bluuebunny/crossref_metadata_2025 huggingface dataset:
Histone Deacetylation Makes an Important Contribution to Chromatin Condensation and Enucleation during Murine Erythroblast Terminal Differentiation.
Authors: Krauss Sharon Wald , Popova Evgenya , Short Sarah A. , Lee Gloria , Villalobos Jonathan , Koury Mark J. , Grigoryev Sergei , Chasis Joel Anne
Published: April 2020

Mammalian erythroid differentiation is characterized by progressive chromatin condensation and culminates in nuclear extrusion. Core histone modifications are known to play a significant role in formation of heterochromatin in several other differentiated cell types. To elucidate molecular mechanisms governing changes in mammalian erythroblast chromatin preceding enucleation, we used a model system in which Friend virus-infected murine spleen erythroblasts undergo terminal differentiation and enucleation in vitro during 0–44 hours. At the conclusion of terminal differentiation of murine erythroblasts, we found that histone H3(K9) dimethylation markedly increased while acetylation of histone H3(K9) and histone H4(K12) dramatically decreased, strongly correlating with nuclear chromatin condensation. To test the effects of altering histone deacetylation on chromatin condensation and enucleation, we added either 1 or 2 mM sodium butyrate, an inhibitor of histone deacetylase, to cultures of mouse erythroblasts and compared specific parameters of differentiation with untreated control cells, which normally terminally differentiate at 44 hrs. Western blotting revealed that control cells had the predicted dramatic decrease in histone H4(K12) acetylation while 1 mM or 2mM butyrate-treated erythroblasts did not show diminished levels of acetylation. Numbers of erythroblasts were 5- and 8-fold higher at 44 hrs in cultures treated with 1 and 2mM butyrate, respectively, compared to untreated control cells and there was a correlated marked reduction in expelled nuclei and young reticulocytes. We also found that nuclei in the butyrate-treated erythroblast populations were 1.5 fold larger relative to nuclei with condensed chromatin in terminally differentiated erythroblasts in control cultures. Evaluation of hemoglobinization of erythroblasts by benzidine/hemotoxin staining showed that hemoglobin synthesis continued after butyrate treatment, indicating cell viability and the absence of significant butyrate-induced apoptosis. In sum, these data show that butyrate exposure maintains erythroblasts in a nucleated state with relatively uncondensed chromatin and inhibits them from normally differentiating into young reticulocytes. Thus widespread deacetylation of histones appears to be necessary to achieve extensive chromatin condensation during mammalian erythroid maturation. Our data add support to the attractive idea that during normal mammalian erythropoiesis chromatin condensation may be essential for the transition from nucleated erythroblasts to enucleated reticulocytes.
Inhibition of p38 MAPK Reduces Ionizing Radiation-Induced Hematopoietic Cell Injury.
Authors: Liu Lingbo , Zhou Daohong , Wang Yong
Published: April 2020

Our previous studies demonstrated that exposure to ionizing radiation (IR) causes long-term bone marrow (BM) damage by induction cellular senescence in hematopoietic cells. The senescent hematopoietic cells exhibited an elevated activity of the senescence associated β-galactosidase (SA-β-gal), a biomaker for senescent cells, and expressed increased levels of p16Ink4a (p16), whose expression has been implicated in the establishment and maintenance of cellular senescence. Activation of the p38 MAPK pathway has been implicated in the induction of cell cycle arrest and senescence in response to a variety of stressors in part by up-regulating the expression of p16. Therefore, in the present study we examined the role of the p38 MAPK pathway in IR-induced hematopoietic cell senescence. The results showed that exposure of male C57BL/6 mice to a sublethal dose (6.5 Gy) of total body irradiation (TBI) caused a sustained activation (>4 weeks) of p38 in Lin− c-kit+ Sca-1+ (LKS+) hematopoietic stem cells (HSCs). This activation was associated with a persistent up-regulation of p16 and quantitative and qualitative reduction of LKS+ HSCs. Inhibition of p38 activity with SB202190 (a specific p38 inhibitor) attenuated IR-induced inhibition of the hematopoietic function of BM hematopoietic cells in an in vitro colony forming cell (CFC) assay. Moreover, the production of hematopoietic progenitor cells (HPCs) in SB202190-treated BM cells was 7.5 fold higher than that of irradiated cells without SB202190 treatment after five weeks of long-term BM cell culture (LTBMC); and the cells treated with SB202190 after IR retained their clonogenic function while the control irradiated cells lose their ability to form colonies. These findings suggest that p38 may play an important role in mediating IR-induced suppression of hematopoietic cell function and pharmacological inhibition of the p38 pathway may potentially be developed as novel therapeutic strategy to ameliorate IR- and chemotherapy-induced BM toxicity.
Increased Unfolded Protein Response in Hematopoietic Cells from Cyclic Hematopoietic Dogs.
Authors: Meng Ronghua , Upchurch David , Niemeyer Glenn P. , Lothrop Clinton D.
Published: April 2020

Canine cyclic hematopoiesis, a model of human cyclic neutropenia/congenital neutropenia (CN), is caused by a mutation in the AP3b1 gene. Human CN is caused by mutations in the Ela2 gene. The exact mechanism(s) whereby mutations in the Ela2 and AP3b1 cause CN or CH is not well understood. Elastase is directed to the primary granules by AP3 in myeloid progenitor cells, suggesting abnormal trafficking of elastase as a cause for CN/CH. Discontinuous percoll gradients on PMN cells from normal and CH dogs were performed. Fractions corresponding to the cytosol, plasma membrane, nuclei, primary, secondary and tertiary granules were collected and assayed for elastase and myeloperoxidase (MPO) enzyme activity and protein levels by Western blot using canine specific antibodies. Percoll density gradient fractionation results indicated that MPO, a primary granule protein that is not an AP3 cargo protein, is present in the primary granules in approximately equivalent amounts in both normal and CH dog PMN’s. Elastase was also localized in the primary granule fraction from both normal and CH dog PMN, but with a lower amount in CH dogs. Elastase was not present in the plasma membrane fraction in either CH or normal dog PMN’s. Quantitatively the CH dog primary granules had 10–20% of normal dog primary granule elastase. These results suggest PMN’s from CH dogs correctly sort elastase to the primary granules but at a lower level compared to PMNs from dogs without AP3b1 defect. Glucose-regulated protein (GRP78/BiP) is an indicator of ER stress. ER stress leads to activition of the unfolded protein response (UPR) and is cytoprotective. However, prolonged UPR leads to apoptosis. Analysis of GRP78 expression in PMN’s and bone marrow cell cultures from normal and CH dogs stimulated with SCF and G-CSF demonstrated 3–4 fold increase of GRP78 in CH cells compared to normal dog cells. These results indicate mistrafficking or accumulation of misfolded elastase induces the UPR in myeloid precursor cells and disrupts normal PMN production in CH dogs. These results are consistent with in vitro studies in which over-expression of mutant but not normal human elastase induces the UPR in myeloid cells and cell death. These results suggest that induction of the UPR is a common event, in myeloid progenitor cells from patients and animal models with either Ela2 or AP3b1 mutations, which ultimately results in congenital and/or cyclic neutropenia.
Directed Differentiation: Evolution towards Human Application.
Authors: Colvin Gerald A. , Dooner Gerri J. , Dooner Mark S. , Aliotta Jason , Politopoulou Galatia , Abedi Mehrdad , Quesenberry Peter J.
Published: April 2020

Directed differentiation is defined as the ability to program a stem cell at the most primitive level while it still has its reproductive and full proliferative potential. This is in contrast to ex-vivo expansion where the stem cells are forced into specific lineage commitments, limiting the overall therapeutic utility. We have reproducibly induced directed stem cell differentiation towards megakaryopoiesis by capitalizing on inherent changes in sensitivities to inductive cytokine signals in the context of cell cycle position. Murine experiments have been performed on highly purified quiescent G0–1 lineagenegative rhodaminelowHoeschtlow (LRH) marrow stem cells. When exposed to thrombopoietin, FLT3-ligand and steel factor (TFS), they synchronously pass through cell cycle. Megakaryopoiesis is focused at early to mid S-phase, returning to baseline before initial cell division. Population based differentiation cultures after 14-days produced up to 49% megakaryocytes with stem cells sub-cultured during early-mid S-phase with little to no production with colonies cultured from stem cells in G0–1 or G2 phase at time directed differentiation signaling. Gene expression showed over 2 fold increases in FOG, Nfe2 and Fli1. Clonal studies confirm the results. In one experiment, 33% of clonally derived colonies that grew from early-S phase cells and 10% of colonies that grew from mid-S phase cells had megakaryocytes present compared with 0% for G0–1 and G2 cells. We have now worked with human lineagenegative double-effluxed-rhodaminelow double-effluxed-Hoeschtlow G0–1 stem cells. When expose to TFS cytokines, there initial cell cycle lasts more than 80 hours opposed to CD34+ cells and murine LRH cells which have divided by 44–48 hours. This human population of stem cells comprises approximately 0.01% of CD34+ cells and has tremendous promise in replicating our murine work, elucidating opportunities for human translational work targeting patients that have a block of differentiation toward megakaryopoiesis i.e. sub-sets of autologous transplant or myelodysplastic syndrome patients.
Perturbing Hemostasis by Altering the Kinetics of the Interactions between GPIb Apha and the A1 Domain of VWF: A Mouse Tale.
Authors: Diacovo Thomas G. , Tan Kui , Liddington Robert C. , Chen Jianchun
Published: April 2020

Effective hemostasis relies on ability of circulating platelets to be rapidly deposited at sites of vascular injury. Critical to this process is the formation of adhesive bonds between the platelet receptor GPIb alpha and the A1 domain of VWF, an interaction that is rapid and reversible due to the relatively high rate of bond formation and dissociation. The importance of maintaining the kinetic properties of this interaction within a critical range is suggested by the bleeding phenotype that results from gain- or loss-of function points mutations associated with VWD. To demonstrate the an alteration in bond kinetics can have a dramatic effect of hemostasis, we generated a mouse in which a single point mutation was introduced into the vWF-A1 domain that results in a shortening of the time it can maintain contact with GPIb alpha (aka - increase in off-rate). The mutation was chosen based on our crystal structure data of the recombinant mouse A1 domain protein and its effect on mouse platelet adhesion to the immobilized mutant protein under flow conditions. Homozygous animals were viable and exhibited normal Mendelian ratios. Both VWF antigen levels as determined by ELISA and multimer banding pattern were identical to WT littermate controls. Moreover, transcription of the A1-A2-A3 region of VWF was comparable to controls as determined by RT-PCR. Despite the similarities in biochemical and molecular parameters to WT littermates, mutant A1 domain mice had a significant prolongation in bleeding time as measured by severing a small portion of their tail. Moreover, perfusion of whole blood from these animals over surface-immobilized collagen resulted in >50% reduction in platelet accumulation at 1600 s-1. This abnormality in platelet adhesion was also observed in vivo. Although mutant A1 domain mice were able to form thrombi upon laser-induced injury to arterioles located within the cremaster muscle, these thrombi were unstable and rapidly dissolved. In conclusion, we are the first to demonstrate that an alteration in the kinetic properties of a receptor-ligand bond can have a significant effect on biological processes such as hemostasis. Our results also provide insight into mechanisms by which point mutations associated with certain types of VWD may result in bleeding in afflicted individuals.
Mitochondrial Proteome from Human Peripheral Blood Cells.
Authors: Sarry Jean-Emmanuel , Danet-Desnoyers Gwenn-ael , Carroll Martin , Emerson Stephen G. , Daldal Fevzi , Dancis Andrew
Published: April 2020

Mitochondria play a special role in iron metabolism as the site of heme synthesis for hemoglobin. Mitochondria also function in cellular respiration, apoptosis, amino acid synthesis, Fe-S cluster formation and repair, and redox homeostasis; different blood cell lineages depend on some or all of these diverse mitochondrial functions. Mitochondrial abnormalities in hematopoietic stem cells might manifest themselves in proteomes of all the hematopoietic lineages. Therefore, we have begun characterization of mitochondria from different peripheral blood cell populations: platelets, lymphocytes, neutrophils and reticulocytes with the objective of comparing their function and proteomes in normals and in certain disease states. The procedures utilized as starting material a blood draw of approximately 80 ml from normal volunteers. The peripheral blood samples were separated by centrifugation and Hypaque density gradient into platelet, mononuclear cell, neutrophil and red cell populations. The red cells were further sorted by density gradient and magnetic cell sorting with specific CD71 microbeads to obtain enrichment of reticulocytes (reticulocytes retain their mitochondria and lose these upon maturation into mature red cells). The various cell fractions were evaluated by cell counting, flow cytometry and staining for morphology and identification. In accordance with differences in size and surface characteristics of these cell types, different procedures for cell rupture were utilized: shearing with a home-made device using ball bearings (mononuclear cells, neutrophils), nitrogen cavitation (platelets) and hypotonic shock (reticulocytes). Mitochondria were prepared by differential centrifugation and Percoll density gradient separation. The mitochondria were evaluated by fluorescence microscopy, flow cytometry, marker enzyme activity (succinate dehydrogenase) and Western blotting with compartment-specific antibodies. Mitochondrial protein profiles were obtained using 2-dimensional gel electrophoresis coupled to mass spectrometry. From 80 ml blood, 50 million lymphoctes were obtained equivalent to 150 microgram mitochondrial protein and 10 fold enrichment of succinate dehydrogenase activity. In parallel, K562 cell mitochondria were studied. The imaging analysis revealed significant differences in the protein patterns due to hematopoietic cell lineage. This work seeks to establish a proteomic database of shared and distinct erythroid, myeloid and lymphoid mitochondrial proteins that will form the basis of future studies of blood diseases in which perturbations of mitochondrial proteins are expected to occur. We are especially interested in examining the mitochondrial proteome and correlating with mitochondrial function in myelodysplasia and sideroblastic anemia.
Identification of Transcription Factor AP-1 Bound to Untranslated 5′ Regulatory Region DNA of mdr1 Gene with Atomic Force Microscopy.
Authors: Chen Bao-An , Xiong Jie , Ba Long , Gao Feng , Gao Chong , Ding Jia-Hua , Sun Yun-Yu , Cheng Jian , Wang Jun , Zhao Gang , Lu Zu-Hong , Chen Ning-Na , Fei Fei
Published: April 2020

Multidrug resistance(MDR) phenotype of cancer cells is a major obstacle for cancer chemotherapy, this phenotype is main due to the overexprssion of the mdr1 gene. Transcription factor AP-1, which is one of important regulate proteins in the promoter region of mdr1 gene. To date, no direct data of AP-1-DNA regulating complexes on mdr1 gene. A novel method for identifying DNA-binding proteins from image analysis using atomic force microscopy(AFM) was developed. This study is to image and map the structure of Untranslated 5′ regulatory region DNA of mdr1gene, identificate and analysis of transcription factor AP-1 bound to Untranslated 5′ regulatory region DNA of mdr1 gene complex with atomic force microscopy, to find out the molecule mechanism of multidrug resistance.

Human leukemia adriamycin resistant strain K562/A02 was used as a target cells, transcription factor AP-1 was used as a target protein. Cultivate and PCR technique was used to amplify K562/A02 cells with the 769bp 5′regulatory region DNA of mdr1 gene, which fragment from −755 to +14. The amplified DNA products were purified by the PCR product purification kit, AP-1 of hela nuclear extract were purified by Sephadex spin-column filled with a bed of Sephadex G-100. The target DNA fragments were incubated with the target protein AP-1 at a 1:2 molar ratio in binding buffer and then immobilized the the AP-1-DNA complexes on a freshly cleaved mica surface which treated by MgCl2. AFM was used to idificate image of the structure of target DNA and the AP-1-DNA complexes.

We show here the applicability of AFM in the quantitative analysis of the molecular mechanisms of DNA-protein interaction:

The optimum DNA concentration to yield well-absorbed DNA molecular on the mica surface was found to be 10ng/ul. the contour of target DNA AFM image :the length of the DNA fragment measured by AFM image was 260.13± 2.29nm, the width was 11.88 ± 0.92nm, the mean height was 1.2nm(mean±SD, N = 50). Sephadex spin-column (Ultrafree- MC 0.22) can be used to purificate DNA and protein-DNA complex, the contour data of AP-1 protein AFM image:: the width of proteinlarge was 30±3.2nm, protein small was 19±2.8nm; the height of proteinlarge was 3.8±1.4nm, proteinsmall was 3.2±1.8nm (mean±SD, N = 30). we got the contour data of pro- tein-DNA complexes: the width of protein large was 28±2.7nm, the height was 3.4± 0.94nm (mean±SD, N = 8), the width of proteinsmall was 18±1.7nm, the height was 3.1±2.2nm (mean±SD, N = 22), and deduced the possible AP-1 site on mdr1DNA located between bases −126 and −115bp, this result is in close agreement with the expected −121 and −115 bp values.

the overall connection efficiency of protein was about 10%. The AFM method to visualize individual biomolecules allows us to investigate the conformation of protein-DNA complexes.
Genome-Wide Computational Screen for Blood Stem Cell Enhancers Integrates RUNX1 and the BMP Signaling Pathway into the SCL Transcriptional Network.
Authors: Pimanda John E. , Donaldson Ian J. , Bruijn Marella F. , Kinston Sarah J. , Knezevic Kathy , Huckle Liz , Piltz Sandie , Landry Josette-Renee , Green Anthony R. , Tannahill David , Gottgens Berthold
Published: April 2020

Hematopoietic stem cell (HSC) development is regulated by several signaling pathways and a number of key transcription factors, which include Scl/Tal1, Runx1 and members of the Smad family. However, it remains unclear how these various determinants interact. Using a genome-wide computational screen based on the well-characterized Scl +19 HSC enhancer, we have identified a related Smad6 enhancer that also targets expression to blood and endothelial cells in transgenic mice. We show that at E10.5, Smad6 and Bmp4 transcripts are concentrated along the ventral pole of the dorsal aorta and resemble Runx1 expression in the aorta-gonad-mesonephros (AGM) region where HSCs originate. Moreover, Smad6 binds and inhibits Runx1 activity whereas Smad1, a mediator of Bmp4 signaling, transactivates the Runx1 promoter. Taken together, our results integrate three key determinants of HSC development; the Scl network, Runx1 activity and the Bmp4/Smad signaling pathway.
Specific Regulation of NFAT (Nuclear Factors of Activated T Cells) Expression in CD34+ Cells Differentiating into Diverse Hematopoietic Lineages.
Authors: Kiani Alexander , Kuithan Hanna , Kuithan Friederike , Kyttaelae Satu , Habermann Ivonne , Bornhaeuser Martin , Ehninger Gerhard
Published: April 2020

NFAT (Nuclear Factor of Activated T cells) transcription factors are a family of five proteins that are primarily known for their central role in the regulation of inducible gene expression in activated T cells. It is now clear that NFAT proteins are also expressed in various non-immune cell types, where they regulate the expression of genes involved in such diverse cellular processes as proliferation, apoptosis and differentiation. We have previously shown that NFATc2 is strongly expressed in human CD34+ cells and megakaryocytes, but not in purified peripheral blood neutrophil granulocytes and monocytes. Furthermore, granulocytic differentiation of CD34+ cells in vitro was paralleled by the rapid and profound suppression of NFATc2 mRNA and protein. The function of NFATc2 in CD34+ cells, however, is unknown, and no information exists on the expression or regulation of other NFAT family members in CD34+ cells or during heamtopoietic differentiation. To provide a systematic basis for further functional analysis, we established in the present study a comprehensive expression profile of all five NFAT family members in CD34+ cells and during their in vitro differentiation into neutrophil, eosinophil, erythroid and megakaryocytic lineages. CD34+ cells were purified from umbilical cord blood and cultured in the presence of cytokines or cytokine combinations inducing differentiation of the respective lineages. At several time-points during the culture, the efficacy and specificity of the differentiation was monitored by morphological examination of cytospin preparations as well as by analysis of lineage-specific cell surface markers. By quantitative RT-PCR, NFATc3 and NFAT5 were the NFAT family member found to be most prominently expressed in CD34+ cells of both peripheral blood and umbilical cord blood, as well as in the immature CD34+CD38− subpopulation of cells. NFAT expression during the differentiation of umbilical cord blood CD34+ cells into the diverse hematopoietic lineages followed a family member- and lineage-specific pattern. Neutrophil differentiation was accompanied by a rapid suppression of transcript level for all NFAT family members. In contrast, eosinophil, erythrocyte and megakaryocyte differentiation was paralleled by an upregulation of NFATc3, NFATc1/NFATc3 and NFATc1 mRNA, respectively. The most obvious lineage-specific pattern was observed for NFATc4, where transcript levels were low in CD34+ cells and either not or only transiently increased in neutrophil, eosinophil and erythrocyte differentiation; in contrast, they were specifically upregulated about 10-fold in the megakaryocytic lineage. The expression profile of NFAT family members in developing hematopoietic cells of diverse lineages presented here will allow predicting and directly assessing the role of individual NFAT family members in hematopoietic differentiation.
32D TPO, a TPO-Dependent Subclone of the 32d Cell Line, Expresses CXCR4 and Proliferates in Response to SDF-1.
Authors: Amabile Giovanni , Botta Rosanna , Di Noia Antonella , Alfani Elena , Migliaccio Anna Rita , Migliaccio Giovanni
Published: April 2020

Both Thrombopoietin (TPO) and Stromal Derived Factor 1 (SDF-1) have been reported to regulate megakaryocyte differentiation in vivo and in vitro. Using growth factors [G-CSF, M-CSF, GM-CSF, erythropoietin (EPO) or thrombopoietin (TPO)], as selective pressure, factor-dependent clones were consistently isolated from the murine multipotential IL-3-dependent cell line 32D. The different clones acquired a phenotype consistent with the growth factor for which they were dependent. In particular, the EPO-dependent clones showed erythroblast morphology and expressed high levels of CD71 and TER119, on the other hand, the TPO-dependent clones were megakaryocytes in morphology and expressed CD41 and CD61 (see Figure). The TPO-dependent subclones retain the capacity to grow in IL-3, but respond to neither EPO, G-CSF or GM-CSF. Here it is shown that the TPO-dependent clone, but not its EPO-dependent sister (see figure) or parental 32D cell lines, express robust level of CXCR4 on their surface. 32D TPO cells are also capable to respond to SDF-1 (the CXCR4 ligand). In fact, these cells die in 3 days in the absence of growth factors but proliferate equally well (from 5x104 to 6–9x105 cells/mL) in the presence of TPO (100 ng/mL) and/or SDF-1 (100 ng/mL), retaining a robust megakaryocytic phenotype in both culture conditions. By expression profile 32D TPO cells growing in TPO express the same level of Gata1 (both ~2−ΔCt = 3.6 ± 0.5x10−2 ) and Platelet Factor-4 (both 2−ΔCt = 3.5±1x10−1) more Gata2 (2−ΔCt = 2.3±0.1x10−1 vs 1.3±0.2x10−1, respectively), and Fog1 (2−ΔCt = 4.5±1x10−2 vs 3.1±0.5x10−2, respectively), and less Acetylcholinesterase (2−ΔCt = 4.96±1x10−5 vs 6.68±2x10−5, respectively), and Von Willebrand Factor (2−ΔCt = 2.7±0.3x10−2 vs 3.4±0.5x10−2, respectively), than those growing in SDF-1. These data indicate that a TPO-dependent cell line acquires the ability to respond to SDF-1, supporting the notion that megakaryocytic differentiation can be sustained equally well by TPO or SDF-1. To our knowledge, the 32D TPO cell line is the first megakaryocytic cell line that is dependent for growth on the presence of SDF-1. As such, it might represent a suitable model to compare TPO and SDF-1 signalling in megakaryocytes.

Figure Figure
Differential Expression of Hematopoietic Genes in Murine Embryonic E14 Stem Cells upon G-CSF and IL-3 Stimulation.
Authors: Stoelting Stephanie , Fiedler Anja , Wagner Thomas , Peters Stefan O.
Published: April 2020

Introduction:

G-CSF is used widely in humans as in stem cell mobilization protocols for clinical transplantation. Even though it’s mechanisms of action on differentiated cells are well described, it’s effects on stem cells are widely unknown. IL-3 acts as a survival factor without differentiational effects. The presence of G-CSF and IL-3 receptors on embryonic stem cells have been described. We here use murine embryonic E14 stem cells (ESC) to study cellular and molecular events induced by G-CSF and IL-3 during early hematopoietic development.

Methods:

Cells were precultured in standard medium (DMEM, 20% FCS) for 6 days and then kept as embryoid bodies (EBs) for 0, 5, 10, 15, 20, 25, 30 days either in standard or medium continuously supplemented with G-CSF (50 ng/ml) and/or IL-3 (10 ng/ml). Suspended cells were analyzed for CD 90 (thy1) positivity by flow cytometry. CD13 stained cells of embryoid bodies were examined by fluorescent microscopy. cDNA was analyzed for PU.1, GATA-2, Flk-1, SCL, Epo, HoxB4, LMO2, LMO4 and cyclin D1 activation by Taqman PCR.

Results:

First hematopoietic specific CD90 positivity is found on embryoid bodies at day 6+5, either with or without cytokine stimulation. There is a marked increase in hematopoietic development at day 6+10 consisting in 5% CD90 positivity declining at later time points in unstimulated EBs. Interestingly the fraction of CD90 positive cells could not be augmented by G-CSF alone. Upon IL-3 stimulation the fraction of CD90 positive EBs increased twofolded as compared to unstimulated controls. This remained for the entire duration until day 6+30. Double stimulation with IL-3 and G-CSF abrogated this stimulatory effect. We selected a number of genes of interest in hematopoietic development based on published gene expression profiling studies. Under G-CSF stimulation there is a continuous minor upregulation of some of these genes. Interestingly, under IL-3 stimulation most of these genes (GATA-2, SCL, PU.1, Epo and HoxB4) are markedly downregulated while CD90 positive cell fractions are stimulated. This downregulation is most prominent at day 6+10 and 6+15.

Conclusion:

G-CSF activates key genes of hematopoietic development in E14 ESCs without causing differentiation. IL-3 activation of hematopoiesis in EBs parallels downregulation of important genes known as activators of adult hematopoiesis. This could mean that causally determined effects of growth factors may differ at early and committed progenitor cell levels.
Effects of Darbepoetin Alfa Administered Once Per Cycle for the Prevention of Anemia on the Incidence of Transfusions, Neurocognitive Functions, and Symptom Assessment.
Authors: Vadhan-Raj Saroj , Trent Jon , Patel Shreyaskumar , Collard Mindy , Bueso-Ramos Carlos E. , Cleeland Charles , Benjamin Robert , Meyers Christina
Published: April 2020

Background: Chemo-naïve sarcoma patients (pts) experience severe anemia during front-line chemotherapy (CT) with doxorubicin and ifosfamide (AI). In our prior study with AI, vast majority of pts (65 of 68, 93%) who received >1 cycle of CT, required PRBC transfusions. Anemia has significant negative impact on pts quality of life (QOL) and neurocognitive (NC) functions. Prior studies have shown that cancer pts receiving CT experience decline in NC functions and QOL. Preclinical studies suggest that treatment with erythropoietic stimulating agents (ESA) may protect NC functions. The purpose of the present study was to evaluate the effects darbepoetin alfa (DA) administered once per cycle as prophylaxis on the prevention of anemia, need for transfusions, symptom burden and NC functions in sarcoma pts receiving AI.

Methods: DA (initial dose 500 mcg SC) was administered once per cycle before CT (day 0) starting from cycle-1(n=26) or cycle 2 (n=23) and continued up to max 6 cycles. Pts underwent a complete battery of neuropsychological tests and assessment of symptoms via the MD Anderson Symptom Inventory (MDASI) and quality of life with the Functional Assessment of Cancer Therapy-Anemia (FACT-An) Scale at baseline, during CT (after 3 cycles) and at the end of study. At baseline, the median hemoglobin (Hb) was 13.2 g/dL, and only 5 of 49 pts were anemic to a level <11 g/dL. However, significantly more NC deficits or symptoms, including memory loss (29% of pts), fatigue (24%), pain (21%), distress (16%), and sadness (16%) were seen in pts at baseline as compared to normal populations. Pts with higher baseline Hb values had significantly less sadness (p=0.008) and less shortness of breath (p=0.003). Of the 45 pts that received > 1 cycle of CT (median, 4; range, 2–6 cycles), 38% avoided PRBC transfusions. Patients who avoided transfusions had less distress (p=0.032) and less sadness (p=0.01) during treatment. Patients with higher Hb nadir during treatment had less numbness and tingling (p=0.006). Overall, there was a significant decline in verbal fluency, non-dominant hand dexterity (p<0.001), and memory recall (p=0.018). Interestingly, there was a significant improvement in several symptoms including distress (p=0.001), sadness (p=0.032), and pain (p=0.004) during treatment as compared to baseline. These findings suggest that cancer pts can have NC deficits prior to CT. Prevention or early intervention with ESA may avoid the need for transfusions, reduce symptom burden and NC deficit.
Stem Cell Numbers and Quality in a Group of Patients Undergoing Hip Replacement Surgery: Increasing Marrow Fat and Plasma IL-6 Concentrations with Aging.
Authors: McGuire Timothy R. , Brusnahan Susan K. , Jackson John , Berger Ann M. , Garvin Kevin L. , Lane Judy , Murphy Barbara J. , Kessinger Anne , Sharp John Graham
Published: April 2020

Diseases such as anemia which increase with age may be indicative of a decline in stem cell numbers and quality. Anemia in many elderly patients is thought to be mediated by inflammatory cytokines. Interleukin-6 (IL-6) has been reported to increase with age and to have negative effects on myelopoiesis. Another change which may correlate with age and a related decline in stem cell quality is an increase in marrow fat content. The present investigation was designed to evaluate stem cell numbers and quality in a group of patients undergoing hip replacement surgery and correlate stem cell characteristics with the percentage of fat in the marrow and plasma IL-6 concentrations. Marrow stem cells were obtained from the femoral head and trochanter at surgery. Quality was assessed by measuring the proportion of cells in the lower tip of the marrow side population compared to the upper side population by flow cytometry. Fat content in the marrow was measured as a percentage of total volume after sedimentation. A sandwiched-type ELISA was used to measure plasma IL-6 concentrations (R and D Systems). A total of 30 patients were analyzed (36–87 years of age). The number of side-population cells (stem cell numbers) declined with increasing fat content of the marrow. No difference was seen in the ratio between lower and upper side populations (stem cell quality) with age or fat content. Plasma IL-6 concentrations significantly increased with age confirming prior reports (r=0.37; p=0.03). IL-6 concentrations did not correlate with fat content of the marrow. This data suggests that marrow conversion from red (low fat) to yellow (high fat) increases with age along with plasma IL-6 concentrations but these are independent processes. Other inflammatory cytokines are currently under evaluation as potential mediators of the decline in the stem cell compartment with age with particular emphasis on various adipokines since the decline appears to correlate with increased marrow fat content.
A Phase II Study (Biov-121) of Clofarabine Monotherapy First Line in Patients Aged 65 Years or Older with Acute Myeloid Leukemia for Whom Standard Intensive Chemotherapy Is Not Considered Suitable.
Authors: Burnett Alan K. , Baccarani M. , Johnson Peter , Yin John , Saunders Andrew , Russell Nigel , Hills Robert K.
Published: April 2020

Background: The treatment of older patients with AML is unsatisfactory and not improving. A substantial proportion of patients are not offered a standard intensive (“3+7”) chemotherapy approach because they are not considered medically fit (Juliusson et al, Leukemia, 2006 (20):42-7). Such patients are usually treated with best supportive care or low-dose Ara-C with a median survival of approximately 3 months. BIOV-121 is an open-label non-randomised multicentre international study of clofarabine, a next generation purine nucleoside analogue, in elderly patients (≥ 65 yrs) with previously untreated acute myeloid leukaemia (AML) who are unsuitable for standard (“3+7”) treatment.

Methods: 66 patients aged ≥ 65 with untreated AML (WHO classification) were enrolled in study BIOV-121. Clofarabine was administered at a dose of 30mg/m2 for 5 days repeated every 28–42 days (1 course) for a maximum of 3 courses. All patients were considered unsuitable for standard intensive treatment based primarily on age, comorbidity and/or performance status. The primary endpoint of the study was overall response rate defined as the number of patients achieving a CR, CRi or PR according to the international working group guidelines.

Results: 66 patients were recruited with a median age of 71 (range 64–81). 48 patients had de novo and 16 had secondary AML. 72% had an ECOG performance sore of <2; 74% had one or more comorbidities. 31% had an adverse cytogenetic profile; 69% had intermediate cytogenetics (Grimwade D et al Blood 1998). The principal reasons patients were considered unsuitable for standard intensive chemotherapy were age and comorbidity. Nineteen patients achieved a CR; 10 CRi and 3 PR, giving an overall response rate of 48%. 14 patients died (any cause) within 30 days of study start date. The CR/CRi rate in patients over 70 years (49%) was similar to that observed for younger patients (36%). 42% (8/19) of patients with adverse cytogenetics and 31% (5/16) of patients with secondary AML achieved a CR/CRi. The median times to recovery of neutrophils to 1.0 x 109/L and platelets to 100 x 109/L following course 1 were 18 and 27 days respectively. 26% of patients developed neutropenic sepsis. Liver and renal adverse events (NCI CTC grade 3+) were both observed in 23% of patients. The median duration of remission is 6 months; 12 patients have relapsed to date. The median overall survival for all patients is 5 months with a 1 year survival of 26%.

Conclusion: A complete response rate of 44% achieved with clofarabine in older AML patients unsuitable for standard intensive treatment is encouraging and potentially superior to the current standard of care, low dose Ara-C. Of note a good proportion of patients with an adverse cytogenetic profile and/or secondary disease achieved a CR/CRi when treated with clofarabine.
Low Levels of Circulating EPCs Following Bone Marrow Carcinogenic Stimulus Predict the Onset of Hematological Tumors.
Authors: Fragoso Rita , Igreja Catia , Ferreira Manuela , Dias Sergio
Published: April 2020

Endothelial progenitor cells (EPCs) originate in the bone marrow (BM), proliferate, migrate and differentiate into endothelial cells, contributing to the formation of new blood vessels in normalcy and in malignant tumor growth. EPCs have been detected at increased frequency in the circulation of cancer patients and lymphoma-bearing mice, and tumor VEGF production correlates with EPCs mobilization. In order to clarify the contribution and importance of endothelial progenitor cells during tumor development, we established a murine carcinogenesis model that produces hematological neoplasias, namely thymomas and acute leukemia. This model consisted of administering 3 cycles of irradiation (sub-lethally), within one month of interval between them. Every month after the last irradiation, the levels of circulating endothelial progenitor cells (Flk-1+ and Sca+ cells) and hematopoietic progenitor cells (FLT-1+ and Sca+ cells) in peripheral blood were quantified (by flow cytometry). A first assay was done using 30 mice (FVB/N strain), divided into 2 groups: the control group (not irradiated) with 10 mice and the irradiated group with 20 mice. At the end of the assay (~11 months after last irradiation) the incidence of tumors was approximately 63% in the irradiated group (of these, there was an equal incidence of thymomas and acute leukemia). About 37% of the irradiated mice presented signs of disease 2,5 moths after the last irradiation ( called early disease group) while the other 26% presented signs of disease 7,2 months after the last irradiation ( called late disease group). Surprisingly, when comparing the EPC levels between the two groups that developed tumors and the remaining irradiated mice that did not (called no disease group), the two groups of mice that developed tumors had a decreased number of circulating EPC. This decreased was more evident in the early disease group (p<0.01). An inverse relation was observed for VEGF levels in circulation, since the mice with no disease presented lower VEGF levels (5pg/ml) comparied with the two groups that developed tumors (>10pg/ml). At this point, we hypothesized the BM derived EPC levels somehow modulated the onset of hematological disease in irradiated mice. To modulate EPC levels, we transplanted EPCs into irradiated mice with low levels of circulating EPCs. In a preliminary experiment, mice (n=2) that received exogenous EPCs survived, while the mice that did not succumbed to hematological diseases. Taken together, the data obtained suggest a role for EPCs in modulating the BM microenvironment, thus regulating the onset of hematological malignancies. Ongoing studies are addressing possibility. Finally, these results suggest that detection of reduced circulating BM EPCs levels following cyclic whole body irradiation may predict a worse disease outcome.
Cytokines and Chemokines Involved in the Inflammatory Process Have Potent Chemoattractive Properties on MSC.
Authors: Tondreau Tatiana , Meuleman Nathalie , Dejeneffe Marielle , Massy Martine , Delforge Alain , Bron Dominique , Lagneaux Laurence
Published: April 2020

Background : Recent studies have demonstrated the transmigration capacity of mesenchymal stem cells (MSC) over the endothelial cell barrier into various tissues including brain, bone marrow and many others. Nevertheless, little is know about the agents that promote and control their migration.

Methods : The MSC (5.105 cells) migration potential was evaluated using matrigel invasion chamber assay in the presence/absence of SDF-1 (100ng/mL), PDGFbb (100ng/mL), IGF (500ng/mL), IL-6 (100ng/mL) and 10% of conditioned medium in the bottom chamber. The chemotactic properties of these cytokines/chemokines were compared to the spontaneous migration of MSC in DMEM medium without FCS. After 24 hours, cells that migrated through the membrane were counted. The presence of specific receptors was analysed using Raybio Assay, Western Blot and semi-quantitative RT-PCR.

Results : We observed that PDGFbb has the highest chemoattractive property on the MSC and correlates with the strongest expression of PDGF-bb receptor. We also noted that the migratory response to IL-6, IGF and SDF-1 is correlated with the expression of their respective receptors. However, a weak migration was observed in response to SDF-1 due to the low extracellular expression of CXCR4. Nevertheless, a high intracellular expression of CXCR4 was demonstrated with western blots and FACS analysis. In addition, using Raybio assay, we demonstrated the expression of various receptors and metalloproteinases involved in the migration of MSC including ALCAM, L-selectin, ICAM-1, MMP2 and MMP9.

Conclusions : Our observations suggest that chemokines or cytokines involved in the regulation of immunity or the inflammatory process promotes the migration of MSC into bone marrow or damaged tissues.
Homing, Proliferating and Surviving Sites of Human Leukemia Cells In Vivo in Immunodeficient Mice.
Authors: Naoe Tomoki , Ninomiya Manabu , Abe Akihiro , Katsumi Akira , Xu Jinglan , Kiyoi Hitoshi , Kinoshita Tomohiro
Published: April 2020

The cellular components of the hematopoietic stem cell (HSC) niche have been gradually identified. However, the niche for malignant hematopoiesis remains to be elucidated. Here, using human leukemia cells which could be transplanted to immunodeficient mice, we studied the in vivo homing, proliferating and surviving sites by immunohistopathology, compared with the corresponding sites for cord blood CD34+ (CBCD34+) cells. The human leukemia cells initially localized on the surface of osteoblasts in the epiphysial region, and expanded to the inner vascular and diaphysial regions within 4 weeks. The percentage of CD34+ leukemia cells in the BM was transiently increased up to 50% and associated with the entry to S phase of the cell-cycle. In vivo BrdU-labeling showed that the epiphysis was the most active site for leukemia cell proliferation. CBCD34+ cells show the similar pattern of homing and proliferation to leukemia samples. After high-dose administration of Ara-C, residual leukemia cells were localized in the perivascular endothelium as well as in contact with the trabecular endosteum. These findings suggest that xenotransplantation into immunodeficient mice provides a useful model to study the leukemia niche, and that disruption of this niche can potentially facilitate the anti-leukemia effects of various therapies.
Human Myeloid Leukemia Xenotransplant Model Demonstrates Leukemia Blood Vessels from Both Human and Mouse Origin.
Authors: Fu DongTao , Jorgensen Marda L. , Scott Edward W. , Cogle Christopher R.
Published: April 2020

Leukemia stem cell activity has been extensively studied and has been approached mainly in terms of self-renewal within the hematopoietic compartment. However, given the recent findings that the adult hematopoietic stem cell can provide hemangioblast activity - giving rise to both blood and blood vessels - we questioned whether leukemia also exhibits hemangioblast activity. To test this hypothesis we established a xenotransplant model whereby K562 human myeloid leukemia cells suspended in Matrigel are injected into sublethally irradiated NOD/scid mice. Leukemia loaded Matrigel plugs reliably grew in size in all (n=6) immunocompromised animals. After four weeks of leukemia growth the Matrigel plugs were excised and blood vessels evaluated. Out of six animals, two animals demonstrated human endothelial cells within the Matrigel plug, confirmed by LAMP-1 staining. Interestingly, six out of six animals demonstrated mouse endothelial cells within the Matrigel. This in vivo model demonstrates that human myeloid leukemia can provide hemangioblast activity. Moreover, it appears that within this Matrigel xenotransplant model, host blood vessels are also recruited. The significance of these results highlights the importance in recognizing the hemangioblast potential of leukemia stem cells. Ultimately, complete eradication of leukemia may depend on targeting leukemia-derived blood vessels in addition to cytotoxic therapy directed at the hematopoietic compartment.
The Ubiquitin Specific Peptidase 42 Gene (USP42) Is Recurrently Involved as a Fusion Gene with RUNX1 in Acute Myeloid Leukemia with the t(7;21)(p22;q22) Cryptic Translocation.
Authors: Giguere Amelie , Hazourli Sawcene , Meftah Douja , Fetni Raouf , Chagnon Pierre , Hebert Josee
Published: April 2020

The RUNX1 gene encodes the DNA-binding subunit of the heterodimeric transcription factor core binding factor (CBF) and is involved in the regulation of several genes important for hematopoiesis. RUNX1 is frequently rearranged by chromosomal translocations in a spectrum of hematologic malignancies, generating a RUNX1 fusion gene. Some of these RUNX1 fusion genes have been shown to be important in leukemogenesis, such as the ETV6/RUNX1 fusion in B-lineage acute lymphoblastic leukemia and the RUNX1/RUNX1T1 fusion in M2 acute myeloid leukemia (AML). We here describe the first case of adult AML with the t(7;21)(p22;q22) cryptic translocation. Morphologic analysis showed an AML without maturation (AML M1). The karyotype was 46, XY, del(5)(q22q33),?del(21)(q22) in twenty metaphases and analysis by spectral karyotyping (SKY) revealed a t(7;21). The translocation breakpoints at bands 21q22 and 7p22 were studied using bacterial artificial chromosomes (BACs) and showed RUNX1 and USP42 rearrangements. An in-frame fusion between RUNX1 exon 7 and USP42 exon 3 was confirmed by RT-PCR and sequencing. The RUNX1/USP42 fusion includes both the DNA binding RUNT domain of RUNX1 and the catalytic region of USP42. The RUNX1/USP42 fusion gene has recently been reported in one case of pediatric AML-M0 with normal karyotype at diagnosis (Paulsson et al., Leukemia2006;20:224). A complex karyotype including a deletion of the long arm of chromosome 5 was found at relapse in this case. Interestingly, blast cells in our t(7;21) positive leukemia share similar features with the previously published case. By flow cytometry, leukemic cells were positive for HLA-DR, CD34, CD33, CD13 markers and aberrantly expressed CD7 and CD56 without B-lineage markers. We next analyzed samples of CD7 and CD56 positive AMLs with different karyotypes (normal and complex) by FISH using probes to detect the RUNX1/USP42 fusion gene. The USP42 gene encodes a cysteine protease involved in the ubiquitin pathway as a deubiquitinating enzyme. Ubiquitin-specific proteases (USPs) are a large group of enzymes with highly conserved amino acid sequences that form the catalytic region. Some USPs have been shown to be involved in the stabilization of proteins, such as p53, and in the regulation of gene silencing. USP42 is part of a novel class of genes involved in the pathogenesis of leukemia and its role in hematologic cancers remains to be studied. To elucidate the possible implication of USP42 in hematologic malignancies, the expression of USP42 was also investigated by real-time PCR in our case with t(7;21) and in a series of acute leukemias.