Bone Marrow Nucleated Cells Research Articles

Formaldehyde and co-exposure with benzene induce compensation of bone marrow and hematopoietic stem/progenitor cells in BALB/c mice during post-exposure period

Formaldehyde (FA) is a human leukemogen. Since there is a latency period between initial FA exposure and the development of leukemia, the subsequent impact of FA on hematopoietic stem or progenitor cells (HSCs/HPCs) in post-exposure stage is crucial for a deep understanding of FA-induced hematotoxicity. BALB/c mice were exposed to 3mg/m3 FA for 2weeks, mimicking occupational exposure, and were monitored for another 7days post-exposure. Meanwhile, we included benzene (BZ) as a positive control, separately and together with FA because co-exposure occurs frequently. After 7-day recovery, colonies of progenitors for CFU-GM and BFU-E, and nucleated bone marrow cells in FA-exposed mice were comparable to controls, although they were significantly reduced during exposure. Levels of reactive oxygen species (ROS) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in CFU-GM and BFU-E from FA-exposed mice were higher than controls, although the increase in 8-OHdG was not significant. Granulocyte-macrophage colony stimulating factor (GM-CSF) level in the FA group was lower than controls, but the expression level for the receptor was not upregulated. It suggests that HSCs/HPCs in FA-exposed mice respond to a small amount of GM-CSF and proliferate rapidly, which may cause a possible risk of expansion of abnormal stem/progenitor cell clones. FA co-exposure with BZ was more potent for promoting CFU-GM formation and inducing ROS in BFU-E and 8-OHdG in CFU-GM during the post-exposure period. The compensation of myeloid progenitors with elevated ROS and 8-OHdG may lead to a risk of transforming normal HSCs/HPCs to leukemic stem/progenitor cells. Thus, co-exposure may pose a greater leukemia risk.

Enrichment and Molecular Analysis of Breast Cancer Disseminated Tumor Cells from Bone Marrow Using Microfiltration

PurposeMolecular characterization of disseminated tumor cells (DTCs) in the bone marrow (BM) of breast cancer (BC) patients has been hindered by their rarity. To enrich for these cells using an antigen-independent methodology, we have evaluated a size-based microfiltration device in combination with several downstream biomarker assays.MethodsBM aspirates were collected from healthy volunteers or BC patients. Healthy BM was mixed with a specified number of BC cells to calculate recovery and fold enrichment by microfiltration. Specimens were pre-filtered using a 70 μm mesh sieve and the effluent filtered through CellSieve microfilters. Captured cells were analyzed by immunocytochemistry (ICC), FISH for HER-2/neu gene amplification status, and RNA in situ hybridization (RISH). Cells eluted from the filter were used for RNA isolation and subsequent qRT-PCR analysis for DTC biomarker gene expression.ResultsFiltering an average of 14×106 nucleated BM cells yielded approximately 17–21×103 residual BM cells. In the BC cell spiking experiments, an average of 87% (range 84–92%) of tumor cells were recovered with approximately 170- to 400-fold enrichment. Captured BC cells from patients co-stained for cytokeratin and EpCAM, but not CD45 by ICC. RNA yields from 4 ml of patient BM after filtration averaged 135ng per 10 million BM cells filtered with an average RNA Integrity Number (RIN) of 5.3. DTC-associated gene expression was detected by both qRT-PCR and RISH in filtered spiked or BC patient specimens but, not in control filtered normal BM.ConclusionsWe have tested a microfiltration technique for enrichment of BM DTCs. DTC capture efficiency was shown to range from 84.3% to 92.1% with up to 400-fold enrichment using model BC cell lines. In patients, recovered DTCs can be identified and distinguished from normal BM cells using multiple antibody-, DNA-, and RNA-based biomarker assays.

Myeloprotective effects of C-type natriuretic peptide on cisplatin-induced bone marrow granulocytopenia in mice.

Cisplatin is an effective chemotherapeutic agent used to treat a variety of malignant tumors. The major toxicity associated with cisplatin treatment is granulocytopenia. C-type natriuretic peptide (CNP), a member of the natriuretic peptide family, protects against toxicity in many organs, including the heart, blood vessels, lung, and kidney. The objective of this study was to investigate the myeloprotective effects of CNP in a mouse model of cisplatin-induced granulocytopenia. The mice were divided into two groups: cisplatin with vehicle and cisplatin with CNP. CNP (2.5μg/kg/min via osmotic pump, subcutaneously) or vehicle administration was started two day before cisplatin injection, and continued until the mice were killed. At 0, 2, 4, 8, and 14 days after cisplatin injection (16mg/kg, intraperitoneally as a single dose), we counted total and living cells and granulocyte/macrophage colony-forming units (CFU-GM) in bone marrow. In addition, at 0, 1, 2, and 4 days after cisplatin injection, we measured mRNA levels of CXC chemokine receptor 4 (CXCR4) and chemokine CXC ligand 12 (CXCL12) in bone marrow. CNP significantly attenuated the reduction in bone marrow nucleated cell count and CFU-GM in bone marrow at 4 days after cisplatin injection. Four days after cisplatin injection, CNP significantly decreased the CXCR4 mRNA level in bone marrow, but had no effect on the level of CXCL12 mRNA. CNP exerts myeloprotective effects in cisplatin-induced granulocytopenia and decreases CXCR4 expression.

Distribution and differentiation of myeloid-derived suppressor cells after fluid resuscitation in mice with hemorrhagic shock.

To investigate the distribution and differentiation of myeloid-derived suppressor cells (MDSCs) in hemorrhagic shock mice, which are resuscitated with normal saline (NS), hypertonic saline (HTS), and hydroxyethyl starch (HES). BALB/c mice were randomly divided into control, NS, HTS, and HES resuscitation groups. Three subgroups (n=8) in each resuscitation group were marked as 2, 24, and 72 h. Flow cytometry was used to detect the MDSCs, monocytic MDSCs (M-MDSCs), and granulocytic/neutrophilic MDSCs (G-MDSCs) in peripheral blood nucleated cells (PBNCs), spleen single-cell suspension, and bone marrow nucleated cells (BMNCs). The MDSCs in BMNCs among three resuscitation groups were lower 2 h after shock, in PBNCs of the HTS group were higher, and in spleen of the NS group were lower (all P<0.05 vs. control). The M-MDSC/G-MDSC ratios in PBNCs of the HTS and HES groups were lower (both P<0.05 vs. control). At 24 h, the MDSCs in PBNCs of the NS and HTS groups were higher, while the spleen MDSCs in the HTS group were higher (all P<0.05 vs. control). The M-MDSC/G-MDSC ratios were all less in PBNCs, spleen, and BMNCs of the NS and HTS groups, and were lower in BMNCs of the HES group (all P<0.05 vs. control). At 72 h, the elevated MDSCs in PBNCs were presented in the HTS and HES groups, and in spleen the augment turned up in three resuscitation groups (all P<0.05 vs. control). The inclined ratios to M-MDSC were exhibited in spleen of the NS and HTS groups, and in PBNCs of the NS group; the inclination to G-MDSC in BMNCs was shown in the HES group (all P<0.05 vs. control). HTS induces the earlier elevation of MDSCs in peripheral blood and spleen, and influences its distribution and differentiation, while HES has a less effect on the distribution but a stronger impact on the differentiation of MDSCs, especially in bone marrow.

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.

Effect of acoustic pulses and EHF radiation on multipotent marrow stromal cells in tissue engineering constructs

In this paper, we studied the effects of physical factors, such as, acoustic pulses of laser-induced hydrodynamics (ALIH) and extremely-high frequencies (EHF) radiation, on the formation of heterotopic bone marrow organs. A suspension of precipitated bone marrow cells from CBA mice were exposed to ALIH pulses and EHF radiation separately and in their combination tissue engineering constructs, presenting gelatin sponges 2 by 2 by 2[Formula: see text]mm in size containing 107 nucleated bone marrow cells, were exposed to physical factors and were implanted under the renal capsules of syngeneic mice. The newly formed hematopoietic organs were examined in three and five months later after treatment. The five months old transplants were bigger in size than the three months old transplants. The number of hematopoietic cells in the rest of the groups increased during this period by a factor from 3 to 10, the increase being as high as 20-fold in the ALIH[Formula: see text]EHF group. Maximal concentration of multipotent stromal cells (MSCs) was in the EHF[Formula: see text]ALIH, and minimal concentration was in the ALIH[Formula: see text]EHF. The accumulation rate of bone capsule weight was highest for the transplants of EHF[Formula: see text]ALIH and ALIH-sponge groups during the first three months. These data showed that the combined impact of the EHF[Formula: see text]ALIH on MSCs is the most effective for the formation of bone marrow transplantation.

Protective effect of sugar polyphenols A on radiation injury in mice

Objective To study the radiation protection effect of sugar polyphenols A obtained by structured modification using mouse model of radiation injury induced by 137Cs γ-ray. Methods Total 42 male Kunming mice aged 4-6 weeks were randomly divided into six groups: negative control group (NCG), positive control group (PCG), irradiation control group (ICG) as well as three irradiation experimental groups. The radiation injury model was developed by once whole-body irradiation with 6.0 Gy 137Cs γ-ray. The mice in three experimental groups were fed sugar polyphenols A respectively with doses of 0.05, 0.10 and 0.20 g/kg, for 14 d before irradiation, and continuously fed with the drug for 7 d after the irradiation. After that the mice were sacrificed, and the organ index, hematological parameters, spleen colony number, bone marrow DNA content and nucleated cells in bone-marrow were tested to evaluate the effect of sugar polyphenols A. Results Compared with the ICG, the irradiation experimental groups with the sugar polyphenols A doses of 0.05, 0.10, 0.20 g/kg showed reductions in irradiation injuries of immune system and hematopoietic system, which makes the significant increases of the spleen indexes (Z=-2.364, -3.003 and -2.747, P<0.05) and the number of endogenous spleen colony (Z=-2.458, -2.141 and -2.206, P<0.05). In the groups with the doses of 0.05 and 0.2 g/kg, the number of white blood cells were significantly increased (Z=-2.652 and-2.139, P<0.05). Besides, in the group with the dose of 0.2 g/kg, the level of red blood cells (Z=-2.364, P<0.05), bone marrow DNA content (Z=-2.492, P<0.05) and nucleated cells in bone-marrow (Z=-2.492, P<0.05) were also increased compared with the ICG. Conclusions Sugar polyphenols A has the positive effects on the recovery of immune and hematopoietic function of mice through improving the organ index, endogenous spleen colony number, peripheral white blood cell number, bone marrow DNA content and nucleated cells in bone-marrow, which can reduce the injury induced by ionizing radiation. Key words: Structural modification; Sugar polyphenols A; Ionizing radiation; Hematological parameters; Endogenous spleen colony; Bone marrow DNA

The development and biological characteristics of a novel potentially radioresistant inbred mouse strain.

The growth of biomedical research over the previous decades has been accompanied by an increase in the number, complexity and diversity of experimental animals developed as research tools, and inbred mice are some of the most widely used. However, thus far, no inbred mice have exhibited strong radioresistance for use in radiation-damage research. To develop a radioresistant mouse model, a female Japanese outbreeding strain ICR/JCL mouse was mated with a male Chinese inbred strain 615 mouse. From the F1 generation, the mouse line was maintained by brother-to-sister mating. A novel mouse strain was established over >20 continuous generations and designated the Institute of Radiation Medicine-2 (IRM-2) mouse. The biological characteristics, genetic characteristics and susceptibility to radiation of these mice were determined. The IRM-2 mice inherited traits from the parents, including strong reproductive capacity, stable physiological and biochemical indices and few differences among individuals. According to the genetic results, the IRM-2 mice exhibited homozygosity, isogenicity and consistency, in agreement with international standards for inbred strains. Radiosensitivity studies have previously suggested that the lethal dose (LD)50 values for IRM-2 mice were 7.17 Gy (male) and 7.5 Gy (female), resulting in a dose reduction factor value of 1.39 (male) and 1.37 (female). The mortality of IRM-2 mice irradiated with 8 Gy total body irradiation was 15% at day 9 and 90% at day 15 after radiation. The number of nucleated cells in bone marrow, DNA content and colony-forming unit-spleen counts in IRM-2 mice after exposure to γ-ray irradiation were markedly higher than the corresponding values for the parental strains, suggesting that the IRM-2 mice exhibit high resistance to ionizing radiation. Thus, it is suggested that this novel inbred mouse strain may be developed as an animal model of radioresistance for future use in radiation research.

Bone Marrow Granulocytes Drive Vascular and Hematopoietic Regeneration

In addition to eliminating host hematopoietic cells myeloablation also disrupts the blood vessels that sustain hematopoiesis. Regeneration of the bone marrow (BM) vasculature is necessary for hematopoietic recovery and survival after transplantation (Cell Stem Cell. 2009 Mar 6;4(3):263-74) but the mechanisms that drive vascular regeneration are not clear.We found that, fourteen days after lethal irradiation and transplantation, mice transplanted with 20x106 bone marrow nucleated cells (BMNC) had ~6-fold more CD45-Ter119-CD31+CD105+ endothelial cells (6.9x103 vs 0.96x103 EC/femur, p<0.001), 2-fold more blood vessels (195 vs 87 blood vessels/sternum, p<0.05) and ~2-fold less vascular leakage (4.8 vs 9.3 ng of Evans Blue/ml of BM extracellular fluid, p<0.001) than mice transplanted with 105 BMNC. Transplant experiments using GFP+donor BMNC revealed that all endothelial cells after transplantation were host derived.Because hematopoietic progenitors inhibit vascular regeneration via angiopoietin 1 (Elife 2015 Mar 30;4:e05521) we hypothesized that mature hematopoietic cells mediated vascular recovery. To test this we adoptively transferred, B and T cells, monocytes and macrophages (MO), granulocytes and erythroid cells into lethally irradiated recipients previously transplanted with 105 donor BMNC. Only CD115-Gr1+ granulocytes promoted endothelial cell regeneration (2.5x103 for granulocyte treated mice vs 0.9x103 for PBS, 0.3x103 for B- and T-cell, 0.7 1x103 for MO and 0.3x103 EC/femur for erythroid cell-treated mice; p<0.01). Granulocyte transfer also promoted survival (granulocytes=100%, PBS=50% p<0.05), probably due to faster host platelets and red blood cells recovery (granulocytes= 4.5x107, PBS=2.1x107 platelets/ml of blood, p<0.001; granulocytes=4x109, PBS=6.2x109 RBC/ml of blood, p<0.01). Importantly, competitive BM transplants showed that granulocytes did not exhaust donor HSC. These demonstrate that granulocyte transfer is sufficient to promote survival and drive vascular and hematopoietic recovery after transplantation. We then generated Mrp8-cre:iDTR mice which allowed us to specifically ablate BM granulocytes via diphtheria toxin (DT) injection. We transplanted lethally irradiated WT recipients with 106 BMNC purified from C67BL/6 WT or Mrp8-cre:iDTR mice followed by DT treatment for 7 days. This led to granulocyte depletion (1.6x106 vs 0.4x106 p<0.001) and impaired endothelial cell recovery (5.7x103 vs 2.4.x103 p<0.05) in mice transplanted with Mrp8-cre:iDTR BMNC. These results demonstrate that donor granulocytes are necessary for vascular regeneration.We found that granulocytes produced high levels of the angiogenic cytokine TNFα. This cytokine signals via Tnfrsf1aand Tnfrsf1b. Tnfrsf1a was upregulated specifically in BM endothelial cells. After myeloablation with 5-fluorouracil Tnfa-/- mice have reduced survival (Tnfa-/-= 13% vs WT= 93%; p<0.001) and reduced endothelial cell numbers (WT=9x103, Tnfa-/-=4.1x103 EC/femur; p<0.05) indicating that TNFα is necessary for survival and vascular regeneration after myeloablation. To test whether granulocytes promoted vascular regeneration via TNFα we lethally irradiated and transplanted C57BL/6 recipients followed by treatment with PBS or adoptive transfer of 106 WT or Tnfa-/- granulocytes. Only WT granulocytes induced vascular recovery as demonstrated by quantification of endothelial cells (PBS=0.9 x103, WT granulocytes=5.24x103 and Tnfa-/- granulocytes=3.0x103 cells/femur, p<0.05) and blood vessel numbers (PBS=126, WT granulocytes=186 and Tnfa-/- granulocytes=84 vessels per sternum BM; p<0.05). Further, adoptive transfer of WT granulocytes promoted survival and vascular regeneration (WT+PBS=1.4x103 vs WT+granulocytes=2.6x103, p<0.05; Tnfrsf1a-/-:Tnfrsf1b-/- +PBS=0.8x103 vs Tnfrsf1a-/-:Tnfrsf1b-/-+granulocytes=0.7x103 EC/femur p=0.83) in WT but not Tnfrsf1a-/-:Tnfrsf1b-/-recipients after transplantation. These experiments demonstrate that granulocytes crosstalk directly with stromal cells (likely endothelial cells) via TNFα to drive vascular regeneration.We have identified a new type of cellular crosstalk in the microenvironment that drives regeneration. Our research also provides proof of principle for studies targeting BM granulocytes to enhance vascular recovery and survival after transplantation in patients. DisclosuresNo relevant conflicts of interest to declare.

Comparative Transcriptome Analyses of Laser-Captured Human Primary Megakaryocytes with Platelets from the Same Healthy Donors and from Cord Blood Derived Megakaryocytes

Anucleated platelets have a rich repertoire of RNAs, many of which have provided insights into megakaryocyte-platelet biology and have been proposed as disease biomarkers. Although it is generally believed that the platelet transcriptome is a reflection of the megakaryocyte (MK) RNA content and is transported into platelets during pro-platelet production, this is a poorly studied concept. Healthy human bone marrow (BM) is difficult to access and because MKs represent <0.05% of nucleated cells in BM, making it technically challenging to obtain a pure population. Cultured MKs from CD34-positive umbilical vein cord blood have advantages of being human and initially primary cells, but it is unknown how well the transcriptomes of differentiating MKs in culture correlate with primary BM MKs. Microdissection of BM aspirates by laser capture scanning microscopy can overcome purity limitations by isolating virtually 100% MKs. The goals of this study were to assess the transcriptome correlations between laser-captured primary human MKs (LC-MKs) and both (1) platelets from the same individuals who donated the BM and (2) cord blood derived MKs (CB-MKs).BM aspirates and peripheral blood were collected from three healthy donors. Mature BM MKs were identified by a Board certified Hematologist using standard morphologic criteria, and approximately 200 MKs were microdissected by laser capture scanning microscopy from each aspirate. Peripheral blood platelets were isolated from the same donors followed by leukocyte and erythrocyte depletion. Separately, CD34-positive cells were isolated from umbilical vein cord blood, cultured under standard MK-producing conditions with thrombopoietin and stem cell factor, and CD61-positive CB-MKs were isolated at day 6, 9 and 13 in three independent experiments. Total RNA was extracted from LC-MKs, platelets and CB-MKs and subjected to next-generation RNA-sequencing (total of 15 samples: 5 cell types from 3 independent experiments). The Table summarizes the RNA-seq read numbers generated and the number of protein-coding genes determined for each sample. Notably, the LC-MK RNA yielded fewer reads because the input (estimated in the low picogram range) was less than the other samples. Several known platelet transcripts, including PPBP, PF4, B2M, TMSB4X and OST4 were abundant in our LC-MKs RNA-sequencing analysis. All samples exhibited high expression (1,721-74,923 FPKM) of mitochondrial transcripts.To assess RNA correlations across cell types, we only considered transcripts expressed in both comparison groups. mRNAs from LC-MKs were statistically significantly correlated with mRNAs from both platelets and day 13 CD41a-CD42a-positive CB-MKs. These correlations were highest for the most abundantly expressed (FPKM>50) transcripts (Spearman rank correlation Rho = 0.71 for LC-MKs vs. platelets, and Rho = 0.67 for LC-MKs vs. day 13 CB-MKs). However, even the lower expressed transcripts (using the standard threshold of FPKM>0.3) showed moderate correlation (Rho = 0.37-0.42, p value <E-13). Regardless of transcript abundance, the platelet transcriptome displayed higher correlations with LC-MKs than did CB-MKs. Of the 400 mRNAs common to all LC-MKs samples, 23 were not identified in any of the platelet samples and 9 were not identified in any of the d13 CB-MKs. A secondary goal was to compare transcriptome profiles of CB-MKs during differentiation. Not surprisingly, mRNAs from cultured CB-MKs showed higher correlation when their time in culture was shorter than longer (Rho = 0.87 for d13 vs. d9; 0.81 for d9 vs. d6; and 0.64 for d13 vs. d6). During CB-MK differentiation, 825 transcripts were up-regulated whereas 2725 transcripts were down-regulated.In summary, we provide the first report of transcript profiling from normal, primary human MKs. Peripheral blood platelet RNA transcript levels were representative of MK transcripts from the same subjects, especially if abundantly expressed; cultured MK mRNAs correlated strongly with LC-MKs, while platelet RNAs from the same donor showed an even stronger correlation. mRNA expression differences between platelets and BM MKs could provide insights into thrombopoiesis and may be due to (1) regulated transcript partitioning from MK to platelets, (2) differential RNA endocytosis or exocytosis or (3) RNA degradation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genetic Profile of Acute Erythroid Leukemia

IntroductionAcute erythroid leukemias (AELs) are infrequent acute leukemias characterized by a predominant erythroid population, dysplasticity and ring sideroblasts. The 2016 revision of the World Health Organization (WHO) Classification re-classified previous AELs as myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) not otherwise specified (non erythroid subtype, or pure erythroid type), according to blast percentage in all nucleated bone marrow cells. Recent studies have offered the evidence that genetic profiles are associated with pathophysiology and clinical outcomes in myeloid malignancies. However, characteristic mutation profiles of AEL and its genetic differences or similarities with other-types of AML and MDS have not been fully elucidated due to the lack of a sufficient number of samples, although frequent TP53 mutations have been reported (35-43.5%).MethodWe performed a comprehensive genetic study, in which paired tumor/normal DNA from 24 AEL cases were analyzed by whole-exome sequencing (WES). Subsequently, in total 41 cases were screened for mutations in 69 driver genes associated with myeloid malignancies using targeted-capture sequencing. Genome-wide copy number analysis was also performed based on allele frequency of heterozygous single nucleotide polymorphisms and sequencing depth. Frequencies of driver mutations in AEL were compared to those in other-types of AML analyzed by the Cancer Genome Atlas Network (TCGA) (N = 197).ResultsThe mean number of nonsynonymous mutations identified by WES was 10 per sample (range, 0-21), which was similar to that in other-types of AML or MDS. Most frequently mutated genes were STAG2, TP53, RUNX1 and TET2 (17%, respectively), followed by WT1 and BCOR (13%, respectively). All TP53 mutated cases harbored complex karyotypes. Subsequent targeted sequencing identified 3.4 mutations per sample on average (range, 0-9). TP53 was mutated in 8 cases (20%), and lower frequency of TP53 mutations in our cohort was attributed to the relatively lower proportion (21%) of cases with complex karyotype compared to those in the previous reports. Commonly mutated genes in other-types of AML, such as NPM1, FLT3, and DNMT3A, were relatively infrequent in AEL and affected 7 (17%), 4 (9.8%) and 3 (7.3%) cases, respectively. Genes associated with splicing machinery were mutated in 5 cases (12%), including SRSF2 (9.8%) and U2AF1 (2.4%). Of note, mutations in genes implicated in cohesin complex were observed in as many as 32% of cases, including those in STAG2 (24%), SMC1A (4.9%) and RAD21 (2.4%). Mutations in epigenetic regulators were also frequently identified in AEL, including those in WT1 (20%), TET2 (15%) and IDH1/2 (7%). Mutations in TP53 were mutually exclusive with those in cohesin complex (p = 0.04) and epigenetic regulators (p = 0.05). When we compared the frequencies of recurrent mutations in AEL to those in other-types of AML analyzed by TCGA, mutations in STAG2 (p = 6*105), SRSF2 (p = 7*104), WT1 (p = 0.01), GATA2 (p = 0.008), and BCOR (p = 0.02) were significantly more frequent in AEL, whereas those in FLT3 (p = 0.01) and DNMT3A (p = 0.007) were significantly less frequent in AEL. Mutations in cohesin complex and splicing factors were frequently observed both in AEL and MDS, and higher frequency of these genetic lesion in AEL rather than other-types of AML suggested that AEL and MDS are similar disease entities. Recently, genomic subgroups of AML with mutations in cohesin complex, spliceosome and TP53 have been associated with poor prognosis in AML, and poor prognosis of AEL was thought to be due to higher frequency of these genetic lesions.ConclusionWES and follow-up targeted sequencing revealed the genetic landscape of AEL. Frequent mutations in TP53, splicing factors and cohesin complex were characteristic to AEL, and thought to be involved in its pathophysiology. Our data showed the genetic similarities between AEL and MDS, which were compatible with the partial integration of AEL into MDS in the recent revision of WHO classifications. DisclosuresKataoka:Kyowa Hakko Kirin: Honoraria; Yakult: Honoraria; Boehringer Ingelheim: Honoraria. Ogawa:Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding. Heuser:Pfizer: Research Funding; BerGenBio: Research Funding; Novartis: Consultancy, Research Funding; Tetralogic: Research Funding; Celgene: Honoraria; Karyopharm Therapeutics Inc: Research Funding; Bayer Pharma AG: Research Funding.

Impaired Mitophagy of Erythrocytes Leads to Anemia in Patients with Myelodysplastic Syndromes

Objective: To investigate the change of mitophagy level of bone marrow nucleated red blood cell in anemia patients with myelodysplastic syndromes (MDS), to explore its significance in the anemic pathogenesis of MDS.Methods: Fifty anemia patients with MDS and twenty-six normal controls were enrolled in this study. The mitophagy were observed by transmission electron microscopy (TEM). The level of autophagy-associated protein LC3B, the number of mitochondria, mitochondrial membrane potential, the level of ROS in GlycoA + nucleated red blood cell were measured by Flow cytometry .The expression of AMPK, ULK1, mTOR mRNA of GlycoA + nucleated red blood cell were measured by real time PCR. The expression of the mitochondrial outer membrane protein TOM20 in GlycoA + nucleated red blood cell were detected by Western blotting.Results: (1) Autophagosome or autolysosome including mitochondria were scarcely observed by TEM. (2) The expression of LC3B in GlycoA + nucleated red blood cell in high-risk MDS patients(0.22±0.12) was significantly lower than that in normal controls (0.43±0.22, P<0.01), and lower than that in low-risk MDS patients (0.40±0.16, P<0.01), there were no significant difference in expression of LC3B between normal controls and low-risk MDS patients (P>0.05); (3) The number of mitochondria in GlycoA + nucleated red blood cell in high-risk MDS patients (937.17±707.85) was significantly higher than that in normal controls (513.49±372.33, P<0.05), and higher than that in low-risk MDS patients (461.74±438.02, P<0.01), there were no significant difference in number of mitochondria between normal controls and low-risk MDS patients (P>0.05); (4) The level of mitochondrial membrane potential in GlycoA + nucleated red blood cell in high-risk MDS patients(4.24±3.00)was significantly higher than that in normal controls (2.04±1.11, P<0.01), and higher than that in low-risk MDS patients (2.11±1.10, P<0.01), there were no significant difference in the level of mitochondrial membrane potential between normal controls and low-risk MDS patients(P>0.05); (5) The level of ROS in GlycoA + nucleated red blood cell in high-risk MDS patients(438.65±322.83)was significantly higher than that in normal controls (242.77±136.87, P<0.01), and higher than that in low-risk MDS patients(197.40+95.07, P<0.01), there were no significant difference in the level of ROS between normal controls and low-risk MDS patients(P>0.05); (6) The expression of AMPK mRNA in GlycoA + nucleated red blood cell in high-risk MDS patients(1.06±1.51) was significantly lower than that in low-risk MDS patients (4.35±4.79, P>0.05), there were no significant difference with normal controls(P>0.05). (7) The exception of ULK1 mRNA in GlycoA + nucleated red blood cell in high-risk MDS patients (0.64±0.91) was significantly lower than that in normal controls (2.70±3.27, P<0.01), and lower than that in low-risk MDS patients(4.82±4.81, P<0.01);(8)The level of mTOR mRNA in GlycoA + nucleated red blood cell in high-risk MDS patients (2.81±2.80) was significantly higher than that in normal controls (1.28±0.81, P<0.05),and higher than that in low-risk MDS patients (0.85±0.74, P<0.05), there were no significant difference in expression of AMPK,ULK1and mTOR mRNA between low-risk MDS patients and normal controls (P>0.05). (9) The expression of mitochondrial outer membrane protein TOM20 in high-risk MDS patients was higher than that in normal controls.Conclusion: The pathogenesis of anemia in MDS may be correlated with declining mitophagy level, increasing damaged mitochondria, increasing ROS, DNA damage, and shortening the life of nucleated red blood cells, it may be an effective method to induce mitophagy to correct anemia in MDS. DisclosuresNo relevant conflicts of interest to declare.

Telomere Length and Somatic Mutation in Aplastic Anemia- Correlation with Response to Treatement

Background: Telomere length (TL) of cell reflects the cellular age and short TL suggest the potential susceptibility to DNA damage. We investigated whether 1) the treatment response is associated with TL of hematopoietic cells and 2) whether somatic mutations are associated with TL in patients with aplastic anemia (AA).Methods: We measured the mean TL and heterogeneity of bone marrow nucleated cells at the single cell level by quantitative fluorescence in situ hybridization. A total of 146 AA patients were enrolled; AA (n=146) and normal peripheral blood control (n=147). TL was expressed as telomere/centromere ratio. We analyzed the average and the distribution of TL belonging to the lowest 10th percentile of cell population. Along with measurement of TL, target sequencing for 88 hematopoiesis-related genes was performed to detect somatic mutations.Results: We investigated the frequencies of cytogenetic aberrations and somatic mutations in 437 patients with aplastic anemia (AA) with prognostic relevance. Clonality was determined by G-banding/ fluorescent in situ hybridization (FISH) (n=280 patients), and targeted capture sequencing for 88 hematopoiesis-related genes (n=77). Of 437 patients, 20 (4.6%) showed disease progression and monosomy 7(50%) was the most predominant cytogenetic evolution at disease transformation. One third of patients (25/77, 32.4%) presented at least 1 mutation and the number of mutated genes was 18 and frequently mutated genes was NOTCH1 (3/25, 12%), NF1 (3/25, 12%), SCRIB (2/25, 8%), BCOR (2/25, 8%), DIS3 (2/25, 8%), DNMT3A (2/25, 8%), MED12 (2/25, 8%). In contrast to that 4.6% of AA patients showed disease progression, 4.0% of patients with SM showed disease progression. The telomere length of bone marrow nucleated cells was measured at single cell level by FISH (n=146) patients. Mean TL of AA (T/C ratio 6.99) was significantly shorter than normal control (T/C ratio 11.24). Patients with cytogenetic aberrations (CA) and/or somatic mutation (SM) showed significantly shorter TL compared to AA with normal karyotypes. However, the severity of AA did not correlate with TL. Patients with TL<6.88 showed significantly lower CR to IST (p=0.008) (Fig.1)Conclusion: Patients with TL attrition showed poor response to IST. Short TL can be used not only as a biomarker for AA, but also as a predictive marker for treatment response to IST. In addition, CA and/or SM was strongly associated with adverse prognosis or treatment response, so we strongly suggest to adopt more sensitive test for the detection of minor clonal cell population in AA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Florfenicol induces more severe hemotoxicity and immunotoxicity than equal doses of chloramphenicol and thiamphenicol in Kunming mice

Amphenicols are effective, broad-spectrum antibiotics that function by inhibiting the peptidyl transferase activity of bacteria, while the drugs can also inhibit mitochondrial protein synthesis in eukaryotes through the same mechanism, which leads to multi-organ toxicity. Some side effects of each drug have been studied, while differences in the severity of the hemotoxicities and immunotoxicities of amphenicols have not been reported. Thus, it is important to identify, evaluate, and compare the potential hemotoxicities and immunotoxicities to guide their proper use in humans and animals, which will guarantee food safety and animal welfare. Ovalbumin-immunized Kunming mice were gavaged daily with amphenicols for seven days. Blood samples were collected for hematology analysis, and measuring anti-ovalbumin antibody levels and serum intereukin-2 concentrations. The bone marrow, spleen and thymus were collected for histopathology and apoptosis analyzes. Bone marrow nucleated cells (BMNCs) and splenocytes were harvested to determine their cell cycle stages and to analyze lymphocyte proliferation. The results demonstrated that amphenicols, especially florfenicol (FLO), induced cell cycle arrest and apoptosis of hematopoietic cells, and it changed the bone marrow hematopoietic microenvironment by decreasing the number of peripheral blood cells. Moreover, amphenicols, especially FLO, induced hypoplasia and atrophy of the spleen and thymus, induced cell cycle arrest, as well as splenocyte apoptosis, and decreased the proliferation and viability of lymphocytes and the humoral and cellular immunity of the treated mice. These results suggest that amphenicols induce hemotoxicity and immunotoxicity to some extent, and that FLO induces more severe toxicity than equal doses of chloramphenicol (CAP) and thiamphenicol (TAP).

Bone marrow granulocytes drive hematopoietic and vascular regeneration

Bone marrow (BM) vascular regeneration is absolutely necessary for hematopoietic recovery after myeloablation, yet the mechanisms of vascular regeneration are poorly understood. We found that, two weeks after lethal irradiation and BM transplantation, mice transplanted with 20x106 bone marrow nucleated cells (BMNC) had ∼6-fold more CD45-Ter119-CD31+CD105+ endothelial cells (6.9x103 vs 0.96x103 p < 0.001), increased blood vessels (195 vs 97 vessels per segment, p < 0.05), and reduced BM vascular leakage (4.8 vs 9.3 ng/mL of extravasated Evans Blue dye p < 0.001) than mice transplanted with 105 BMNC. These indicated that hematopoietic cells crosstalk with the vasculature to promote vascular regeneration. Adoptive transfer of the major BM hematopoietic populations into lethally irradiated mice demonstrated that only Gr1+CD115- granulocytes are capable of promoting vascular regeneration (2.52x103 for granulocyte treated mice vs 0.9x103 for PBS p < 0.01, 0.3x103 for B- and T-cells, 0.7x103 for monocyte/macrophages, and 0.3x103 for erythroid lineage treated mice). Granulocyte transfer also promoted survival (granulocytes=100%, PBS=50% p < 0.05), probably due to faster recovery of blood platelets and red blood cells in peripheral blood (granulocytes= 4.5x107 PLT/ml and 6.2x109 RBC/ml; PBS= 2.1x107 PLT/ml and 4x109 RBC/ml, p < 0.01). To investigate the mechanisms of granulocyte-mediated vascular recovery, we profiled expression of angiogenic factors. We found that granulocytes were the main source of TNFα in the BM and Tnfrsf1a (the TNFα receptor) was specifically upregulated in BM endothelial cells. To test whether TNFα is necessary for vascular regeneration, we myeloablated WT or Tnfα-/- mice with 5-fluorouracil. Tnfα-/- mice showed dramatically reduced survival (13% vs 93% p < 0.001) and reduced numbers of BM endothelial cells (4.2x103 vs 9x103 p < 0.05) when compared with WT mice. Taken together these results indicate that TNFα is necessary for survival and vascular regeneration after myeloablation. In adoptive transfer experiments only WT, but not Tnfα-/- granulocytes promoted vascular regeneration (PBS= 0.9x103, WT= 5.24x103, and Tnfα-/- = 3.0x103endothelial cells/femur, p < 0.05). These results demonstrate that BM granulocytes crosstalk with the endothelial cells via TNFα to promote vascular and hematopoietic regeneration.

Short telomere length and its correlation with gene mutations in myelodysplastic syndrome

BackgroundTelomere erosion can lead to genomic instability and cancer progression. It has been suggested that the shortest telomere, not the average telomere length (TL), is critical for cell viability. Some studies have shown shorter TL in myelodysplastic syndrome (MDS) patients but the critically short telomeres, the variability of TL within individual patient has not been evaluated. Thus, we aimed to investigate the TL of MDS patients and assessed the association of TL with recurrent genetic mutations in MDS.MethodsWe measured the TL of bone marrow nucleated cells for diagnostic samples at a single-cell level by quantitative fluorescence in situ hybridization (Q-FISH) for 58 MDS patients and analyzed the minimum, median, average, standard deviation, average of the 0th to 10th percentile TL within a patient, and the proportion of cells with TL that is shorter than the lowest 10th percentile of the normal control (NC). The correlations of TL to clinical parameters, cytogenetic results, and genetic mutations were assessed.ResultsMDS patients showed eroded telomeres and narrow distribution compared to the NC (P < 0.001, P = 0.018, respectively). Patients with mutation showed significantly lesser cells with short TL, below the lowest 10th percentile of the NC (P = 0.017), but no differences in TL were found according to mutations/cytogenetic abnormalities except for CSF3R mutation. However, those patients with a high percentage (≥80 %) of cells with short TL showed poorer overall survival (P = 0.021), and this was an independent prognostic factor, along with TP53, U2AF1 mutation, and high BM blast count (P = 0.044, 0.001, 0.004, 0.012, respectively).ConclusionsThe shortest TL, which determines the fate of the cell, was significantly shorter, and higher burden of cells with short TL were found in MDS, which correlated with poor survival, suggesting the need to measure TL in single cells by Q-FISH.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0287-9) contains supplementary material, which is available to authorized users.

Radioprotective effects of nitroxide compound NHCOCH3-TEMPO in BALB/c mice

Objective To investigate the protective effects of nitroxide compound NHCOCH3-TEMPO against γ-ray irradiation-induced injury in male BALB/c mice. Methods 120 BALB/c male mice were randomly divided into two super group, each super group then were randomly divided into 6 groups: control group, normal saline(NS)+ γ group, low-dose TEMPO+ γ group, middle-dose TEMPO+ γ group, high-dose TEMPO+ γ group and the positive control (WR-2721)+ γ group. BALB/c mice were intraperitoneally injected with nitroxide compound at 0.5 h before whole body 60Co γ-ray irradiation. The drug dosages were as follows: WR-2721 group 200 mg/kg, low-dose TEMPO group 100 mg/kg, middle-dose TEMPO group 200 mg/kg, high-dose TEMPO group 400 mg/kg. The first super group of the mice was given 7 Gy γ-ray whole-body irradiation, and then 30 d survival rate was observed. The second super group of the mice was given 4 Gy γ-ray whole-body irradiation. The number of nucleated cells and the content of DNA in bone marrow, as well as the blood routine were detected. Results At 8th and 15th day after 4 Gy γ-ray irradiation, the number of bone marrow nucleated cells, the content of DNA significantly increased in TEMPO+ γ-ray groups compared with NS+ γ-ray group(t=2.53-6.13, P<0.05). The number of white blood cells significantly increased in middle dose TEMPO+ γ-ray groups compared with NS+ γ-ray group(t=4.34, P<0.05), while there was no significant difference in the number of red blood cells between the TEMPO+ γ-ray group and the NS+ γ-ray group. After 7 Gy γ-ray irradiation, the survival rate of mice on the 30th day significantly increased in low-dose TEMPO+ γ-ray group compared with NS+ γ-ray group(χ2=5.934, P<0.05). Conclusions The nitroxide compound NHCOCH3-TEMPO could provide radio protective effects in BALB/c mice. Key words: Nitroxide compound; BALB/c mice; γ-rays; Radiation protection

Formaldehyde induces toxicity in mouse bone marrow and hematopoietic stem/progenitor cells and enhances benzene-induced adverse effects.

Formaldehyde (FA) is a human leukemogen and is hematotoxic in human and mouse. The biological plausibility of FA-induced leukemia is controversial because few studies have reported FA-induced bone marrow (BM) toxicity, and none have reported BM stem/progenitor cell toxicity. We sought to comprehensively examine FA hematoxicity in vivo in mouse peripheral blood, BM, spleen and myeloid progenitors. We included the leukemogen and BM toxicant, benzene (BZ), as a positive control, separately and together with FA as co-exposure occurs frequently. We exposed BALB/c mice to 3mg/m3 FA in air for 2weeks, mimicking occupational exposure, then measured complete blood counts, nucleated BM cell count, and myeloid progenitor colony formation. We also investigated potential mechanisms of FA toxicity, including reactive oxygen species (ROS) generation, apoptosis, and hematopoietic growth factor and receptor levels. FA exposure significantly reduced nucleated BM cells and BM-derived colony-forming unit-granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-E); down-regulated GM-CSFRα and EPOR expression; increased ROS in nucleated BM, spleen and CFU-GM cells; and increased apoptosis in nucleated spleen and CFU-GM cells. FA and BZ each similarly altered BM mature cells and stem/progenitor counts, BM and CFU-GM ROS, and apoptosis in spleen and CFU-GM but had differential effects on other end points. Co-exposure was more potent for several end points. Thus, FA is toxic to the mouse hematopoietic system, including BM stem/progenitor cells, and it enhances BZ-induced toxic effects. Our findings suggest that FA may induce BM toxicity by affecting myeloid progenitor growth and survival through oxidative damage and reduced expression levels of GM-CSFRα and EPOR.

P38 MAPK Inhibitor Insufficiently Attenuates HSC Senescence Administered Long-Term after 6 Gy Total Body Irradiation in Mice.

Senescent hematopoietic stem cells (HSCs) accumulate with age and exposure to stress, such as total-body irradiation (TBI), which may cause long-term myelosuppression in the clinic. However, the methods available for long-term myelosuppression remain limited. Previous studies have demonstrated that sustained p38 mitogen-activated protein kinases (p38 MAPK) activation in HSCs following exposure to TBI in mice and the administration of its inhibitor twenty-four hours after TBI may partially prevent long-term myelosuppression. However, long-term myelosuppression is latent and identified long after the administration of radiation. In this study, we investigated the effects of SB203580 (a small molecule inhibitor of p38 MAPK) on long-term myelosuppression induced by TBI. Mice with hematopoietic injury were injected intraperitoneally with SB203580 every other day five times beginning 70 days after 6 Gy of 137Cs γ ray TBI. Our results at 80 days demonstrated that SB203580 did not significantly improve the TBI-induced long-term reduction of peripheral blood cell and bone marrow nucleated cell (BMNC) counts, or defects in hematopoietic progenitor cells (HPCs) and HSC clonogenic function. SB203580 reduced reactive oxygen species (ROS) production and p-p38 expression; however, SB203580 had no effect on p16 expression in the HSCs of mice. In conclusion, these findings suggest that treatment with SB203580 70 days after TBI in mice inhibits the ROS-p38 oxidative stress pathway; however, it has no therapeutic effect on long-term myelosuppression induced by TBI.

Hematopoietic stem cell-specific GFP-expressing transgenic mice generated by genetic excision of a pan-hematopoietic reporter gene.

Selective labeling of specific cell types by expression of green fluorescent protein (GFP) within the hematopoietic system would have great utility in identifying, localizing, and tracking different cell populations in flow cytometry, microscopy, lineage tracing, and transplantation assays. In this report, we describe the generation and characterization of a new transgenic mouse line with specific GFP labeling of all nucleated hematopoietic cells and platelets. This new "Vav-GFP" mouse line labels the vast majority of hematopoietic cells with GFP during both embryonic development and adulthood, with particularly high expression in hematopoietic stem and progenitor cells (HSPCs). With the exception of transient labeling of fetal endothelial cells, GFP expression is highly selective for hematopoietic cells and persists in donor-derived progeny after transplantation of HSPCs. Finally, we also demonstrate that the loxP-flanked reporter allows for specific GFP labeling of different hematopoietic cell subsets when crossed to various Cre reporter lines. By crossing Vav-GFP mice to Flk2-Cre mice, we obtained robust and highly selective GFP expression in hematopoietic stem cells (HSCs). These data describe a new mouse model capable of directing GFP labeling exclusively of hematopoietic cells or exclusively of HSCs.