The characterization of distinct populations of murine skeletal cells that have different roles in B lymphopoiesis.

Hematopoiesis is extrinsically controlled by cells of the bone marrow microenvironment, including skeletal lineage cells. The identification and subsequent studies of distinct subpopulations of maturing skeletal cells is currently limited because of a lack of methods to isolate these cells. We found that murine Lin-CD31-Sca-1-CD51+ cells can be divided into 4 subpopulations by using flow cytometry based on their expression of the platelet-derived growth factor receptors ⍺ and ß (PDGFR⍺ and PDGFRß). The use of different skeletal lineage reporters confirmed the skeletal origin of the 4 populations. Multiplex immunohistochemistry studies revealed that all 4 populations were localized near the growth plate and trabecular bone and were rarely found near cortical bone regions or in central bone marrow. Functional studies revealed differences in their abundance, colony-forming unit-fibroblast capacity, and potential to differentiate into mineralized osteoblasts or adipocytes in vitro. Furthermore, the 4 populations had distinct gene expression profiles and differential cell surface expression of leptin receptor (LEPR) and vascular cell adhesion molecule 1 (VCAM-1). Interestingly, we discovered that 1 of these 4 different skeletal populations showed the highest expression of genes involved in the extrinsic regulation of B lymphopoiesis. This cell population varied in abundance between distinct hematopoietically active skeletal sites, and significant differences in the proportions of B-lymphocyte precursors were also observed in these distinct skeletal sites. This cell population also supported pre-B lymphopoiesis in culture. Our method of isolating 4 distinct maturing skeletal populations will help elucidate the roles of distinct skeletal niche cells in regulating hematopoiesis and bone.

Effects of chemotherapy agents used to treat pediatric acute lymphoblastic leukemia patients on bone parameters and longitudinal growth of juvenile mice.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Therapies for pediatric ALL have improved such that more than 80% of patients survive to 5 years post-therapy, and most survive to adulthood. These ALL patients experience long-term side effects that permanently affect their quality of life, with bone loss and reduced longitudinal growth being the most common skeletal complications. To determine the effects of the chemotherapeutic agents used in ALL induction therapy on bone density and longitudinal growth in mice, we treated juvenile mice with doxorubicin, dexamethasone, vincristine, l-asparaginase, or combination therapy. At adulthood, mice were culled and bones collected and scanned by micro-computed tomography (micro-CT). Mice that received doxorubicin and combination therapy exhibited reduced longitudinal growth and significant reductions in trabecular bone volume, trabecular thickness, and trabecular number, with increased trabecular separation. Mean cortical thickness, cortical area, marrow area, endocortical perimeter, and polar moment of inertia were significantly reduced by doxorubicin and combination therapy. Vincristine treatment significantly decreased trabecular bone volume, trabecular number, and increased trabecular separation but had no effects on cortical bone. Dexamethasone treatment increased trabecular bone separation, cortical marrow area, and cortical bone periosteal perimeter. Mice treated with l-asparaginase did not have any bone phenotypes. In conclusion, these data indicate that the majority of the chemotherapy agents used in induction therapy for pediatric ALL have long-term effects on bone in mice. A single dose of doxorubicin in juvenile mice was sufficient to cause the majority of the bone phenotypes, with combination therapy intensifying these effects.

Imaging methods used to study mouse and human HSC niches: Current and emerging technologies.

Bone marrow contains numerous different cell types arising from hematopoietic stem cells (HSCs) and non-hematopoietic mesenchymal/skeletal stem cells, in addition to other cell types such as endothelial cells- these non-hematopoietic cells are commonly referred to as stromal cells or microenvironment cells. HSC function is intimately linked to complex signals integrated by their niches, formed by combinations of hematopoietic and stromal cells. Studies of hematopoietic cells have been significantly advanced by flow cytometry methods, enabling the quantitation of each cell type in normal and perturbed situations, in addition to the isolation of these cells for molecular and functional studies. Less is known, however, about the specific niches for distinct developing hematopoietic lineages, or the changes occurring in the niche size and function in these distinct anatomical sites in the bone marrow under stress situations and ageing. Significant advances in imaging technology during the last decade have permitted studies of HSC niches in mice. Additional imaging technologies are emerging that will facilitate the study of human HSC niches in trephine BM biopsies. Here we provide an overview of imaging technologies used to study HSC niches, in addition to highlighting emerging technology that will help us to more precisely identify and characterize HSC niches in normal and diseased states.

Retinoic Acid Receptor γ Activity in Mesenchymal Stem Cells Regulates Endochondral Bone, Angiogenesis, and B Lymphopoiesis.

Retinoic acid receptor (RAR) signaling regulates bone structure and hematopoiesis through intrinsic and extrinsic mechanisms. This study aimed to establish how early in the osteoblast lineage loss of RARγ (Rarg) disrupts the bone marrow microenvironment. Bone structure was analyzed by micro-computed tomography (µCT) in Rarg-/- mice and mice with Rarg conditional deletion in Osterix-Cre-targeted osteoblast progenitors or Prrx1-Cre-targeted mesenchymal stem cells. Rarg-/- tibias exhibited less trabecular and cortical bone and impaired longitudinal and radial growth. The trabecular bone and longitudinal, but not radial, growth defects were recapitulated in Prrx1:RargΔ/Δ mice but not Osx1:RargΔ/Δ mice. Although both male and female Prrx1:RargΔ/Δ mice had low trabecular bone mass, males exhibited increased numbers of trabecular osteoclasts and Prrx1:RargΔ/Δ females had impaired mineral deposition. Both male and female Prrx1:RargΔ/Δ growth plates were narrower than controls and their epiphyses contained hypertrophic chondrocyte islands. Flow cytometry revealed that male Prrx1:RargΔ/Δ bone marrow exhibited elevated pro-B and pre-B lymphocyte numbers, accompanied by increased Cxcl12 expression in bone marrow cells. Prrx1:RargΔ/Δ bone marrow also had elevated megakaryocyte-derived Vegfa expression accompanied by smaller sinusoidal vessels. Thus, RARγ expression by Prrx1-Cre-targeted cells directly regulates endochondral bone formation and indirectly regulates tibial vascularization. Furthermore, RARγ expression by Prrx1-Cre-targeted cells extrinsically regulates osteoclastogenesis and B lymphopoiesis in male mice. © 2018 American Society for Bone and Mineral Research.

Inhibition of Endosteal Vascular Niche Remodeling Rescues Hematopoietic Stem Cell Loss in AML.

Bone marrow vascular niches sustain hematopoietic stem cells (HSCs) and are drastically remodeled in leukemia to support pathological functions. Acute myeloid leukemia (AML) cells produce angiogenic factors, which likely contribute to this remodeling, but anti-angiogenic therapies do not improve AML patient outcomes. Using intravital microscopy, we found that AML progression leads to differential remodeling of vasculature in central and endosteal bone marrow regions. Endosteal AML cells produce pro-inflammatory and anti-angiogenic cytokines and gradually degrade endosteal endothelium, stromal cells, and osteoblastic cells, whereas central marrow remains vascularized and splenic vascular niches expand. Remodeled endosteal regions have reduced capacity to support non-leukemic HSCs, correlating with loss of normal hematopoiesis. Preserving endosteal endothelium with the small molecule deferoxamine or a genetic approach rescues HSCs loss, promotes chemotherapeutic efficacy, and enhances survival. These findings suggest that preventing degradation of the endosteal vasculature may improve current paradigms for treating AML.

T-cell acute leukaemia exhibits dynamic interactions with bone marrow microenvironments.

It is widely accepted that complex interactions between cancer cells and their surrounding microenvironment contribute to disease development, chemo-resistance and disease relapse. In light of this observed interdependency, novel therapeutic interventions that target specific cancer stroma cell lineages and their interactions are being sought. Here we studied a mouse model of human T-cell acute lymphoblastic leukaemia (T-ALL) and used intravital microscopy to monitor the progression of disease within the bone marrow at both the tissue-wide and single-cell level over time, from bone marrow seeding to development/selection of chemo-resistance. We observed highly dynamic cellular interactions and promiscuous distribution of leukaemia cells that migrated across the bone marrow, without showing any preferential association with bone marrow sub-compartments. Unexpectedly, this behaviour was maintained throughout disease development, from the earliest bone marrow seeding to response and resistance to chemotherapy. Our results reveal that T-ALL cells do not depend on specific bone marrow microenvironments for propagation of disease, nor for the selection of chemo-resistant clones, suggesting that a stochastic mechanism underlies these processes. Yet, although T-ALL infiltration and progression are independent of the stroma, accumulated disease burden leads to rapid, selective remodelling of the endosteal space, resulting in a complete loss of mature osteoblastic cells while perivascular cells are maintained. This outcome leads to a shift in the balance of endogenous bone marrow stroma, towards a composition associated with less efficient haematopoietic stem cell function. This novel, dynamic analysis of T-ALL interactions with the bone marrow microenvironment in vivo, supported by evidence from human T-ALL samples, highlights that future therapeutic interventions should target the migration and promiscuous interactions of cancer cells with the surrounding microenvironment, rather than specific bone marrow stroma, to combat the invasion by and survival of chemo-resistant T-ALL cells.