Marrow Volume Research Articles

Leukemic blasts program bone marrow adipocytes to generate a protumoral microenvironment

AbstractDespite currently available therapies, most patients diagnosed with acute myeloid leukemia (AML) die of their disease. Tumor-host interactions are critical for the survival and proliferation of cancer cells; accordingly, we hypothesize that specific targeting of the tumor microenvironment may constitute an alternative or additional strategy to conventional tumor-directed chemotherapy. Because adipocytes have been shown to promote breast and prostate cancer proliferation, and because the bone marrow adipose tissue accounts for up to 70% of bone marrow volume in adult humans, we examined the adipocyte-leukemia cell interactions to determine if they are essential for the growth and survival of AML. Using in vivo and in vitro models of AML, we show that bone marrow adipocytes from the tumor microenvironment support the survival and proliferation of malignant cells from patients with AML. We show that AML blasts alter metabolic processes in adipocytes to induce phosphorylation of hormone-sensitive lipase and consequently activate lipolysis, which then enables the transfer of fatty acids from adipocytes to AML blasts. In addition, we report that fatty acid binding protein-4 (FABP4) messenger RNA is upregulated in adipocytes and AML when in coculture. FABP4 inhibition using FABP4 short hairpin RNA knockdown or a small molecule inhibitor prevents AML proliferation on adipocytes. Moreover, knockdown of FABP4 increases survival in Hoxa9/Meis1-driven AML model. Finally, knockdown of carnitine palmitoyltransferase IA in an AML patient-derived xenograft model improves survival. Here, we report the first description of AML programming bone marrow adipocytes to generate a protumoral microenvironment.

The Influence of Aspiration Volume on the Number of Osteoblastic Progenitors Obtained From Bone Marrow in Children.

Autologous bone marrow aspirates are utilized to treat various conditions in children. The biological value of bone marrow aspirate depends on the concentration of competent osteoblastic progenitors present in the aspirate. It has been shown in adults that increasing bone marrow aspiration volume beyond 2 mL decreases the concentration of osteoblast progenitor cells because of dilution of the sample with peripheral blood. The effect of varying bone marrow aspiration volumes on the osteoblast cell content has not been determined in children. In total, 21 children (3 male and 18 female patients, age range 8 mo to 14 y) scheduled for pelvic osteotomy were included in the study. Three separate bone marrow aspirates of 1, 5, and 10 mL were obtained from the anterior superior iliac crest. Total number of nucleated cells was counted per aspirate and the prevalence of alkaline phosphatase-positive colony-forming units was determined per million nucleated cells. We measured a significant, proportional increase in the total number of nucleated bone marrow precursor cells between the 1 and 5 mL samples (mean±SD, 27±13 and 152±78 million nucleated cells, respectively; P<0.0001). When the aspiration volume doubled from 5 to 10 mL the total number of nucleated cells was 178±76 million (P=0.17). A proportional increase from 2214 alkaline phosphatase-positive colony-forming units in the 1 mL sample to 14,100 alkaline phosphatase-positive colony-forming units in the 5 mL sample was observed. However, the number of colony-forming units per aspirate decreased to 11,880 in the 10 mL sample. These data demonstrate that in children aspiration up to 5 mL bone marrow from the iliac crest yields a proportional increase in osteoblastic progenitor cells per aspirate. Increasing the aspiration volume beyond 5 mL results in hemodilution, rather than further selection of osteoblastic material. These data provide clinicians with a guideline for optimizing aspiration volume of bone marrow in children. Level II-development of diagnostic criteria on basis of consecutive patients.

Quantitative Assessment of Optimal Bone Marrow Site for the Isolation of Porcine Mesenchymal Stem Cells.

Background. One of the most plentiful sources for MSCs is the bone marrow; however, it is unknown whether MSC yield differs among different bone marrow sites. In this study, we quantified cellular yield and evaluated resident MSC population from five bone marrow sites in the porcine model. In addition, we assessed the feasibility of a commercially available platelet concentrator (Magellan® MAR01™ Arteriocyte Medical Systems, Hopkinton, MA) as a bedside stem cell concentration device. Methods. Analyses of bone marrow aspirate (BMA) and concentrated bone marrow aspirate (cBMA) included bone marrow volume, platelet and nucleated cell yield, colony-forming unit fibroblast (CFU-F) number, flow cytometry, and assessment of differentiation potential. Results. Following processing, the concentration of platelets and nucleated cells significantly increased but was not significantly different between sites. The iliac crest had significantly less bone marrow volume; however, it yielded significantly more CFUs compared to the other bone marrow sites. Culture-expanded cells from all tested sites expressed high levels of MSC surface markers and demonstrated adipogenic and osteogenic differentiation potential. Conclusions. All anatomical bone marrow sites contained MSCs, but the iliac crest was the most abundant source of MSCs. Additionally, the Magellan can function effectively as a bedside stem cell concentrator.

Corrigendum: Region-specific variation in the properties of skeletal adipocytes reveals regulated and constitutive marrow adipose tissues.

Nature Communications 6: Article number: 7808 (2015); Published: 6 August 2015; Updated: 8 December 2016 In Figure 3 of this Article, marrow adipose tissue volumes (in panels b and c) and adipocyte numbers (in panel g) were calculated incorrectly. In Fig. 3b,c, regional MAT volumes in the ‘GP to T/FJ’ group were normalized incorrectly to both regional marrow volume and total marrow volume instead of normalizing to total marrow volume only.

Enrichment of Hematopoietic Stem Cells Using Immunomagnetic Beads to Facilitate Gene Therapy

Lentiviral (LV)-based hematopoietic stem and progenitor cell (HSPC) gene therapy is becoming a promising clinical strategy for the treatment of genetic diseases. Clinical trials are currently underway to treat hemoglobinopathies using a lentivirus expressing anti-sickling β‐ or γ-globin genes. However, clinical scale production of globin vectors has proven difficult due to the large size and complexity of the human β-globin gene expression cassette. Vector products often have low titers and require large manufacturing volumes to treat a single patient. We hypothesized that less vector per patient could be used by further purifying hematopoietic stem cells (HSC) beyond standard CD34+ selection prior to gene modification and transplantation. Here, we have optimized and characterized an immunomagnetic bead (IB) based cell sorting method to enrich CD34+CD38- cells from human bone marrow (BM). Our results suggest we can use clinically available technology (IB-based cell sorting) to achieve a ~10-fold reduction in vector requirements while still retaining the HSC required for clinical benefit after transplant.We first used competitive transplant studies to determine the SCID repopulating activity of different subpopulations of BM CD34+ cells after 2-day ex-vivo culture and LV transduction. The CD34+ BM cells were sorted into 3 intervals of increasing CD38 expression, each marked with a distinct fluorescent LV vector, and competitively transplanted into NSG mice. At >16 weeks post-transplant, >90% of all hCD45+hCD34+ cells in NSG marrow were derived from the lowest ~6% of CD38 negativity.We next optimized small-scale (50-100 mL of BM) enrichment of CD34+CD38- cells using an IB-based cell isolation method. CD38+ cells were first depleted, and CD34+38- cells were subsequently selected. This double-step CD34+CD38- purification non-specifically enriched granulocytes, which represented up to 50% of total isolated cells. Thus, we modified the first step of our protocol to include co-depletion of both CD15+ myeloid cells and CD38+ cells, which increased the purity of CD34+38- cells up to 2-fold. The dual IB-based CD34+CD38- purification and standard CD34+ purification were performed in parallel on 4 independent BM samples. Recovery of CD34+CD38- cells from starting material (MNCs) was assessed by each method. The single-step IB CD34+ purification recovered 91.8±6.5% (mean±SD) of CD34+CD38- cells while double-step IB CD34+CD38- purification recovered 72±1.4% of CD34+CD38- cells. An additional 20±6% of CD34+CD38- cells remained in the CD38+ fraction. When compared to single-step IB CD34+ purification, CD34+CD38- two-step purification reduced the total number of isolated cells by a range of 7.6-17.8-fold.We next compared the long-term engraftment capabilities of IB-purified CD34+ and CD34+CD38- cells transduced with a LV expressing mCitrine. CD34+ or CD34+CD38- cells purified from an equivalent volume of marrow were transplanted into NSG mice at limiting cell doses. Purified cells were transduced at the same cell density and vector concentration, with the CD34+CD38- cell grafts requiring 11.8-fold less LV than CD34+ cells. At 20 weeks, recipients of CD34+ or CD34+CD38- purified cells had similar levels of human chimerism indicating retention of engraftment capacity by the enriched CD34+CD38- cells. Additionally, hCD45+ cells in engrafted NSG mice exhibited similar levels of mCitrine expression (MFI), suggesting that IB enrichment of CD34+CD38- cells does not affect gene transfer into HSC.One potential disadvantage of using purified CD34+CD38- cells in a clinical setting is delayed myeloid recovery in the post-transplant period. NSG mice transplanted with purified CD34+CD38- cells demonstrated reduced circulating human myeloid cells at 3 weeks post-transplant as compared to mice transplanted with CD34+ cells. In order to overcome this potential clinical hurdle, we explored a strategy of adding back non-transduced CD38+ cells, which restored early levels of circulating human myeloid cells. We are currently investigating the effects of adding non-transduced CD38+ cells on long-term engraftment of gene-marked HSC.In summary, we have developed a clinically applicable method to enrich HSCs and reduce vector requirements. This strategy could directly contribute to clinical trials for hemoglobinopathies by addressing the factors currently limiting gene therapy methods. DisclosuresNo relevant conflicts of interest to declare.

Means of enhancing bone fracture healing: optimal cell source, isolation methods and acoustic stimulation.

BackgroundThe human body has an extensive capacity to regenerate bone tissue after trauma. However large defects such as long bone fractures of the lower limbs cannot be restored without intervention and often lead to nonunion. Therefore, the aim of the present study was to assess the pool and biological functions of human mesenchymal stromal cells (hMSCs) isolated from different bone marrow locations of the lower limbs and to identify novel strategies to prime the cells prior to their use in bone fracture healing. Following, bone marrow from the ilium, proximal femur, distal femur and proximal tibia was aspirated and the hMSCs isolated. Bone marrow type, volume, number of mononuclear cells/hMSCs and their self-renewal, multilineage potential, extracellular matrix (ECM) production and surface marker profiling were analyzed. Additionally, the cells were primed to accelerate bone fracture healing either by using acoustic stimulation or varying the initial hMSCs isolation conditions.ResultsWe found that the more proximal the bone marrow aspiration location, the larger the bone marrow volume was, the higher the content in mononuclear cells/hMSCs and the higher the self-renewal and osteogenic differentiation potential of the isolated hMSCs were. Acoustic stimulation of bone marrow, as well as the isolation of hMSCs in the absence of fetal bovine serum, increased the osteogenic and ECM production potential of the cells, respectively.ConclusionWe showed that bone marrow properties change with the aspiration location, potentially explaining the differences in bone fracture healing between the tibia and the femur. Furthermore, we showed two new priming methods capable of enhancing bone fracture healing.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0318-1) contains supplementary material, which is available to authorized users.

Dosimetric Predictors of Treatment-related Lymphopenia induced by Palliative Radiotherapy: Predictive Ability of Dose-volume Parameters based on Body Surface Contour.

BackgroundRadiation-related lymphopenia has been associated with poor patient outcome. Our aim was to identify predictors of lymphopenia after palliative radiotherapy, with a focus on dose-volume parameters.Patients and methodsTo retrospectively assess patients with various cancers who had undergone palliative radiotherapy, we delineated three organs at risk: the volume enclosed by the body surface contour (body A), the volume left after excluding air, pleural effusion, ascites, bile, urine, and intestinal content (body B), and the volume of the bone marrow (BM). We then noted the absolute volume of the three organs at risk that had received 5-30 Gy, and assessed the predictive value for post-treatment lymphopenia of grade 3 or higher (LP3+).ResultsOf 54 patients, 23 (43%) developed LP3+. Univariate logistic regression analysis showed that body A V5, body A V10, body B V5, body B V10, the number of fractions, and splenic irradiation were significant predictors of LP3+ (p < 0.05). By multivariate analysis, body A V5, body A V10, body B V5, body B V10, and the number of fractions retained significance (p < 0.05). BM dose-volume parameters did not predict lymphopenia.ConclusionsHigher body A and body B dose-volume parameters and a larger number of fractions may be predictors of severe lymphopenia after palliative radiotherapy.

Cortical bone deficit and fat infiltration of bone marrow and skeletal muscle in ambulatory children with mild spastic cerebral palsy

IntroductionNonambulatory children with severe cerebral palsy (CP) have underdeveloped bone architecture, low bone strength and a high degree of fat infiltration in the lower extremity musculature. The present study aims to determine if such a profile exists in ambulatory children with mild CP and if excess fat infiltration extends into the bone marrow. Materials and methodsAmbulatory children with mild spastic CP and typically developing children (4 to 11years; 12/group) were compared. Magnetic resonance imaging was used to estimate cortical bone, bone marrow and total bone volume and width, bone strength [i.e., section modulus (Z) and polar moment of inertia (J)], and bone marrow fat concentration in the midtibia, and muscle volume, intermuscular, subfascial, and subcutaneous adipose tissue (AT) volume and intramuscular fat concentration in the midleg. Accelerometer-based activity monitors worn on the ankle were used to assess physical activity. ResultsThere were no group differences in age, height, body mass, body mass percentile, BMI, BMI percentile or tibia length, but children with CP had lower height percentile (19th vs. 50th percentile) and total physical activity counts (44%) than controls (both p<0.05). Children with CP also had lower cortical bone volume (30%), cortical bone width in the posterior (16%) and medial (32%) portions of the shaft, total bone width in the medial-lateral direction (15%), Z in the medial-lateral direction (34%), J (39%) and muscle volume (39%), and higher bone marrow fat concentration (82.1±1.8% vs. 80.5±1.9%), subfascial AT volume (3.3 fold) and intramuscular fat concentration (25.0±8.0% vs. 16.1±3.3%) than controls (all p<0.05). When tibia length was statistically controlled, all group differences in bone architecture, bone strength, muscle volume and fat infiltration estimates, except posterior cortical bone width, were still present (all p<0.05). Furthermore, a higher intermuscular AT volume in children with CP compared to controls emerged (p<0.05). ConclusionsAmbulatory children with mild spastic CP exhibit an underdeveloped bone architecture and low bone strength in the midtibia and a greater infiltration of fat in the bone marrow and surrounding musculature compared to typically developing children. Whether the deficit in the musculoskeletal system of children with CP is associated with higher chronic disease risk and whether the deficit can be mitigated requires further investigation.

Hematologic Toxicity During Anal Cancer Treatment: The Importance of Pelvic Bone Marrow Volume and Limiting Radiation Dose to a Critical Marrow Volume

Hematologic Toxicity During Anal Cancer Treatment: The Importance of Pelvic Bone Marrow Volume and Limiting Radiation Dose to a Critical Marrow Volume

Assessment of Baseline Active Bone Marrow Volume and Correlation to Baseline Hematologic Parameters for Pelvic Malignancies

Assessment of Baseline Active Bone Marrow Volume and Correlation to Baseline Hematologic Parameters for Pelvic Malignancies

A Comparison of Dosimetric Parameters in PET-Based Active Bone Marrow Volume and Total Bone Volume in Prediction of Hematologic Toxicity in Cervical Cancer Patients Treated with Chemoradiation

Objective The purpose of this study is to evaluate whether dose to PET-defined active regions of the bone marrow is a better predictor of hematologic toxicity than dose to the total bone as a surrogate of the bone marrow.

Height is an indicator of vascular maintenance capacity in older men.

Bone-derived circulating CD34-positive cells are reported to play an important role in vascular maintenance. Additionally, height might influence age-related hematopoietic bone marrow decline, as it positively correlates with total bone marrow volume. As hypertension should mask the beneficial effects of circulating CD34-positive cells, hypertension status should account for this correlation. The present study aimed to clarify the clinical importance of height on vascular maintenance in older Japanese men. We carried out a cross-sectional study of 343 older men aged 65-69 years who underwent a general health checkup from 2013 to 2015. Independent of known cardiovascular risk factors, height was found to be slightly, but significantly, positively correlated with the log number of circulating CD34-positive cells in systolic, but not non-systolic, hypertensive men. Multilinear regression analysis showed a parameter estimate (B) and standardized parameter estimate (β) of 3.23 × 10-2 , 0.28 (P = 0.003) for systolic hypertensive men and -0.49 × 10-2 , -0.04 (P = 0.495) for non-systolic hypertensive men. Height positively correlates with circulating CD34-positive cells in systolic, but not non-systolic, hypertensive men. As the beneficial effects of circulating CD34-positive cells on endothelial repair might be masked by hypertension where the production of CD34-positive cells is stimulated by hypertension-induced vascular damage, among individuals with systolic hypertension, circulating CD34-positive cells should indicate the limits of endothelial repair. Therefore, height might indicate the capacity for adequate vascular maintenance in older men. Geriatr Gerontol Int 2017; 17: 1729-1736.

Processing Volumes and Therapeutic Cellular Doses of Point of Care Bone Marrow Concentrates

AIMS: Bone Marrow Concentrates (BMC) applied to treat several osteo-articular pathologies had reported positive clinical outcomes at short- to medium-term follow up. However, the diversity of indications reported and the lack of consensus describing the formulation of BMC make it difficult to circumscribe basic processing variables to generate them. We analyzed the influence of processing different Bone Marrow (BM) volumes over the formulation and deliverable cell dose of BMC. MATERIALS AND METHODS: Main cellular populations were characterized in BMC manufactured and applied for autologous use during the same surgical procedure. To do this, Flow Cytometry and Fibroblastic Colony Forming Units (CFU-Fs) assays were used. RESULTS: Cell concentration of aspirates was not statistically influenced in the range of volumes analyzed. Consequently the quality of BM seems to be conserved in the range of volumes assayed. By using the protocol described, the quality of BMC traditionally defined as CFU-F per mL did not differ in the range of volumes assayed whereas total dose of CFU-F and other differentiated cells effectively changed. CONCLUSIONS: The volume of BM defines cellular doses arriving to the patient with statistically significant differences. Parameters currently used to describe the quality of BMC as CFU-F/mL appear to be directly influenced by working volumes and thus total cellular doses applied might better characterize these products. Finally, since it is uncertain what cells within BMC form part of its active substance, the quantification of main cell subsets could be helpful to better understand where, when and how these medicinal products work.

AN AUTOMATIC 3D CT/PET SEGMENTATION FRAMEWORK FOR BONE MARROW PROLIFERATION ASSESSMENT.

Clinical assessment of bone marrow is limited by an inability to evaluate the marrow space comprehensively and dynamically and there is no current method for automatically assessing hematopoietic activity within the medullary space. Evaluating the hematopoietic space in its entirety could be applicable in blood disorders, malignancies, infections, and medication toxicity. In this paper, we introduce a CT/PET 3D automatic framework for measurement of the hematopoietic compartment proliferation within osseous sites. We first perform a full-body bone structure segmentation using 3D graph-cut on the CT volume. The vertebrae are segmented by detecting the discs between adjacent vertebrae. Finally, we register the bone marrow CT volume with its corresponding PET volume and capture the spinal bone marrow volume. The proposed framework was tested on 17 patients, achieving an average accuracy of 86.37% and a worst case accuracy of 82.3% in automatically extracting the aggregate volume of the spinal marrow cavities.

A comparison of dosimetric parameters in PET-based active bone marrow volume and total bone volume in prediction of hematologic toxicity in cervical cancer patients treated with chemoradiation

The purpose of this study is to evaluate whether dose to PET-defined active regions of the bone marrow is a better predictor of hematologic toxicity than dose to the total bone as a surrogate of the bone marrow. Twenty-one patients with stage IB-IVB cervical cancer who had baseline PET imaging and underwent definitive cisplatin-based chemoradiation from 2010 to 2015 were reviewed. Total bone marrow (TBM) volume was defined as the bone from L4 to the coccyx, the pelvic bones, and proximal femoral heads. Active bone marrow (ABM) was defined by PET as the volume of the bone within the TBM greater than or equal to the mean total body standard uptake value (SUV). Dosimetric parameters evaluated were mean dose, V10, V20, and V40, which were compared by a t test. Hematologic toxicity was graded using Common Terminology Criteria for Adverse Events (CTCAEv4). Receiver operating characteristic (ROC) analysis was used to assess predictors of grade 3 or higher (grade 3+) hematologic toxicity. ABM volume (mean = 1227 mL, range 793–1671 mL) was significantly smaller than the TBM volume (mean = 1553 mL, range 1117–1920 mL) [p = 0.0001]. ROC analysis identified ABM volume (AUC = 0.800, p = 0.002) and V40 to the ABM (AUC = 0.800, p = 0.008) as the best predictors for grade 3+ hematologic toxicity. All ten patients with an ABM volume 1201 mL (Fisher’s p = 0.012). A lower volume of ABM at a baseline (<1201 mL) was highly predictive of grade 3+ hematologic toxicity.

Abstract 355: Heart Failure Impairs Bone Marrow Mesenchymal Stem Cell Renewing Capacity and Migration

Background: Clinical trials have shown a sustained beneficial effect of bone marrow mesenchymal stem cells (MSCs) as a therapy for acute and chronic heart failure (HF). Clinical grade cell manufacturing of autologous and allogeneic MSCs is becoming a standard procedure. This study investigates the differences of bone marrow MSC isolation and expansion in favorable in vitro conditions. We compared MSC production from both healthy young donors and chronic HF patients. Methods: We analyzed MSC manufacturing records from five clinical trials: TAC-HFT (Ischemic cardiomyopathy (CMP), patient and donors), POSEIDON (Ischemic CMP, donors), POSEIDON DCM (Dilated CMP, donors), TRIDENT (Ischemic CMP, donors), and CRATUS (Frailty, donors). Results: All cells expressed CD105, CD90 and lacked hematopoietic marker CD45. The age of healthy donors (25.5 ± 0.7 y.o., N=24) and HF patients (56.1 ± 2.5, y.o., N=23), was significantly different (P&lt;0.0001). Collectively, the number of mononuclear cells (MNCs) isolated from bone marrow (volume range 58-125ml) didn’t differ between the groups. However, plated MNCs from healthy adults generated more MSCs than MNCs from HF patients (p0: 5.9x10^6±0.5x10^6, N=23 vs 3.8x10^6±0.4x10^6, N=24, respectively, P=0.003 and p1: 15.4x10^6±2.5x10^6, N=23 vs 10.8x10^6±1.1x10^6, N=24, respectively, P=0.036). No correlation was found between stem cell growth and age (35 to 78y.o.). Left ventricular ejection fraction (LVEF) in patients with HF had a direct correlation with MSC growth rate (P=0.03), particularly, in patients with severely depressed LVEF (&lt;30%), which had a tendency to generate less MSCs overall. Moreover, MSCs from HF patients demonstrated less migration compared to MSCs from healthy donors at 6, 10 and 24 hours (h) relative to baseline (6 h: 9.5±3.0% vs 30.7±2.1%; 10 h: 19.9±13.7% vs 53.1±2.5% and 24 h: 41.5±15.8% vs 88.9±1.6%, N=4, respectively. P=0.03). Conclusions: Despite equivalent numbers of MNCs, healthy young donors generate significantly more MSCs than HF patients, due to increased growth rate and higher number of resident stromal bone marrow stem cells. MSC migration was impaired in HF patients compared to healthy donors. HF appears to be an independent factor for MSC renewal capacity and culture phenotype.

Prepubertal Di-n-Butyl Phthalate Exposure Alters Sertoli and Leydig Cell Function and Lowers Bone Density in Adult Male Mice.

Phthalate exposure impairs testis development and function; however, whether phthalates affect nonreproductive functions is not well understood. To investigate this, C57BL/6J mice were fed 1-500 mg di-n-butyl phthalate (DBP) in corn oil, or vehicle only, daily from 4 to 14 days, after which tissues were collected (prepubertal study). Another group was fed 1-500 mg/kg·d DBP from 4 to 21 days and then maintained untreated until 8 weeks for determination of adult consequences of prepubertal exposure. Bones were assessed by microcomputed tomography and dual-energy X-ray absorptiometry and T by RIA. DBP exposure decreased prepubertal femur length, marrow volume, and mean moment of inertia. Adult animals exposed prepubertally to low DBP doses had lower bone mineral content and bone mineral density and less lean tissue mass than vehicle-treated animals. Altered dynamics of the emerging Leydig population were found in 14-day-old animals fed 100-500 mg/kg·d DBP. Adult mice had variable testicular T and serum T and LH concentrations after prepubertal exposure and a dose-dependent reduction in cytochrome p450, family 11, subfamily A, polypeptide 1. Insulin-like 3 was detected in Sertoli cells of adult mice administered the highest dose of 500 mg/kg·d DBP prepubertally, a finding supported by the induction of insulin-like 3 expression in TM4 cells exposed to 50 μM, but not 5 μM, DBP. We propose that low-dose DBP exposure is detrimental to bone but that normal bone mineral density/bone mineral content after high-dose DBP exposure reflects changes in testicular somatic cells that confer protection to bones. These findings will fuel concerns that low-dose DBP exposure impacts health beyond the reproductive axis.

Process automation in manufacturing of mesenchymal stromal cells.

Process automation in manufacturing of mesenchymal stromal cells.

Dosimetric predictors of acute hematologic toxicity during concurrent intensity-modulated radiotherapy and chemotherapy for anal cancer

This study aimed at investigating whether the irradiated volume of pelvic bone marrow (PBM) and specific subsites may predict the occurrence of acute hematologic toxicity (HT) in anal cancer patients undergoing concurrent chemo-radiation. 50 patients, submitted to IMRT and concurrent chemotherapy, were analyzed. Several bony structures were defined on planning-CT: PBM and lumbar-sacral (LSBM), lower pelvis (LPBM) and iliac (IBM) bone marrow. On dose-volume histograms, dosimetric parameters were taken. Endpoints included white blood-cell-count (WBC), absolute-neutrophil-count (ANC), hemoglobin (Hb) and platelet nadirs and acute hematologic toxicity (HT) according to RTOG scoring scale. Generalized linear modeling was used to find correlations between dosimetric variables and blood cell nadirs, while logistic regression analysis was used to test correlation with≥G3 HT. Receiver Operating Characteristic (ROC) curve analysis was used to evaluate the optimal cut-off points for predictive dosimetric variables with the Youden method. Maximum detected acute HT comprised 38% of≥G3 leukopenia and 32% of≥G3 neutropenia. Grade 2 anemia was observed in 4% of patients and≥G3 thrombocytopenia in 10%. On multivariate analysis a higher PBM-V 20 was associated with lower WBC nadir. Increased LSBM-V 40 was correlated with a higher likelihood to develop≥G3 HT. A cut-off point at 41% for LSBM-V 40 was found. Patients with LSBM-V 40≥41% were more likely to develop≥G3 HT (55.3 vs. 32.4%; p<0.01). Increased low-dose to pelvic bony structures significantly predicted for WBC decrease. Medium-high dose to specific osseous subsites was associated with a higher probability of HT. LSBM-V 40 was a strong predictor of≥G3 HT. A threshold at 41% for LSBM-V 40 could be used to limit HT.

Identity, proliferation capacity, genomic stability and novel senescence markers of mesenchymal stem cells isolated from low volume of human bone marrow.

Human bone marrow mesenchymal stem cells (hBM-MSCs) hold promise for treating incurable diseases and repairing of damaged tissues. However, hBM-MSCs face the disadvantages of painful invasive isolation and limited cell numbers. In this study we assessed characteristics of MSCs isolated from residual human bone marrow transplantation material and expanded to clinically relevant numbers at passages 3-4 and 6-7. Results indicated that early passage hBM-MSCs are genomically stable and retain identity and high proliferation capacity. Despite the chromosomal stability, the cells became senescent at late passages, paralleling the slower proliferation, altered morphology and immunophenotype. By qRT-PCR array profiling, we revealed 13 genes and 33 miRNAs significantly differentially expressed in late passage cells, among which 8 genes and 30 miRNAs emerged as potential novel biomarkers of hBM-MSC aging. Functional analysis of genes with altered expression showed strong association with biological processes causing cellular senescence. Altogether, this study revives hBM as convenient source for cellular therapy. Potential novel markers provide new details for better understanding the hBM-MSC senescence mechanisms, contributing to basic science, facilitating the development of cellular therapy quality control, and providing new clues for human disease processes since senescence phenotype of the hematological patient hBM-MSCs only very recently has been revealed.