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Summary One-year treatment of osteoporotic postmenopausal women with transdermal estrogen resulted in significant decreases in bone marrow adipocyte volume and prevented increases in adipocyte number as compared to placebo-treated controls. Estrogen treatment also prevented increases in mean adipocyte size over 1 year. Introduction Aging is associated not only with bone loss but also with increases in bone marrow adipocytes. Since osteoblasts and adipocytes are derived from a common precursor, it is possible that with aging, there is a preferential “switch” in commitment of this precursor to the adipocyte over the osteoblast lineage. We tested the hypothesis that the apparent “age-related” increase in marrow adipocytes is due, at least in part, to estrogen (E) deficiency. Methods Reanalysis of bone biopsies from a randomized, placebo-controlled trial involving 56 postmenopausal osteoporotic women (mean age, 64 years) treated either with placebo (PL, n = 27) or transdermal estradiol (0.1 mg/d, n = 29) for 1 year. Results Adipocyte volume/tissue volume (AV/TV) and adipocyte number (Ad#) increased (by ∼20%, P < 0.05) in the PL group, but were unchanged (Ad#) or decreased (AV/TV, by −24%, P < 0.001) in the E group. E treatment also prevented increases in mean adipocyte size over 1 year. Conclusions These findings represent the first in vivo demonstration in humans that not only ongoing bone loss, but also the increase in bone marrow adipocyte number and size in postmenopausal osteoporotic women may be due, at least in part, to E deficiency.

Apoptosis and DNA oxidative damage serve significant roles in the pathogenesis of steroid-induced femoral head necrosis. Vitamin E demonstrates anti-apoptotic and anti-oxidant properties. Therefore, the present study investigated the effects of vitamin E on osteocyte apoptosis and DNA oxidative damage in bone marrow hemopoietic cells at an early stage of steroid-induced femoral head osteonecrosis. Japanese white rabbits were randomly divided into three groups (steroid, vitamin E-treated, and control groups), each comprising 12 rabbits. Those in the steroid group (group S) were initially injected twice with an intravenous dose of 100 µg/kg Escherichia coli endotoxin, with a 24 h interval between the two injections, and then with an intramuscular dose of 20 mg/kg methylprednisolone, three times at intervals of 24 h in order to establish a rabbit model of osteonecrosis. The vitamin E treated group (group E) received the same treatment as group S, and were administered 0.6 g/kg/d vitamin E daily from the beginning of modeling. The control group (group C) was injected with normal saline at the equivalent dosage and times as the aforementioned two groups. Two time points, weeks 4 and 6 following the completion of modeling, were selected. Osteonecrosis was verified by histopathology with hematoxylin-eosin staining. The apoptosis rate of osteonecrosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The apoptosis expression levels of caspase-3 and B-cell lymphoma 2 (Bcl-2), and DNA oxidative damage of bone marrow hematopoietic cells were analyzed by immunohistochemistry. At weeks 4 and 6 following the completion of modeling, the vacant bone lacunae rates of group E were 15.87±1.97 and 25.09±2.67%, respectively, lower than the results of 20.02±2.21 and 27.79±1.39% for group S; and the osteocyte apoptosis indexes of group E were 20.99±2.95 and 33.93±1.62%, respectively, lower than the results of 26.46±3.37 and 39.90±3.74% from group S. In addition, the Bcl-2 expression at week 4 in the femoral head tissues of group E was higher compared with group S; and the proportion of Bcl-2-positive cells of group E was 9.81±1.01%, higher compared with group S at 8.26±1.13%. The caspase-3 staining data at week 4 in femoral head tissues demonstrated that in the 12 femoral heads of group S, four were negative (32%) and eight were positive (68%); in group E, five were negative (45%) and seven were positive (55%); and in group C, 11 were negative (95%) and one was positive (5%). In addition, the DNA oxidative damage rate at week 4 in the bone marrow hemopoietic cells of group E was (7.24±1.44%), lower compared with group S (11.80±1.26%), and higher compared with group C (5.75±1.47%). Vitamin E is effective in intervening in apoptosis through decreasing caspase-3 expression and upregulating Bcl-2 expression, and by alleviating DNA oxidative damage in bone marrow hemopoietic cells at the early stage of steroid-induced femoral head necrosis in rabbit models.

IntroductionBone marrow aspiration and biopsy is an invasive procedure associated with morbidity and mortality risk. We compared a powered bone marrow aspiration and biopsy device to the traditional method by relatively assessing pain scores, procedure times, biopsy capture rates, quality of material retrieved, and safety and operator satisfaction.MethodsTwo large academic medical centres participated in this trial. Patients were randomised to have procedures carried out using the powered system or the manual technique. A visual analogue scale pain score was recorded immediately following skin puncture and once again at the end of the procedure for each patient. Procedure time was measured from skin puncture to core specimen acquisition. Pathologic assessment of 30 randomised samples was carried out. Operator satisfaction with devices was measured on a scale of 0–10, with 10 as the highest rating.ResultsFive operators from two sites enrolled 50 patients (powered, n=25; manual, n=25). Groups were evenly matched, with no significant differences in the means for age, weight and height. The powered system was superior to the manual system with respect to patient perceived pain from needle insertion (2.6±2.0 vs 4.1±2.5, p=0.022) and procedural time (100.0±72.8 s vs 224.1±79.0 s, p<0.001). Overall pain scores at the end of both procedures were comparable (3.2±2.2 vs 3.8±3.0, p=0.438). No complications were observed in either arm of the study. Blinded pathologic analysis of the specimens retrieved revealed that cores obtained using the powered system were longer and wider than those obtained using the manual technique (25.4±12.3 mm2 vs 11.9±5.6 mm2, p=0.001). For marrow aspiration, no difference was seen between groups for clot/particle spicules or smear spicules. Operator assessment favoured the use of the powered device.ConclusionsResults of this trial suggest that the use of a powered bone marrow biopsy device significantly reduces needle insertion pain and procedural time when compared to a manual technique. The superior size and overall quality of core specimens retrieved by the powered device provides more material for pathologic evaluation, thereby increasing diagnostic yield and reducing the need for repeat procedures.

Background Treatment of acute myocardial infarction with stem cell transplantation has achieved beneficial effects in many clinical trials. The bone marrow microenvironment of ST-elevation myocardial infarction (STEMI) patients has never been studied even though myocardial infarction is known to cause an imbalance in the acid-base status of these patients. The aim of this study was to assess if the blood gas levels in the bone marrow of STEMI patients affect the characteristics of the bone marrow cells (BMCs) and, furthermore, do they influence the change in cardiac function after autologous BMC transplantation. The arterial, venous and bone marrow blood gas concentrations were also compared. Methods Blood gas analysis of the bone marrow aspirate and peripheral blood was performed for 27 STEMI patients receiving autologous stem cell therapy after percutaneous coronary intervention. Cells from the bone marrow aspirate were further cultured and the bone marrow mesenchymal stem cell (MSC) proliferation rate was determined by MTT assay and the MSC osteogenic differentiation capacity by alkaline phosphatase (ALP) activity assay. All the patients underwent a 2D-echocardiography at baseline and 4 months after STEMI. Results As expected, the levels of pO 2 , pCO 2 , base excess and HCO 3 were similar in venous blood and bone marrow. Surprisingly, bone marrow showed significantly lower pH and Na + and elevated K + levels compared to arterial and venous blood. There was a positive correlation between the bone marrow pCO 2 and HCO 3 levels and MSC osteogenic differentiation capacity. In contrast, bone marrow pCO 2 and HCO 3 levels displayed a negative correlation with the proliferation rate of MSCs. Patients with the HCO 3 level below the median value exhibited a more marked change in LVEF after BMC treatment than patients with HCO 3 level above the median (11.13 ± 8.07% vs. 2.67 ± 11.89%, P = 0.014). Conclusions Low bone marrow pCO 2 and HCO 3 levels may represent the optimal environment for BMCs in terms of their efficacy in autologous stem cell therapy in STEMI patients.

Lower numbers of progenitor cells (PCs) in peripheral blood (PB) have been associated with cardiovascular events in high-risk populations. Therapies aiming to increase the numbers of PCs in circulation have been developed, but clinical trials did not result in better outcomes. It is currently unknown what causes the reduction in PB PC numbers: whether it is primary depletion of the progenitor cell reserve, or a reduced mobilization of PCs from the bone marrow (BM). In this study, we examine if PB and BM PC numbers predict Amputation-Free Survival (AFS) in patients with Severe Limb Ischemia (SLI). We obtained PB and BM from 160 patients enrolled in a clinical trial investigating BM cell therapy for SLI. Samples were incubated with antibodies against CD34, KDR, CD133, CD184, CD14, CD105, CD140b, and CD31; PC populations were enumerated by flow cytometry. Higher PB CD34+ and CD133+ PC numbers were related to AFS (Both Hazard Ratio [HRevent] = 0.56, p = 0.003 and p = 0.0007, respectively). AFS was not associated with the other cell populations in PB. BM PC numbers correlated with PB PC numbers and showed similar HRs for AFS. A further subdivision based on relative BM and PB PC numbers showed that BM PC numbers, rather than mobilization, associated with AFS. Both PB and BM PC numbers are associated with AFS independently from traditional risk factor and show very similar risk profiles. Our data suggest that depletion of the progenitor cell reserve, rather than decreased PC mobilization, underlies the association between PB PC numbers and cardiovascular risk.

Single-cell genomics technology has transformed our understanding of complex cellular systems. However, excessive cost and a lack of strategies for the purification of newly identified cell types impede their functional characterization and large-scale profiling. Here, we have generated high-content single-cell proteo-genomic reference maps of human blood and bone marrow that quantitatively link the expression of up to 197 surface markers to cellular identities and biological processes across all main hematopoietic cell types in healthy aging and leukemia. These reference maps enable the automatic design of cost-effective high-throughput cytometry schemes that outperform state-of-the-art approaches, accurately reflect complex topologies of cellular systems and permit the purification of precisely defined cell states. The systematic integration of cytometry and proteo-genomic data enables the functional capacities of precisely mapped cell states to be measured at the single-cell level. Our study serves as an accessible resource and paves the way for a data-driven era in cytometry. Haas, Velten and colleagues use single-cell multiomics of human blood and bone marrow to generate a reference map allowing the quantitative linking of cytometry and proteo-genomic information.

Acute myeloid leukaemia (AML) is distinguished by the generation of dysfunctional leukaemic blasts, and patients characteristically suffer from fatal infections and anaemia due to insufficient normal myelo-erythropoiesis. Direct physical crowding of bone marrow (BM) by accumulating leukaemic cells does not fully account for this haematopoietic failure. Here, analyses from AML patients were applied to both in vitro co-culture platforms and in vivo xenograft modelling, revealing that human AML disease specifically disrupts the adipocytic niche in BM. Leukaemic suppression of BM adipocytes led to imbalanced regulation of endogenous haematopoietic stem and progenitor cells, resulting in impaired myelo-erythroid maturation. In vivo administration of PPARγ agonists induced BM adipogenesis, which rescued healthy haematopoietic maturation while repressing leukaemic growth. Our study identifies a previously unappreciated axis between BM adipogenesis and normal myelo-erythroid maturation that is therapeutically accessible to improve symptoms of BM failure in AML via non-cell autonomous targeting of the niche. Boyd et al. monitored the effects of patient-derived acute myeloid leukaemia (AML) cells on human HSPCs in vivo and found that AML impairs bone marrow adipocyte differentiation, and this in turn impedes healthy endogenous haematopoiesis.

ZBTB7A is frequently mutated in acute myeloid leukemia (AML) with t(8;21) translocation. However, the oncogenic collaboration between mutated ZBTB7A and the RUNX1–RUNX1T1 fusion gene in AML t(8;21) remains unclear. Here, we investigate the role of ZBTB7A and its mutations in the context of normal and malignant hematopoiesis. We demonstrate that clinically relevant ZBTB7A mutations in AML t(8;21) lead to loss of function and result in perturbed myeloid differentiation with block of the granulocytic lineage in favor of monocytic commitment. In addition, loss of ZBTB7A increases glycolysis and hence sensitizes leukemic blasts to metabolic inhibition with 2-deoxy-d-glucose. We observed that ectopic expression of wild-type ZBTB7A prevents RUNX1-RUNX1T1-mediated clonal expansion of human CD34+ cells, whereas the outgrowth of progenitors is enabled by ZBTB7A mutation. Finally, ZBTB7A expression in t(8;21) cells lead to a cell cycle arrest that could be mimicked by inhibition of glycolysis. Our findings suggest that loss of ZBTB7A may facilitate the onset of AML t(8;21), and that RUNX1-RUNX1T1-rearranged leukemia might be treated with glycolytic inhibitors.

Tumours most often arise from progression of precursor clones within a single anatomical niche. In the bone marrow, clonal progenitors can undergo malignant transformation to acute leukaemia, or differentiate into immune cells that contribute to disease pathology in peripheral tissues 1 – 4 . Outside the marrow, these clones are potentially exposed to a variety of tissue-specific mutational processes, although the consequences of this are unclear. Here we investigate the development of blastic plasmacytoid dendritic cell neoplasm (BPDCN)—an unusual form of acute leukaemia that often presents with malignant cells isolated to the skin 5 . Using tumour phylogenomics and single-cell transcriptomics with genotyping, we find that BPDCN arises from clonal (premalignant) haematopoietic precursors in the bone marrow. We observe that BPDCN skin tumours first develop at sun-exposed anatomical sites and are distinguished by clonally expanded mutations induced by ultraviolet (UV) radiation. A reconstruction of tumour phylogenies reveals that UV damage can precede the acquisition of alterations associated with malignant transformation, implicating sun exposure of plasmacytoid dendritic cells or committed precursors during BPDCN pathogenesis. Functionally, we find that loss-of-function mutations in Tet2 , the most common premalignant alteration in BPDCN, confer resistance to UV-induced cell death in plasmacytoid, but not conventional, dendritic cells, suggesting a context-dependent tumour-suppressive role for TET2. These findings demonstrate how tissue-specific environmental exposures at distant anatomical sites can shape the evolution of premalignant clones to disseminated cancer. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) arises from clonal (premalignant) haematopoietic precursors in the bone marrow, and BPDCN skin tumours first develop at sun-exposed anatomical sites and are distinguished by clonally expanded mutations induced by ultraviolet radiation.

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are prevalent liver conditions that underlie the development of life-threatening cirrhosis, liver failure and liver cancer. Chronic necro-inflammation is a critical factor in development of NASH, yet the cellular and molecular mechanisms of immune dysregulation in this disease are poorly understood. Here, using single-cell transcriptomic analysis, we comprehensively profiled the immune composition of the mouse liver during NASH. We identified a significant pathology-associated increase in hepatic conventional dendritic cells (cDCs) and further defined their source as NASH-induced boost in cycling of cDC progenitors in the bone marrow. Analysis of blood and liver from patients on the NAFLD/NASH spectrum showed that type 1 cDCs (cDC1) were more abundant and activated in disease. Sequencing of physically interacting cDC-T cell pairs from liver-draining lymph nodes revealed that cDCs in NASH promote inflammatory T cell reprogramming, previously associated with NASH worsening. Finally, depletion of cDC1 in XCR1 DTA mice or using anti-XCL1-blocking antibody attenuated liver pathology in NASH mouse models. Overall, our study provides a comprehensive characterization of cDC biology in NASH and identifies XCR1 + cDC1 as an important driver of liver pathology. Single-cell analyses reveal cDC1 as conserved immunological drivers of non-alcoholic steatohepatitis in mice and humans