone and Mineral Research (ASBMR).

Stem and progenitor cells in skeletal development.

Accumulating evidence supports the idea that stem and progenitor cells play important roles in skeletal development. Over the last decade, the definition of skeletal stem and progenitor cells has evolved from cells simply defined by their in vitro behaviors to cells fully defined by a combination of sophisticated approaches, including serial transplantation assays and in vivo lineage-tracing experiments. These approaches have led to better identification of the characteristics of skeletal stem cells residing in multiple sites, including the perichondrium of the fetal bone, the resting zone of the postnatal growth plate, the bone marrow space and the periosteum in adulthood. These diverse groups of skeletal stem cells appear to closely collaborate and achieve a number of important biological functions of bones, including not only bone development and growth, but also bone maintenance and repair. Although these are important findings, we are only beginning to understand the diversity and the nature of skeletal stem and progenitor cells, and how they actually behave in vivo.

Withdrawal of parathyroid hormone after prolonged administration leads to adipogenic differentiation of mesenchymal precursors in vivo.

Intermittent PTH-like drugs are the only approved so-called anabolic agent that increases bone mass in both mice and humans. It is well documented that PTH targets mature cells of the osteoblast lineage, with only indirect evidence of its actions on early cells of the osteoblast lineage. Using a triple transgenic mouse model that allowed labeling of very early cells of the osteoblast lineage, we traced the progeny of these into osteoblast lineage in adult mice. These early cells expressed PTH1R and multiplied when PTH (1-34) was administered daily. We also showed that the early mesenchymal cells showed accelerated differentiation into mature osteocalcin-positive osteoblasts and osteocytes. Rather surprisingly, when teriparatide administration was stopped, these early mesenchymal precursors differentiated into adipocytes. We showed that the adipogenic differentiation is accompanied by a decrease in wnt signaling in osteoblast precursors. In this review, we discuss the possible clinical relevance of this finding and the possible molecular mechanisms that contribute to this phenotype in vivo.

Parathyroid hormone regulates fates of murine osteoblast precursors in vivo.

Teriparatide, a recombinant form of parathyroid hormone (PTH), is the only approved treatment for osteoporosis that increases the rate of bone formation. Teriparatide increases osteoblast numbers by suppressing osteoblast apoptosis and activating bone-lining cells. No direct evidence for teriparatide's actions on early cells of the osteoblast lineage has been demonstrated. Here, we have employed a lineage-tracing strategy that uses a tamoxifen-dependent, promoter-driven cre to mark early cells of the osteoblast lineage in adult mice. We show that teriparatide increases the numbers of osteoblast precursors and drives their differentiation into mature osteoblasts. Unexpectedly, following withdrawal of teriparatide therapy, bone marrow adipocytes increased dramatically in number. Some of these adipocytes derived from cells marked by Sox9-cre expression weeks earlier. Continued therapy with teriparatide prevented the appearance of adipocytes. Selective, inducible deletion of the PTH receptor in Sox9-cre cells demonstrated that PTH receptor expression is required for teriparatide-mediated increases in early osteoblast precursors. The increase in early precursors after teriparatide administration was associated with robust suppression of precursor apoptosis without affecting their rate of proliferation. Thus, teriparatide increases the numbers of early cells of the osteoblast lineage, hastens their differentiation into osteoblasts, and suppresses their differentiation into adipocytes in vivo.

Bone CLARITY: Clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow.

Bone tissue harbors unique and essential physiological processes, such as hematopoiesis, bone growth, and bone remodeling. To enable visualization of these processes at the cellular level in an intact environment, we developed "Bone CLARITY," a bone tissue clearing method. We used Bone CLARITY and a custom-built light-sheet fluorescence microscope to detect the endogenous fluorescence of Sox9-tdTomato+ osteoprogenitor cells in the tibia, femur, and vertebral column of adult transgenic mice. To obtain a complete distribution map of these osteoprogenitor cells, we developed a computational pipeline that semiautomatically detects individual Sox9-tdTomato+ cells in their native three-dimensional environment. Our computational method counted all labeled osteoprogenitor cells without relying on sampling techniques and displayed increased precision when compared with traditional stereology techniques for estimating the total number of these rare cells. We demonstrate the value of the clearing-imaging pipeline by quantifying changes in the population of Sox9-tdTomato-labeled osteoprogenitor cells after sclerostin antibody treatment. Bone tissue clearing is able to provide fast and comprehensive visualization of biological processes in intact bone tissue.

Sclerostin Antibody Administration Converts Bone Lining Cells Into Active Osteoblasts.

Sclerostin antibody (Scl-Ab) increases osteoblast activity, in part through increasing modeling-based bone formation on previously quiescent surfaces. Histomorphometric studies have suggested that this might occur through conversion of bone lining cells into active osteoblasts. However, direct data demonstrating Scl-Ab-induced conversion of lining cells into active osteoblasts are lacking. Here, we used in vivo lineage tracing to determine if Scl-Ab promotes the conversion of lining cells into osteoblasts on periosteal and endocortical bone surfaces in mice. Two independent, tamoxifen-inducible lineage-tracing strategies were used to label mature osteoblasts and their progeny using the DMP1 and osteocalcin promoters. After a prolonged "chase" period, the majority of labeled cells on bone surfaces assumed a thin, quiescent morphology. Then, mice were treated with either vehicle or Scl-Ab (25 mg/kg) twice over the course of the subsequent week. After euthanization, marked cells were enumerated, their thickness quantified, and proliferation and apoptosis examined. Scl-Ab led to a significant increase in the average thickness of labeled cells on periosteal and endocortical bone surfaces, consistent with osteoblast activation. Scl-Ab did not induce proliferation of labeled cells, and Scl-Ab did not regulate apoptosis of labeled cells. Therefore, direct reactivation of quiescent bone lining cells contributes to the acute increase in osteoblast numbers after Scl-Ab treatment in mice. © 2016 American Society for Bone and Mineral Research.