bluuebunny/crossref_metadata_2025_split huggingface dataset:
Automated Quantitative Assessment of Bone Marrow in Normal Samples: Correlation with the Reference Method.
Authors: Zini Gina , Garzia Mariagrazia , Di Mario Antonella , Rossi Elena , Farina Giuliana , d’Onofrio Giuseppe 
Published: April 2020

Abstract
Bone marrow (BM) analysis is conventionally performed by microscope examination on films of about 0.3ml of aspirated bone marrow fluid, stained with Romanowsky dyes. Until 1996 the simple automated screening of marrow composition was made very difficult by a number of factors, mainly the lack of the erythroblasts quantitation and the fat interference. From 1996 last generation automated hematology analyzers provide accurate and precise erythroblasts counts; moreover same systems have improved their software reducing the problem of fat interference.
We have analyzed data from 100 normal BM samples from patients submitted for diagnostic and/or follow up purposes in our Hematology Day Hospital. BM fluid was harvested from the superior posterior iliac crest. The first 0,3–0.5 ml were used for smears, while the next 1–2 ml of BM, collected into K3-EDTA, were analysed with Coulter LH 750, a fully automated hematology analyzer which provides Complete Blood Count, White Blood Count Differential included Nucleated Red Blood Cells (NRBC) and Reticulocytes count. We used the microscope examination conventionally performed on films stained with Romanowsky dyes as reference method. Quantitative detection BM cellularity was obtained by semi quantitative evaluation based on the evaluation of hematopoietic cells in several marrow particles: physiological differences age related were also taken in account. If hematopoietic cells occupy less than 25% or more than 85% the sample is defined respectively hypocellular or hypercellular (none of our sample was as). Differential cell count was usually performed on two different slides counting 500 cells (1000 when hypercellular, but none of our sample was as).
We found a strict correlation between microscope semi-quantitative cellularity evaluation and the instrumental cell count as sum of WBC plus NRBC, the Total Nucleted Cell Count (TNCC).
The mean value of the TNCC in normal PM samples was 29,48 x109/L with a range 25,9–54,9 x109/L. These results are in good agreement with normal BM cell count reported in the literature using a cytofluorimetric method, which is 34,5 x109/L (SD28.0).
The instrumental mean percentage of BM granulocytes corrected for TNNC was 62% (range: 23,5–93,7) versus a mean microscope percentage of 58,42% (range: 40–72).
The automated NRBC BM count corrected for TNCC was 11,38% (range: 2,7 – 39,17) versus a microscopic mean value of 28% (range: 9–45). These results, including the slight NRBC underestimation probably due to partial mature cell lysis, are in line with the data of the literature.
This study confirms the feasibility of routine automated cell count using a hematology in normal BM fluid samples. Automated methods will support morphologists quickly providing accurate and precise quantitative information such as TNCC and myeloid/erythroid ratio.
Development of New Small Molecule Bone Marrow Radioprotectors.
Authors: Niu Yunyun , Kanai Anthony J. , Wipf Peter , Zhang Xichen , Greenberger Emily E. , Franicola Darcy , Epperly Michael W. , Kagan Valerian E. , Fink Mitchell P. , Greenberger Joel S. 
Published: April 2020

Abstract
New ways to protect the bone marrow from irradiation damage are required. We are currently developing new small molecules that can be given orally to protect the marrow from irradiation damage. One small molecule is EUK-134, a manganese superoxide dismutase (MnSOD) mimetic. A nitric oxide synthase inhibitor, and a nitroxide homologue which have been both conjugated to a mitochondrial localization peptide signal, delivers the NOS inhibitor (another radioprotector) and nitroxide to the mitochondria. In addition, we tested ethyl pyruvate (EP), which has been demonstrated to protect tissues from ischemia-reperfusion injury at the level of the mitochondria. Incubation of 32D cl 3 hematopoietic cells 1 hour before irradiation in the presence of 20 μM EUK-134 or 100 μM of NOS inhibitor demonstrated an increased shoulder on the irradiation survival curve in one case (n = 5.55 ± 0.17 for EUK-134 compared to 1.60 ± 0.38 for control irradiated, respectively, p = 0.0044) and increase in the D0 for the mitochondrial targeted NOS inhibitor compared to irradiated control 32D cl 3 cells (D0 = 3.04 ± 0.27 compared to 1.29 ± 0.01 Gy, p = 0.0236). In experiments with 32D cl 3 cells in EP before irradiation or following irradiation, there was an increase in the D0 to 2.2 ± 0.25 and 2.21 ± 0.15, respectively, compared to irradiated controls 1.42 ± 0.09 Gy for the 32D cl 3 cells (p = 0.0447 and 0.0119, respectively). Furthermore, incubation of 32D cl 3 cells in EP both before and after irradiation significantly increased the shoulder on the survival curve compared to irradiated control 32D cl 3 cells (n = 4.14 ± 1.59 compared to 1.70 ± 0.6 for the control irradiated cells, respectively, p=0.0485). To determine whether EP protected the bone marrow from irradiation in vivo, C57BL/6J mice were injected with EP intraperitoneally (70 mg/kg) 30 minutes before irradiation, daily for 5 days after irradiation, or both before and following irradiation to the LD 50/30 TBI dose of 900 cGy. Mice injected with EP either after irradiation or both before and after irradiation had prolonged survival compared to irradiated control mice or those injected with EP only before irradiation. (Percent survival at 50 days 100% and 100%, respectively, compared to 60%, p&lt;0.05). These results demonstrate the potential effectiveness of new small molecule bone marrow radioprotectors.
Age-Related Transcription Levels of KU70 and BIK in CD34+ Hematopoietic Stem and Progenitor Cells.
Authors: Prall Wolf C. , Czibere Akos , Grall Franck , Zerbini Luiz F. , Jaeger Markus , Spentzos Dimitrios , Libermann Towia A. , Gattermann Norbert , Aivado Manuel , Haas Rainer 
Published: April 2020

Abstract
There is a higher incidence of hematological clonal stem cell disorders in elderly persons. Age-related alterations of hematopoietic stem and progenitor cells (HSC) may represent one factor underlying this observation. However, the molecular changes related to stem cell aging are largely unknown. Therefore, we scrutinized gene expression patterns of HSC from umbilical cord blood (CB) as well as bone marrow from young (BM-Y, mean age 32,8, SD 12,4) and old (BM-O, mean age 88,8, SD 4,4) healthy donors. CD34+ HSC were isolated via immuno-magnetic separation and CD34+ purity was evaluated by FACS analysis. Thereafter we performed cDNA array analyses on a first set of samples (n=18). We found that the BCL2-interacting killer gene (BIK) and the gene encoding KU Antigen 70kD (KU70) show age-related mRNA expression levels. BIK is a proapoptotic gene and its expression was positively correlated with donor’s age, i.e. lowest in CB, 1.8-fold higher in BM-Y and 4.2-fold higher in BM-O. KU70 is a DNA repair gene and part of the DNA dependent protein kinase (DNA-PK). Its expression was negatively correlated with donor’s age showing highest expression levels in CB, 2.2-fold lower levels in BM-Y and 5.3-fold lower levels in BM-O. These findings were confirmed with a second set of samples (n=16) by means of quantitative RT-PCR. Elucidation of age-dependent molecular alteration in healthy HSC might facilitate a better understanding of hematological malignancies in elderly persons.
Development of a Model for the Study of PU.1 Function in Primary Human Hematopoietic Cells.
Authors: Durual Stephane , Rideau Alexandra , Wiznerowics Maciej , Ruault Sylvie , Beris Photis , Piguet Vincent , Matthes Thomas 
Published: April 2020

Abstract
PU.1 is one of the best-studied transcription factors governing hematopoiesis and has been shown to regulate positively differentiation of B-lymphocytes and granulocytes. PU.1 is also expressed in early erythroid progenitors and its interaction with GATA-1 was described to directly inhibit erythroid differentiation, since GATA-1 is the key regulator of erythropoiesis. In addition, the binding of GATA-1 to PU.1 was found to repress PU.1 dependent myeloid gene expression. In order to study more in detail the effect of PU.1 in primary human hematopoietic cell differentiation, we designed lentiviral vectors which allow PU.1 overexpression and PU.1 inhibition.
For PU.1 overexpression, we cloned the PU.1 cDNA into the pWPIR-ires-GFP bicistronic plasmid and verified by transient transfection in 293T cells the production of PU.1 mRNA and of right sized protein. We analyzed PU.1 function by co-transfection assays into 293T cells using the pWPIR-PU.1 vector and CAT reporter genes governed by PU.1 responsive elements. By CAT ELISA assay we observed a dramatic increase in OD. The production of PU.1 mRNA and protein in Hela cells was verified by stable transduction with complete lentivectors.
For PU.1 inhibition, we constructed a lentiviral vector encoding a siRNA specific for PU.1. After transduction of the K562 erythroleukemic cell line, both PU.1 mRNA and protein became undetectable, as verified by RT-PCR and Western blot, respectively, whereas GATA-1 mRNA and protein expression remained unchanged.
We tested both viral constructs in an in vitro culture system, in which CD34+ hematopoietic precursors obtained from bone marrow aspirates, differentiate into mature red cells under the influence of SCF, IL-3 and Epo or into mature granulocytes by stimulation with thrombopoietin, SCF and Flt-3L. Results for cultures with PU.1 transduced cells showed inhibition of erythroid cell differentiation by 40% ± 10% (mean of three experiments) and increased myeloid proliferation, whereas cultures with siPU.1 transduced cells showed no influence on erythroid cells and strong decrease of myeloid cell proliferation (50 – 60x) and differentiation (90 % decrease of CD13+ cells).
In conclusion, our model gives us the opportunity to test the function of PU.1 overexpression and/or inhibition in primary hematopoietic cells, to test the effect on target genes in various stages of differentiating precursors and the interaction with other transcription factors like GATA-1, and to analyze pathologic conditions like some forms of acute myeloid leukemia, where PU.1 was described to be mutated or downregulated.
Upregulation of HOXB4 Message in Human CD34+ Cells Transduced with TAT-NF-YA Fusion Protein.
Authors: Domashenko Alevtina D. , Heyward Christa , Hexner Elizabeth , Zhu Jiang , Emerson Stephen G. 
Published: April 2020

Abstract
We have recently found that the trimeric transcription factor NF-Y is a direct stimulator of hematopoietic stem cell (HSC) renewal, activating the transcription of several genes involved in HSC self-renewal and differentiation including the Hox4 paralogs HoxB4, HoxC4, HoxD4 as well as LEF1, Notch1, p27 and telomerase (Zhu J. et al. PNAS, 102:11728–11733; 2005). HIV-TAT peptide transduction offers a distinct therapeutic advantage over viral vectors due to the temporary and regulatable nature of the induction. We have previously modeled the delivery of NF-YA protein fused with the protein transduction domain of the HIV-1 TAT protein to K562 cells, demonstrating upregulated HOXB4 to levels similar to that achieved by retroviral overexpression (Domashenko A. et al. Blood, 104:964a; 2004). We have now asked whether TAT-NF-YA protein induction can be modified for direct application to normal human CD34+ cells. Recombinant protein was designed by fusion of the human NF-YA sequence with the TAT protein transduction domain and a GST-tag at the N-terminus, and purified under native conditions. Binding activity of fusion protein to HOXB4 promoter probe was confirmed by electrophoretic mobility shift assay (EMSA). Human peripheral blood CD34+ cells were cultured with GST-TAT-NF-YA for 1.5h and displayed bright fluorescence in the cytoplasm and nucleus after staining with anti-GST-Alexa Fluor 488 Ab. To evaluate the effect of TAT-NF-YA fusion protein on HOXB4 promoter activity in primary hematopoietic cells, the level of HOXB4 mRNA was measured by real-time PCR with normalization to GAPDH. It was found that delivered NF-YA was able to increase endogenous HOXB4 message up to 3.5-fold in 2h after incubation with the protein. To minimize the recombinant protein toxicity and maximize the efficiency of delivery we investigated the possibility of having the same functional effect at low protein concentration using lysosomotropic agents (chloroquine and sucrose), to facilitate fusion protein nuclear uptake. The effect of varying concentrations of each agent on protein delivery and cell survival was analyzed by determination of endogenous HOXB4 message using quantitative PCR and trypan blue staining. Both chloroquine and sucrose increased the effect of upregulation of the HOXB4 message by GST-TAT-NF-YA 1.5 – 2.5 fold. Chloroquine exhibited substantial toxicity to the cells whereas sucrose was improving cell survival; therefore we have used sucrose in subsequent experiments. A sustained several fold activation of HOXB4 expression in CD34+ cells exposed to GST-TAT-NF-YA (40 nM) and sucrose (250 mM) has been observed for at least 24 h following exposure to the protein. In conclusion, our results showed the ability to transduce functionally active NF-YA protein into human primary hematopoietic cells. These findings support the further employment of protein transduction techniques for development of new therapeutic strategies.
Gene Regulation during the Erythrocytic Differentiation of Embryonic Stem Cells.
Authors: Carrier Ewa , Kausal Shermila , Srivastava Anand S. 
Published: April 2020

Abstract
We have studied the in vitro differentiation of murine embryonic stem cells (ES cells) towards erythropoiesis and expression of genes during this process. It has been reported that dexamethasone directs ES cells towards erythrocytic differentiation but the mechanism of gene regulation induced by dexamethasone is not well understood. We hypothesized that dexamethasone induces upregulation of erythropoietic genes such as GATA-1, FLK-1, EPO-R and directs ES cells towards erythropoietic differentiation. Murine ES cells (129 CCE) obtained from Dr. Nagy laboratory, Canada (Nagy et al., Histochem Cell Biol., 2001; 115:49–58) were subjected to the in vitro primary hematopoietic differentiation media containing methylcellulose, IMDM, IL -3, IL-6 and SCF (stem cell factor) without LIF (leukemia inhibitory factor) to promote embryoid body (EB) formation. Total RNA was collected on day 3, 5 and 9 EBs for gene expression studies using RT-PCR. On day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combination 1) SCF, EPO, dexamethasone, IGF, 2) SCF, IL-3, IL-6, TPO, 3) SCF IL-3, IL-6, TPO, EPO. Total RNA from day12 of secondary differentiated ES cells was collected to study cytokines and growth factors dependent erythrocytic differentiation and gene regulation, using RT-PCR. Our results demonstrate upregulation of Gata-1, Flk-1, HoxB-4, Epo-R and globin genes (α-globin, BH-1 globin, β-major globin, e -globin and z-globin) in the 9 days old EBs, whereas, RNA collected from 5 days old EBs showed expression of HoxB-4, e-globin, γ-globin, BH1-globin and FLK-1. Three days old EBs showed only HoxB-4 and FLK-1 gene expression and lack of expression of globin genes, indicating that erythtropoiesis-specific genes activate later. Gene expression studies of RNA collected from secondary differentiated ES cells and media containing dexamethasone showed downregulation of GATA-3 and upregulation of GATA-1, Flk-1 and Epo-R in comparison to the two other cytokines and growth factors media combination. These results confirm our hypothesis that dexamethasome induces erythropoiesis by down regulating GATA -3 and upregulating erythropoietic-related genes such as GATA-1, Flk-1 and Epo-R. The morphological characteristics of cells after secondary differentiation showed enhanced production of erythrocytic precursors in dexamethasone containing media, which corresponded with molecular studies. Further studies will address the role of wnt/β-catenin and E-cadherin in this process.
The Combination of Stem Cell Factor and Oncostatin M Maintains Cord Blood-Derived NOD/SCID-Repopulating Cells.
Authors: Gilfillan Siv , Kraner Simone , Goetze Katharina S. , Schiemann Matthias , Marz Stefanie , Peschel Christian , Oostendorp Robert A.J. 
Published: April 2020

Abstract
We here investigated whether receptor tyrosine kinase signaling (ckit, flt3) was sufficient to maintain hematopoietic stem cells (HSC) in culture, or whether, like in embryonic stem cells, signaling through gp130 is also required. Sorted CD34+ AC133+ (CD33/ CD38/ CD71)- cells from human umbilical cord blood (CB) were cultured in the presence of combinations of ckit-ligand stem cell factor (SCF), flt3-ligand (FL) and different stimulators of gp130 (IL-6, IL-11, LIF, OSM, CT1 and CNTF). Of these cytokines, SCF stimulated cell division by itself. Gp130-stimulating cytokines did not give any additional cell division activity. When SCF was included in the cytokine cocktail, progenitor levels (CFC, CAFCw2, CAFCw6) was very similar for all combinations of cytokines tested after this initital 6-day culture. To study more immature progenitors, cells were harvested after 6 days of serum-free culture and cultured for another 6 weeks on FBMD-1 stromal cells (LTC-CAFC). These experiments showed that in initial 6 day cultures with FL or gp130-stimulators alone, very little hematopoietic activity remained. SCF maintained CFC, and LTC-CAFCw2 and LTC-CAFCw6 at about half the original level. However, combinations of SCF with OSM consistently increased the number of LTC-CAFCw2 and w6 about two-fold. This finding suggests that very immature hematopoietic progenitors were at least maintained in serum-free cultures. To study the most immatore cells, we performed in transplantations into sublethally irradiated NOD/SCID mice. When CB cells were cultured with SCF alone, NOD/SCID-repopulating cell (CRU) frequency (1 in 900) dropped to about 20% in 6 days (1 in 5500). Similarly, cultures without growth factors or with FL or OSM showed a decrease to less than 10% of the original CRU number. In contrast, the combination of SCF and OSM maintained CRU at about half the original level (1 in 1900). This study shows that signals through ckit and gp130 synergize to maintain CRU level. However, to achieve expansion of human NOD/SCID-repopulating cell numbers, additional signaling pathways need to be stimulated.
Chemokines and Cytokines Required for Migration of Mesechymal Stem Cells into Marrow or Injured Tissues.
Authors: Tondreau Tatiana , Nathalie Meuleman , Alain Delforge , Marielle Dejeneffe , Martine Massy , Christine Mortier , Dominique Bron , Laurence Lagneaux 
Published: April 2020

Abstract
Background: Intravenous infusion of mesenchymal stem cells (MSC) after chemotherapy are currently used to accelerate the hematopoietic recovery but their potential of migration and homing to bone marrow is poorly understood.
Methods: Bone marrow MSC (BM-MSC) were isolated with the classical plastic adhesion method. Cultured MSC were identified through their expression of SH2, SH3, CD44, CD105 but lacked typical hematopoietic antigens: CD14, CD31, CD34, CD45 and CD62-e. The MSC migration was evaluated using matrigel invasion chamber assay. MSC were used at 5.105 cells/mL in the upper chamber. In the bottom chamber, different cytokines were tested for their chemoattractive properties: SDF-1 (100ng/mL), PDGF-bb (100ng/mL), IGF (0,5 μg/mL) and IL-6 (100ng/mL) in a serum free medium. BM-MSC conditionned medium (CM) at 10% was also used as chemoattractant. After 24 hours, cells that had not migrated were removed from the top of the insert by scrubbing with a cotton swab and cells that migrated through the membrane were counted after staining with 0,5% crystal violet.
Results: We observed that MSC migration was greater in the presence of PDGF-bb, IL-6 or with 10% CM with respectively an increase of 6,6; 5,6 and 5 fold compared to spontaneous migration. Only a small number of MSC migrated in response to SDF-1, and this observation correlates with the very low expression of CXCR4 receptor (0,67±0,08%). Indeed, we demonstrated that CXCR4, the SDF-1 receptor was mainly intracytoplasmic expressed (74,9±1,3%).
Conclusions: IL-6 and PDGF-bb are important cytokines produced by the bone marrow microenvironment or by injured tissues. In our study, we thus demonstrated that these chmokines could be involved in the migration of MSC to bone marrow as well as to damaged tissues.
Mesenchymal stem cells migration Ctrl SDF-1 IGF-1 10%CM IL-6 PDGF-bb *number of cells evaluated/3 fields of view. Migrated MSC * 13,7±2,7 24,9±5 42,2±12,6 69±17,9 77,1±14,1 90,5±5,3
Identification of Erythroid Cell Stimulating Factor as a Unique Protein, Quiescin Q6: Role in Erythropoiesis.
Authors: Udupa Kodetthoor B. , Bose Chhanda 
Published: April 2020

Abstract
We demonstrated earlier the presence of an erythroid cell stimulating factor (ESF) in mouse serum that enhanced the erythropoietin-dependent erythroid cell proliferation in vitro. Employing a highly specific monoclonal antibody generated in our laboratory, we showed further that ESF had an essential role in normal erythropoiesis in vivo [Blood98, 296a, 2001, Exp Hematol.31, Supp. 1, 165, 2003]. In order to clone and express this protein, ESF was purified from mouse serum and on Western blotting using the monoclonal anti-ESF antibody the purified protein was stained as bands of ~62/64 kDa. These bands were analyzed by Mass Spectrometry. The data were processed using Protein Lynx 2.1 (Waters) and the resulting peak list was submitted for database searching on Mascot. One of the peptide sequences had a highest similarity with Quiescin Q6 (Q6). This protein was then BLAST searched for mouse DNA sequences in the NCBI database for nucleotides corresponding to the mouse Q6. We designed the primers to reclone Q6 ORF (Open reading frame) having restriction sites NCO1 and ECOR1. Amplified product was sequenced to confirm DNA fragment. Fragment was cloned in pGEMT vector and subsequently re-cloned in expression vector pET30a(+). After each cloning step, the orientation of the insert was analyzed using restriction enzyme digestion. His-tagged Q6 protein was over-expressed in E.coli BL21 Codon Plus (DE3)-RP cells. Western blot analysis using anti-ESF antibody identified 62/64 kDa-bands of Q6 in the soluble fraction of the bacterial extract. Further characteristics of this recombinant protein and its CFU-E enhancing properties are being investigated and will be reported. Thus we have identified ESF to be a unique protein, quiescin Q6 and successfully cloned and expressed it. Expressed protein is similar to ESF as regards its antigenic property.