bluuebunny/crossref_metadata_2025 huggingface dataset:
Membrane Protein and Lipid Properties of Freeze-Dried Human Red Blood Cells.
Authors: Banerjee Mitali , Bali Rachna , Torok Zsolt , Satpathy Gayana R. , Little Erika , Tablin Fern , Crowe John H. , Tsvetkova Nelly M.
Published: April 2020

Post-rehydration membrane properties are an important indicator of effective freeze-drying of mammalian blood cells. In general, cellular membranes are extremely susceptible to damage during freezing and drying. The disaccharide trehalose, naturally occurring in anhydrobiotic organisms, has been shown to prevent such damage through direct interactions with the phospholipids and proteins, and by forming an amorphous glassy matrix around the membranes. Thermal properties and lipid composition of plasma membranes from freeze-dried trehalose loaded RBCs were studied using Fourier Transform Infrared Spectroscopy (FTIR) and analytical thin layer chromatography (TLC). FTIR results showed that plasma membranes from freeze-dried and rehydrated RBCs have major phase transitions between 10 and 20°C, and between 30 and 40°C, similarly to membranes isolated from fresh RBCs. Analysis of the lipid composition of freeze-dried RBCs showed profiles of the freeze-dried RBCs that are very similar to the ones of membranes from freshly isolated RBCs, suggesting that the freeze-drying procedure did not affect the lipid composition. SDS-PAGE and immunoblotting analysis showed that the cytoskeletal components of the membrane such as spectrin, ankyrin, band 3 and glycophorin A which form a flexible meshwork of proteins underlying the lipid bilayer are also well preserved in the freeze-dried RBCs. The study demonstrates that freeze-drying of trehalose loaded human erythrocytes does not result in significant changes in the lipid and protein structure. These findings have immediate applications in clinical medicine for long-term storage of RBCs. Supported by DARPA grant N66001-03-1-9827
Severe Acute Hemolytic Transfusion Reaction Following ABO-Mismatched Platelet Transfusion: Should ABO-Mismatched Platelet Transfusion Policy Be Evaluated.
Authors: Zubair Abba , Grant Rhonda , Black Neta , Bertholf Marsha , Roy Archana , Roy Vivek
Published: April 2020

Transfusion of ABO-mismatched platelets is common practice. Although a unit of single donor apheresis platelet (SDP) contains as much plasma and passive anti-A or/and anti-B antibodies (allo-agglutinins) as a unit of fresh frozen plasma, ABO- mismatched platelet transfusions are considered safe and hemolytic transfusion reactions have only rarely been reported. We report a case of a 67-year old male, blood group A, with mantle cell lymphoma who received 1 SDP for chemotherapy induced thrombocytopenia. Several prior platelet transfusions had been uneventful. After 150 ml of irradiated, leukoreduced, SDP from blood group O donor had been infused, he developed nausea, light-headiness, chills, and back pain. He became bradycardic and hypotensive. Platelet transfusion was discontinued and intravenous dexamethasone and rapid saline infusion was administered. Later, his hemoglobin dropped from 10.6 to 7.4 g/dl and total serum bilirubin increased by 9 fold. A direct antiglobulin test (DAT) on post-transfusion sample demonstrated 1+ reactivity at room temperature. An elution test performed on the patient’s circulating red blood cells contained anti-A antibodies. These results are consistent with a severe acute hemolytic transfusion reaction. The donor anti-A and anti-B antibodies titers at room temperature and at 37°C were 512 and 2048 with A1 cells and 128 and 256 with B cells, respectively.This case illustrates the rare possibility of hemolytic transfusion reaction occurring as a result of ABO-mismatched platelets from group O donor with high-titer anti-A antibodies. SDP (in contrast to pooled platelets) offers the advantages of reduced risk of infection transmission and alloimmunization by limiting exposure to one donor only. However, it increases the opportunity for hemolytic transfusion reaction to occur if the donor has high-titer antibodies which would have been diluted if pooled platelets were used. As SDP use becomes more widespread, the risk of clinically significant hemolytic reactions is likely to increase, especially in seriously ill patients who tolerate these reactions particularly poorly. Moreover, increasingly, apheresis platelets are being donated by a small group of highly motivated donors, raising the chances of repeated transfusions from the same donors. We propose that the safety of ABO-mismatched platelet transfusions be re-evaluated. Effectiveness (and costs) of potential preventive strategies such as screening all SDP units for high-titer antibodies and/or plasma volume reduction of all mismatched SDP units should be studied.
Sustained Suppression of Hepatitis C by Pegylated Interferon and Ribavirin in Hemophilic Patients.
Authors: Santagostino Elena , Mancuso Maria E. , De Filippi Francesca , Rumi Maria G. , Colombo Massimo , Mannucci Pier M.
Published: April 2020

Pegylated interferon (PegIFN) and ribavirin therapy is the standard treatment option for patients with chronic hepatitis C. In a multicenter open trial, we assessed the efficacy and tolerability of PegIFN and ribavirin in 72 HIV negative hemophiliacs (age: 21–64 years) with persistently high ALT values. HCV genotype was 1 in 72%, 3 in 14%, 2 in 12% and 4 in 3%. Treatment: PegIFN alpha2b 1.5 mcg/Kg/week for 48 weeks in genotypes 1/4 or 24 weeks in genotypes 2/3 plus ribavirin 800–1200 mg/day based on viral type. Treatment was stopped in patients lacking virological response at month 6. All patients were assessed for 6 months post-treatment. Results: 11 patients withdrew for side-effects (6) or non-compliance (5). Sixteen patients (22%) did not achieve a primary virological response and 4 (6%) had a virological breakthrough. Therefore, 41 patients (57%) had had an end-of-treatment response. Post-treatment follow-up is ongoing in 7 patients. In 65 patients who completed the study, 2 were relapsers and 32 (49%) had a sustained virological response (SVR, 82% of genotypes 2/3 and 38% of genotypes 1/4). Neutropenia (<800 cells), high ALT and diabetes were reasons for treatment interruption. The median fall in hemoglobin levels was 3.1 g/dL. Leucopenia (40%), weight loss (39%), and fatigue (35%) were frequent side-effects. Thirty patients (42%) required dose reduction of ribavirin (21, 29%) and PegIFN (22, 30%). SVR rates were similar in patients who adhered to protocol and those requiring dose reduction (56% vs 45%). Conclusions: combination therapy with PegIFN and ribavirin improves SVR in HIV negative hemophiliacs with chronic hepatitis C.
Extracellular UTP Affects In Vitro Migration and In Vivo Homing and Engraftment of CD34+ Hematopoietic Stem Cells.
Authors: Rossi Lara , Fogli Miriam , Ferrari Davide , Pizzirani Cinzia , Vaselli Diletta , Bertolini Francesco , Di Virgilio Francesco , Baccarani Michele , Lemoli Roberto M.
Published: April 2020

Homing and engraftment of hematopoietic stem cells (HSCs) depend on the complex interplay between chemokines, cytokines, growth factors and adhesion molecules in the intricate architecture of bone marrow (BM) microenvironment. We have recently reported that CD34+ HSCs express P2Y and P2X receptors (P2Rs) for extracellular nucleotides. P2Rs activation by ATP and UTP produced stimulatory effects, in synergy with other hematopoietic cytokines, on clonogenic CD34+ and lineage-negative CD34− progenitor cells. ATP and UTP also expanded the more primitive CD34+- derived long-term culture-initiating cells. Extracellular nucleotides are also emerging as chemotactic factors for different cell types, including vascular endothelial cells, arterial smooth muscle cells and neutrophils, and regulate the trafficking of specific dendritic cell populations. In this study we asked if the extracellular nucleotide UTP modulates the migration of HSCs in response to the chemotactic peptide CXCL12/SDF-1α. Very low concentrations of UTP (10 μM) significantly improved stem cell migration, assayed with the dual-chamber migration system and evaluated by cell counting and clonogenic assays. Phenotypical analysis of CXCR4 antigen showed that SDF-1α gradients mainly attract HSCs expressing high levels of this receptor. Conversely, UTP induced the migration of HSCs expressing low levels of CXCR4. Furthermore, we evaluated actin polymerization as index of HSCs capacity to respond to chemotactic stimulation and to home to the BM microenvironment. The combined stimulation of CD34+ cells with UTP and SDF-1α produced a synergistic increase in actin polymerization, as indicated by the enhanced fluorescence intensity of FITC-phalloidin. Adhesion assays, performed in fibronectin-coated wells, also showed that UTP can stimulate the adhesion of HSCs to fibronectin, a key component of the extracelluar bone marrow microenvironment, without any substantial change in VLA-4 and VLA-5 expression levels. Moreover, xenogenic transplant studies showed that short-term pre-incubation with UTP significantly increased the engraftment efficiency of CD34+ HSC in nonobese diabetic / severe combined immunodeficiency mice. Taken together, our data suggest that UTP may affect HSC migration and homing to the BM, in synergy with the chemotactic peptide SDF-1α, perhaps activating a CXCR4-indipendent pathway.
Hematopoietic Stem Cells “Stemness” Genes Contain Many Genes Mutated or Abnormally Expressed in Hematopoietic Malignancies.
Authors: Toren Amos , Bielorai Bella , Jacob-Hirsch Jasmine , Fisher Tamar , Zelikovsky Sharon , Givol David , Domany Eitan , Itskovitz-Eldor Joseph , Kventsel Iris , Rosenthal Ester , Amariglio Ninette , Rechavi Gideon
Published: April 2020

Umbilical cord blood (CB) and mobilized peripheral blood (PB) are relatively new sources of hematopoietic stem cells (HSC) that have been increasingly used in clinical transplantations. The genetic basis of the underlying mechanism of HSC self- renewal and differentiation has not been elucidated yet.

Several groups have recently used microarray technology to study the common characteristics of stem cells from different tissues (“stemness”) and the typical features of each source of stem cell (“specificity”). Most groups focused their study on mice, used relatively small cDNA microarrays, and used CD34 as the cell surface marker for stem cells isolation. We studied human stem cells characterized by expression of the more primitive CD133 antigen, and used the Affymetrix Human Hu133A oligonucleotide arrays containing 22,215 probe sets to study the expression profile of these cells. An unsupervised hierarchical clustering of 14,025 “valid” probe sets showed a clear distinction between the CD133 + cells representing the stem cell population, and CD133 − cells that represent various stages of cell differentiation. CD133+ cells isolated from CB were compared to CD133− cells identifying 304 genes that were up regulated by at least two folds and the comparison between PB CD133+ cells and CD133− cells identified 218 genes which were up regulated by at least two-folds. These genes were considered as source specific and maybe relevant to the unique properties of CB and PB derived HSC.

We were concentrated in the 244 genes that were found to be up regulated by at least two folds in the CD133 positive cells as compared to the CD133 negative cells and were common to both CB and PB. Comparison of these “stemness” genes, to the lists of “stemness” genes that were identified by 2 recent studies that analyzed mainly murine HSC identified 39 (Ramalho et al.) and 83 (Ivanova et al.) common genes.

Twenty-four genes were common to another study that analyzed human HSC (Georgantas et al.).

Among these common “stemness” genes we identified 4 groups of genes that have an important role in hematopoiesis: Growth factor receptors (Receptor thyrosine kinases (RTK) and c-mpl), a group of transcription factors which includes several homeobox genes and TGF-β targeted genes, genes that have an important role in the process of development and genes involved in cell growth.

Among these 4 groups we identified 16 “stemness” genes (MPL, FLT3, HOXA9, MEIS 1, MLLT3, KIT, TIE, GATA-2, HOXA5, BAALC, HLF, MYCN, EVI1, MYB, FHL1, and HMGA2) that are known to be mutated or abnormally regulated in acute leukemias. It can be suggested that perturbation of expression of key hematopoietic stemness machinery genes may lead to abnormal proliferation and leukemia.]
The Higher CD34+ Cell Counts in Cord Blood Than Those in Neonatal Peripheral Blood Correlate with Increased Expression of Cytokine Receptors and Cell Adhesion Molecules on the CD34+ Cells in Cord Blood.
Authors: Lee Young-Ho , Kim Joong-Pyo , Sung Myung-Hwa , Ko Ri-Young , Han Jin-Yeong
Published: April 2020

The hematologic values of neonatal peripheral blood show a characteristic series of changes during neonatal period. The hematopoietic stem cells in cord blood (CB) are more primitive and abundant than those in neonatal peripheral blood (NB). However, there have been no studies regarding the reasons for those differences. Thus, We investigated the kinetics of nucleated cells and CD34+ cells in CB and neonatal peripheral blood. We obtained the CB and NB samples from 14 normal full-term babies for this study, which were collected immediately after delivery as well as at 24 hours and 48 hours after delivery. We analysed the expressions of CD34, CD34/CXCR4, CD34/CD49d and CD34/CD44 as well as CD3, CD19 and CD16/56 on the isolated mononuclear cells by using FACSort. We also performed CFU-GM counts and apoptotic analysis with isolated mononuclear cells. The total white blood cell counts as well as absolute neutrophil counts of NB at delivery were higher than those of CB (p=0.04) and increased until 24 hours, when decreased until 48 hours after delivery. There were no differences of absolute lymphocyte counts, CD3+ cell and CD19+ cell counts among CB and NB at 0, 24 and 48 hours postnatally, however, the CD16/56+ cell counts were lower in CB than in NB and gradually decreased until 48 hours after delivery (p=0.011). The number of CFU-GM counts (p=0.02) and CD34+ cells (p=0.049) as well as CD34+CXCR4+ cells (p=0.002), CD34+CD49d+ (p=0.001) and CD34+CD44+ cells (p=0.001) in CB were more significantly higher than those of NB at delivery, when decreased gradually until 48 hours after delivery. The incidence of apoptosis were statistically not significant among CB and NB at 0, 24 and 48 hours postnatally. In conclusion, the higher leukocyte counts for several days after delivery were correlated with neutrophilic increment which may be influenced by stressful events during delivery. The more higher CFU-GM counts and CD34+ cell counts in CB than those in NB might be correlated with increased expression of cytokine receptors and cell adhesion molecules on the CD34+ cells in CB.
Differential Gene Expression Profiles of Cord Blood Lineage-Negative Cells Prior to and Following Selective Amplification.
Authors: Shi Meng-jiao , Hermann Jason , Laning Joseph , Hoppe Henry , Kraus Morey , Visser Jan W.M.
Published: April 2020

Using a selective-amplification system, we have ex vivo expanded umbilical cord blood (UCB) derived lineage-negative− (Lin−: CD2−, CD3−, CD14−, CD16−, CD19−, CD24−, CD56−, CD66b−, and Glycophorin A−) cells. To assess at the molecular level the effect of the ex vivo expansion on properties of UCB Lin−-cells, we compared gene expression profiles between cells at day 0 and cells cultured in serum-free medium supplemented with rhFlt-3L, rhSCF and rhTPO (100ng/ml each) for 14 days with a reselection using the same lineage depletion antibody cocktail at the 7th day of culture. In this study, we utilized a human stem cell gene cDNA array (SuperArray Bioscience Corp.), which include genes encoding surface markers, growth factor/cytokines, extracellular matrix molecules and cell cycle regulators for embryonic, neural, mesodermal and hematopoietic stem/progenitor cells. Of the 266 total genes in the array, we detected 85 genes expressed above background in either of the two populations. Among these expressed genes, 19 were found only in the day_0 (pre-culture) population and 11 were found only in the day_14 cultured cells. Ex vivo expansion under these conditions had no significant effect on the majority of the genes, which include highly expressed homing-related (CXCR4, CD44) and cell adhesion (CD31) molecules. Further analysis revealed that, compared to the levels in the original day_0 Lin− population, 7 genes showed more than 3-fold increases and 13 genes showed more than 3-fold decreases in their levels of expression in the cultured day_14 cell population. The up-regulated genes included those involved in cell cycling, proliferation and migration or anti-apoptosis (cyclin E1, IGFR-2, alpha(4)beta(1)-integrin, Mdm2, Cystatin C, ALK-5). The down-regulated genes included those involved in embryological or neural development (GCMb, FGF11), a subset of integrin molecules (integrin alpha E, beta 3 and 5) and an inhibitor of BMP signaling (CER1). In conclusion, our results indicate that selective ex vivo expansion conditions promote expression of genes related to the growth and expansion of targeted hematopoietic precursor cells while retaining expression of homing-related genes. Further studies are slated to link, where possible, these transcriptional expression patterns with expression and functions of these genes at the protein and cellular levels.

Figure Figure
Nicotinamide, a Potent SIRT2 Inhibitor, Delays Differentiation of Hematopoietic Progenitor Cells.
Authors: Peled Toni , Adi Sophie , Glukhman Elina , Grynspan Frida , Nagler Arnon , Fibach Eitan , Daniely Yaron
Published: April 2020

CD38, originally described as a differentiation marker, has emerged as an important multifunctional transmembrane protein. Its most intriguing and well-characterized function is its ability to catalyze the synthesis of cyclic ADP-ribose (cADPR) from NAD. Of particular interest is its presence on the inner membrane of the nucleus, suggesting that CD38/cADPR may play a direct role in mediating nuclear activation and gene expression. Our studies on ex vivo expansion of Hematopoietic Stem Cells (HSCs) have led us to test whether alteration of CD38 function carries the potential of affecting cell fate decisions of HSCs. Inhibition of CD38 enzymatic activity was achieved by treating CD34+ cell cultures with nicotinamide (NA), a well-known base-exchange inhibitor demonstrated to inhibit the synthesis of cADPR from NAD.

We report here that exogenously added nicotinamide (5–10 mM) to CD34+ cell cultures supplemented with cytokines (SCF, TPO, IL-6, FLt3, +/− IL-3) resulted in significant enrichment of CD34+CD38− (79±9.3%, n=9) and CD34+CD38−Lin− (19±3%, n=8) cells, as compared with control cultures treated only with cytokines (6.3±1.8%, n=9, and 0.7±0.06%, n=8, respectively, p<0.01). The functionality of these early progenitor subsets was demonstrated using the extended LTC-CFC assay, performed in the absence of NA. These results raised the intriguing possibility that cADPR production may have a pivotal role in regulation of CD34+ cell fate. However, inhibition of cADPR downstream signal transduction pathways by its specific antagonist, 8-amino-cADPR did not yield any effect on CD34+ cell cultures, excluding the possibility that nicotinamide modulates CD34+ cell fate solely by inhibition of cADPR synthesis.

Nicotinamide is also a well-known potent inhibitor of SIRT2, a unique NAD(+)-dependent type III histone deacetylase (HDAC) with mono-ADP-ribosyltransferase activity involved in gene silencing, metabolism, apoptosis and aging. NA blocks NAD(+) hydrolysis by binding to an adjacent conserved pocket, and is therefore suggested as the physiologically relevant regulator of SIRT2 enzymes. This additional function of nicotinamide raises the intriguing possibility that HSC enrichment achieved by nicotinamide treatment may be related to specific inhibition of SIRT2 deacetylase activity and modulation of chromatin architecture leading to re-activation of previously silenced genes.

In line with this hypothesis, Milhem et all. recently reported that addition of trichostatin A, a specific HDAC (type I and II) inhibitor, along with a DNA hypomethylating agent, modulated HSC fate ex vivo resulting in the retention of stem cell phenotype, number, and function (Blood, 2004; 103; 4102).

Ongoing work is aimed at elucidating whether inhibition of SIRT2 is specifically involved in NA mechanism of activity leading to modulation of hematopoietic stem cell fate in ex vivo conditions.
Effect of Serum on Long-Term Cultures of Human Umbilical Cord Blood Megakaryocyte Progenitors.
Authors: Smith Stephen L.
Published: April 2020

Platelet recovery following umbilical cord blood (CB) transplants can be delayed for several months. In vitro expansion and transplantation of CB megakaryocyte progenitor cells (MPC) may be one way to increase circulating platelets thereby shortening the period of thrombocytopenia. The in vitro expansion and differentiation of CB MPC derived from enriched CB CD34+ cells was studied after immuno-magnetic bead isolation. Isolated CB CD34+ cells were incubated in serum-free culture medium supplemented with a Tpo peptide agonist (TpoA), Flt-3L, Stem Cell Factor (SCF) and Vascular Endothelial Growth Factor (VEGF). At week 2, the cultures were divided and 1) maintained in serum-free medium, 2) supplemented with 5% human serum and, 3) supplemented with 5% mouse serum. Using flow cytometry and histocytochemistry, the cultured cells were evaluated for MPC using CD41a, CD36, von Willebrand factor (VWF) and also for myeloid progenitors using CD15 and CD33. The mean total percent MPC (CD41a+ and CD36+) at week 2 was 53 ±17% and 49 ±12%, respectively. After 2–4 weeks of additional culture the %MPC declined to less than 30% in cultures with or without serum supplements. However, at weeks 5–8 of additional culture the %MPC increased in the cultures supplemented with human or mouse serum. The mean %MPC was 45 ±19% (serum-free), 55 ±16% (human serum) and, 71 ±5% (mouse serum) in cultures at week 5–8 of additional culture. The remaining cells in the cultures were myeloid progenitors (CD15+ or CD33+). Total cell numbers at week 5–8 were inhibited 3 to 5x-fold in mouse serum supplemented cultures compared to serum free and human serum supplemented cultures. In addition, VWF+ cells could be detected after 5 weeks of additional culture when either human or mouse serum was present. These data help to explain the delay in platelet recovery typically associated with cord blood transplants but, indicate that additional serum factors are present to enhance megakaryocyte differentiation. These results also suggest that the transplant of cultured CB MPC may prove beneficial for the treatment of thrombocytopenic patients and normal platelet recovery.
Genome-Wide Transcriptional Regulatory Networks Downstream of TAL1/SCL in T-Cell Acute Lymphoblastic Leukemia.
Authors: Palomero Teresa , Odom Duncan T. , O’Neil Jennifer , Ferrando Adolfo A. , Winter Stuart S. , Larson Richard S. , Young Richard A. , Look A. Thomas
Published: April 2020

Aberrant expression of transcription factor oncogenes is a common feature in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL), however, oncogenic transcriptional programs downstream of T-ALL oncogenes are mostly unknown. We used chromatin immunoprecipitation combined with promoter microarrays (ChIP on chip) to identify direct transcriptional targets of TAL1/SCL, a bHLH transcription factor aberrantly expressed in 60% of patients with T-ALL. Using the Hu12K arrays that contain over 12,000 human promoter regions, we have identified 71 direct targets of TAL1 in the Jurkat T-ALL cell line by ChIP on chip, and have been able to validate 80% of them by conventional chromatin immunoprecipitation followed by quantitative real time PCR. It has previously been demonstrated that TAL1 needs to bind to E proteins (E2A, HEB or E2-2) to achieve an efficient interaction with DNA. We verified that promoters occupied by TAL1 are bound by both the E-proteins E2A and HEB in at least 50% of TAL1-bound promoter regions, suggesting that TAL1/E2A as well as TAL1/HEB heterodimers play a role in the transformation of T-cell precursors. Using RNA interference to knock down TAL1, we have demonstrated functional regulation through TAL1-binding of a significant percentage of the targets identified by ChIP on chip. Results demonstrate that some TAL1 direct targets are repressed by the binding of this transcription factor, while others seem to be activated or are not altered, indicating a high level of complexity in the organization of TAL1-mediated transcriptional regulatory networks. Also, specific association of the expression levels of TAL1 targets in TAL1 positive primary leukemia samples has been shown by comparing gene expression arrays from the different T-ALL subsets. Our results indicate that TAL1 acts as a master transcriptional regulator at the top of a complex regulatory network that contributes to leukemogenesis by regulating T-cell differentiation, proliferation and survival.
Gene Expression Profiles for Normal Human Bone Marrow-Derived CD34+ Stem Cells and CD34−/CD33+ Myeloid-Committed Progenitor Cells in Response to Daily Granulocyte-Colony-Stimulating Factor (G-CSF) Treatment.
Authors: Aprikyan Andrew A.G. , Pritchard David , Liles Conrad W. , Schwartz Steve , Dale David C.
Published: April 2020

G-CSF is widely used to accelerate marrow recovery after cancer chemotherapy, to facilitate collection of hematopoietic progenitor cells, and to treat severe chronic neutropenia. Although G-CSF was originally defined as a stimulus for myeloid cell proliferation, it has potent anti-apoptotic properties, affects synthesis of proteins stored in neutrophil granules, and has many other effects on cells of the myeloid lineage. To improve understanding of the molecular and cellular effects of G-CSF, particularly related to its use for the treatment of severe chronic neutropenia, we performed gene expression profile studies using Affymetrix oligonucleotide arrays and purified bone marrow cell sub-populations from normal volunteers treated with daily subcutaneous G-CSF (300 mcg/sc/qd) for five days. Under local anaesthesia, paired marrow aspirates were obtained from the posterior iliac crest before and after 5 daily doses of G-CSF. CD34+ and CD34−/CD33+ cells were purified using Miltenyi immunomagnetic beads. Two rounds of amplification of total RNA isolated from purified CD34+ or CD33+cells was used to obtain sufficient cRNA for hybridization. Expression data from scanned chips were first analyzed using the RMA algorithm. The limma package of the Bioconductor project was used to identify differentially expressed genes. Limma uses an empirical Bayes method to moderate the standard errors of the estimated log-fold changes. The statistical analysis of CD33+ cells revealed that 150 of more than 12,000 genes examined were up- or down-regulated >2-fold in response to G-CSF treatment. The top 10 genes with up- or down-regulated level of expression include clusterin, neutrophil elastase, two transcription factors, gelsolin, Grb2, phospholipase D3, protein kinase C, the major vault protein, and serine-threonine kinase. In the myeloid-committed CD34-/CD33+ progenitor cells, genes with altered expression level represent those with gene products involved in the cell cycle, regulation of apoptosis, the cytoskeleton, the inflammatory response, or serine proteases and transcription factors. Most of the genes up-regulated in CD33+ cells (e.g. neutrophil elastase, phospholipase D, protein kinase C) were down-regulated in CD34-positive cells in response to G-CSF. The results of the comparative analyses revealed the normal signature gene expression profiles for CD34+ and CD34−/CD33+ cells and identified genes that may mediate specific G-CSF effects.
Natural Interferon Producing Cells Develop from Murine CD31+(high)/Ly6C- Marrow Progenitors.
Authors: Kreisel Friederike , Blasius Amanda , Colonna Marco , Cella Marina
Published: April 2020

Dendritic cells (DC) play a critical role in the initiation and regulation of immune responses. Based on immunophenotypic and functional charcateristics, murine CD11c+ DC can be classified into CD11c+, CD11b+, and B220− “conventional” DC and CD11c+, CD11b−/dim, B220+, and Gr-1+ interferon-producing cells (IPC) or plasmacytoid dendritic cells (pDC). The developmental origin of IPC remains controversial. In this study, we investigated the capacity of different murine bone marrow subpopulations to acquire immunophenotypic, morphologic and functional characteristics of IPC after in vitro culture with Flt-3L. Total bone marrow (BM) from 129/SvJ mice was sorted into six distinct BM subsets based on surface expression of the myeloid markers CD31 and Ly6C and subsequently cultured with Flt-3L for 8 days. Purified CD31high/Ly6C− BM cells were the only subset that consistently developed characteristics closely resembling that of IPC. Specifically, these cells expressed high levels of CD11c, low levels of CD11b, the lymphoid marker B220, and the IPC-specific marker 440C. Furthermore, these cells displayed the typical plasmacytoid morphology with eccentrically located nucleus showing mature lymphoid chromatin and lack of nucleoli. Functionally, these cells showed a high proliferation rate as demonstrated by the [3H] thymidine release assay and secreted detectable levels of IFN-alpha when stimulated with CPG ODN 2216. In contrast, all other sorted subsets failed to proliferate in the presence of Flt-3L. Similar results were obtained using BM from B6-RAG−/− mice. These findings indicate that, although both the CD31high/Ly6C− and CD31int/Ly6C− BM subsets contain early progenitor cells corresponding to “cobblestone area forming cells”, only the CD31high/Ly6C− is able to give rise to IPC when cultured with Flt-3L. Interestingly, CD31high/Ly6C− BM cells have been previously shown to generate also conventional DC if cultured with GM-CSF. Thus, IPC and DC may derive from common or closely related progenitors. Moreover, our results demonstrate that IPC development, in contrast to that of lymphocytes, does not require RAG proteins.
More Evidence That the Phenomenon of “Plasticity” of Hematopoietic Stem Cells (HSC) and Potential Involvement of BM-Derived Cells in Organ Regeneration Could Be Explained by the Presence of a Heterogenous Population of Tissue Committed Stem Cells (TCSC) Residing/Hiding out in Bone Marrow.
Authors: Kucia Magda , Wysoczynski Marcin , Reca Ryan , Majka Marcin , Ratajczak Janina , Ratajczak Mariusz Z.
Published: April 2020

Recently we identified a mobile, SDF-1 responsive population of CXCR4+ bone marrow mononuclear cells that express mRNA for various markers of early tissue committed stem cells (TCSC) in bone marrow (BM) by employing chemotactic isolation to an SDF-1 gradient combined with real time RT-PCR and immunohistochemical analysis (Leukemia2004:18;29-40). In the current work we report new data showing that murine and human TCSC (i) are highly mobile and respond robust to HGF and LIF gradients, in addition to an SDF-1 gradient, (ii) are released (mobilized) from BM into peripheral blood during organ injury (e.g., stroke, heart infarct, partial body irradiation), (iii) are highly enriched in mRNA for pluripotent stem cell transcription factors such as Oct-4, Rex-1 and Nanog, iv) reside in a population of BM-derived Sca-1+ (mice) and CD34+AC133+ (human) non-hematopoietic CD45 negative cells, v) are small (~7 mm in diameter), vi) contain supercoiled DNA, vii) rapidly adhere to/undergo emperipolesis in fibroblasts and thus are enriched in a fraction of BM-derived adherent cells, viii) their number is highest in BM from young (1–2 month-old) mice and decreases in 1-year-old animals, and similarly ix) these cells are significantly diminished in short living DBA/2J mice as compared to long living B6 mice. Thus, these findings further support our theory of the BM as a “hideout” of TCSC and we suggest that their presence in BM tissue should be considered before experimental evidence is interpreted simply as trans-dedifferentiation/plasticity of HSC. Since we noticed that not only SDF-1 but also HGF and LIF are upregulated in damaged tissues (e.g., brain, heart or liver), we postulate that TCSC that express CXCR4, c-Met, and LIF-R after being mobilized from the BM into PB, may be subsequently chemoattracted to damaged organs where they play a role in tissue repair/regeneration. Moreover, our observation that the number of TCSC is the highest in BM of young animals and decreases with age and is significantly lower in short-living murine strains provides a novel insight into aging and may explain why the regeneration process becomes less effective in older individuals. Based on our observation, we conclude that BM-derived HSC are not plastic but BM in addition to HSC also contains heterogenous populations of TCSC.
Cellular Aggregates Induced from Monolayer Cultures of Human Pancreatic Cells Correct Hyperglycemia in Diabetic Mice and Demonstrate Physiological Production of Human C-Peptide.
Authors: Wu Fred D. , Jagir Michael R. , Luu Thomas T. , Powell Jerry S.
Published: April 2020

Diabetes is due to loss of appropriate insulin production by pancreatic islet cells, resulting in hyperglycemia and significant morbidity. We have developed cell cultures of pancreas-derived precursor cells that can be maintained for more than 20 population doublings in culture, growing as monolayers of undifferentiated cells. These monolayer cells can then be induced to differentiate into cellular aggregates that resemble islets when grown on different extracellular matrix components, including matrigel, or poly-d-lysine. The induced cellular aggregates, but not monolayer cells, correct hyperglycemia in diabetic (streptozotocin) SCID (severe combined immunodeficiency) mice after implantation under the kidney capsule. This animal model can be used to follow the process of differentiation from undifferentiated precursor cell to fully functional insulin producing beta cells, and to identify key proteins and genes involved in differentiation of pancreatic islet cells. Here we report that in the mice, after implantation of cellular aggregates, human c-peptide concentrations are controlled physiologically appropriately in response to glucose tolerance testing (challenged with 2 grams of glucose injected intraperitoneally) in the treated diabetic SCID mice (n = 6) as well as in normal mice that received implanted cellular aggregates (n = 6). Human c peptide is not detected in any of the control animals, nor in animals implanted with monolayer cells. Comparative RNA microarray data analyses, using U133 arrays (Affymetrix), were compared between the monolayer cells in culture and the cellular aggregates 48 hours after induction with poly-d-lysine. We consider of particular interest the up-regulation of BMP, CXCR4, and HGF in the cell aggregates, and the down-regulation of VCAM-1. We believe that these genes are necessary for either aggregation or for physiologically functional insulin secretion. When examined for greater than two-fold gene expression differences between monolayer and aggregated cells, 424 gene sequences were upregulated and 690 genes down regulated. In addition, RAGE display analysis for tyrosine kinases also showed that the kinases Erb-B2 and PDGFR-B were upregulated at 48 hours in the aggregating process. In contrast, DDR2, a collagen I receptor tyrosine kinase, was expressed equally in both monolayer and aggregated cells. In summary, the results suggest: 1) that a population of pancreatic stem cells can be isolated and cultured in vitro, 2) that these cells can be induced to form functional islet cells that correct hyperglycemia in diabetic mice, 3) that human c-peptide is physiologically regulated in aggregate implanted mice in response to glucose challenge, and 4) HGF, along with other genes of potential interest, is upregulated in the process of differentiation from monolayer to aggregated cell phenotype.
Ubiquitin-Proteasome-Dependent Degradation of GATA-2 and Its Contribution to the UV-C-Induced Down-Regulation.
Authors: Minegishi Naoko , Suzuki Norio , Kawatani Yukie , Yamamoto Masayuki
Published: April 2020

Transcription factor GATA-2 is crucial for the development of hematopoietic stem cells. GATA-2 regulates proliferation and survival of hematopoietic stem/progenitor cells. We analyzed the degradation process of GATA-2, assuming that GATA-2 may be under the strict controls of protein degradation like many factors regulating cellular proliferations and apoptosis. The half-life of endogenous GATA-2 in hematopoietic and neuroblastoma cells was short (within 30 min in P815 cells) after cycloheximide treatment. The UV-C irradiation also induced the reduction of endogenous GATA-2 expression. Treatment with a proteasome inhibitor MG132 abrogated the effects of cycloheximide and UV-C irradiation, and increased the expression levels of both endogenous and transfected GATA-2 protein. Experiments with GATA-2 deletion mutants and their GBD-fusion proteins revealed that degradation elements of GATA-2 are within the transactivation domains and within a region containing putative protein modification motifs. We detected poly-ubiquitinated forms of GATA-2 after MG132 treatment, implying that the rapid degradation of GATA-2 is ubiquitin-dependent. Together, these results suggest that the ubiquitination/proteasome-dependent degradation pathway regulates the GATA-2 protein expression and this process may be important for the GATA-2 function.
An Establishment of a System for Conditional Overexpression of Genes in Megakaryocytes and Platelets In Vivo.
Authors: Nguyen Hao G. , Yu Guangao , Makitalo Maria , Jones Matthew , Ravid Katya
Published: April 2020

Expression of foreign or endogenous genes in vivo is an essential tool for following regulatory processes, including the control of gene expression. Constitutive expression of such genes throughout development often results with lethality due to adverse effects during embryogenesis. Several such examples exist with regard to the megakaryocyte (MK)/platelet lineage. Here, we employed the tetracycline (Tet)-off system to conditionally overexpress genes in MKs and platelets in vivo. We generated three transactivator transgenic lines expressing the Tet transactivator element (tTA), under the control of the megakaryocyte-specific, platelet factor 4 promoter (PF4-Tet). Three responder lines were simultaneously generated, each with a bidirectional minimal CMV-tTA responsive promoter upstream of prokaryotic ß-Galactosidase gene (ß-Gal) (as a marker), with an insertion site in the opposite side for a gene of interest. A transactivator founder line that strongly expressed tTA was cross-bred to the three other responder lines. The resulting double transgenic mouse lines exhibited tetracycline-dependent transgene overexpression in the megakaryocytic lineage. Lineage specificity was confirmed by immunohistochemistry and western blot analyses. By changing tetracycline concentration in the drinking water, we could turn on/off the expression of the gene at any desirable time during the mouse lifespan, and showed that in adult mice under inducing conditions, ß-Gal expression was restricted to MKs and platelets. Hence, we generated a mouse line, PF4-Tet that is available to conditionally overexpress any gene of interest in the MK/platelet lineage in vivo. This system should be valuable for the study of effects of selected proteins on an array of genes expressed in vivo and on consequent megakarypoiesis and/or thrombosis and hemostasis.
Identification of Genes Affected during Myeloid Differentiation Induced by Valproic Acid and Retinoic Acid: The Choice of Array Pre-Processing and Normalization Algorithm Has a Critical Effect on Results.
Authors: Berkofsky-Fessler Windy D. , McConnell Melanie-Jane , Neuberg Donna S. , Licht Jonathan D.
Published: April 2020

Valproic acid is an anti-epileptic drug with histone deacetylase inhibitor activity currently in clinical trial for the treatment of leukemia. In order to understand the effects of this agent in myeloid differentiation and leukemia therapy we determined the changes in gene expression that occurred when the myeloid cell line ML-1 was treated with valproic acid (VPA), all-trans retinoic acid (ATRA) or the combination for 24 or 96 hours. Each condition was compared to no drug treatment at each time point, as full biological triplicates. RNA samples were labeled and hybridized to Affymetrix HGU133A genechips. The data was processed using the Affymetrix MAS5.0 calculated intensity signals or was pre-processed using three different algorithms to normalize data: GC-RMA (GeneChip-Robust Microarray Analysis), RMA (Robust Microarray Analysis) or MBEI (Model-Based Expression Index), using the original Affymetrix CEL files. From the resulting normalized data sets, we then identified genes that were up or down regulated at least two-fold by the combination VPA + ATRA treatment when compared to the no drug condition or either drug alone. If any of these genes were affected by either ATRA or VPA, we selected only those with an additional change of 50% or more with the combination therapy. There were striking differences in the gene sets obtained from each normalization algorithm with respect to both list size and content. GC-RMA identified 100 genes significantly differentially expressed according to the above criteria at the 24 hour time point, MBEI found 19 and MAS5.0 found 39. Using the RMA algorithm, 33 genes were determined to be significantly affected by the combination treatment and all of these were also found with GC-RMA suggesting that GC-RMA has a greater sensitivity than RMA. This may be because GC-RMA, a refinement of RMA, takes into account mismatched probe set signals and probe set sequence content. Only 12 genes were common to all lists, including endothelin receptor B, myosin binding protein H, IL8, and G0S2. We are currently using real-time PCR to validate the extent of differential regulation of the commonly identified genes in reponse to VPA+ATRA. This will be contrasted with the levels of regulation of gene sets found by only one or two algorithms in order to determine the sensitivity and specificity of these methods. These data indicate the potential power of new bioinformatic tools for microarray analysis and the need for systematic validation to ensure biological relevance.
Interferon-gamma Has Dual Potential in Inhibiting or Promoting Survival and Growth of Haematopoietic Progenitors: Interactions with Stromal Cell-Derived Factor-1.
Authors: Jo Deog-Yeon , Hwang Jin-Hee , Kim Seong-Woo , Yun Hwan-Jung , Kim Samyong
Published: April 2020

Despite many apparently contradictory reports, IFN-γ is well known as a hematopoiesis-inhibitory cytokine and is implicated in the pathogenesis of aplastic anemia. It has been shown that IFN-γ intrinsically and simultaneously induces specific and conflicting signalling pathways and transcriptional programs that contribute to the potential dual effects of IFN-γ in promoting or inhibiting proliferation of murine pro-B cells (Asao et al, J Biol Chem275: 867, 2000). We explored the dual roles of IFN-γ in the inhibition or promotion of the survival and growth of hematopoietic progenitors, especially with regard to interactions with stromal cell-derived factor-1 (SDF-1). IFN-γ partially rescued normal bone marrow CD34+ cells and colony-forming cells from apoptosis induced by serum- and growth factor-deprivation. SDF-1 further enhanced cell survival, in synergy with IFN-γ. Short-term IFN-γ treatment of CD34+ cells enhanced the clonal growth of the cells in synergy with SDF-1. In contrast, IFN-γ inhibited the clonal growth of hematopoietic progenitor cells in a standard methylcellulose clonogenic assay and inhibited the growth factor-mediated survival of normal CD34+ cells, CD34+ cells from patients with chronic myeloid leukaemia, and MO7e cells. The addition of SDF-1 did not alter these outcomes. IFN-γ did not activate PI3K/Akt, enhance SDF-1-induced activation of PI3K/Akt, or up-regulate the expression of CXCR4 or its chemotactic function in bone marrow CD34+ cells. IFN-γ up-regulated the expression of Socs1 mRNA in CD34+ cells and MO7e cells. Referring to a previous report describing that Socs1 binds to multiple signalling proteins and suppresses stem cell factor-dependent proliferation of bone marrow-derived mast cells (De Sepulveda et al, EMBO J 18: 904,1999), it is suggested that IFN-γ-induced inhibition of the growth factor-dependent survival of CD34+ cells might be mediated, at least in part, through the induction of Socs1. Paradoxically, IFN-γ down-regulated SDF-1 production in primary human bone marrow stromal cells. These results indicate that IFN-γ, partly in concert with SDF-1, exerts dual effects on the survival and growth of hematopoietic progenitor cells; the effects of IFN-γ on hematopoietic progenitor cells can differ depending on the particular in vitro experimental conditions, especially the presence of hematopoietic growth factors.
DNA Damage Induced by DNA Topoisomerase I- and Topoisomerase II- Inhibitors Detected by Histone H2AX Phosphorylation in Leukemic Cells.
Authors: Halicka Dorota H. , Huang Xuan , Ozkaynak Fevzi M. , Seiter Karen , Traganos Frank , Darzynkiewicz Zbigniew
Published: April 2020

Histone H2AX is phosphorylated on Ser-139 by ATM kinase in response to damage that induces dsDNA breaks. Immunocytochemical detection of phosphorylated H2AX (γH2AX), thus, reveals the presence of dsDNA breaks in chromatin. Multiparameter cytometry was presently used to correlate the appearance of γH2AX with: (a) cell cycle phase; (b) caspase-3 activation; and (c) apoptosis-associated DNA fragmentation in individual human leukemic HL-60 cells treated with the DNA topoisomerase I (topo1) inhibitors topotecan (TPT) and camptothecin (CPT) or with the topo2 inhibitor mitoxantrone (MTX). In response to TPT or CPT maximal increase of γH2AX immunofluorescence was seen in S-phase cells by 90 min. In contrast, following MTX treatment the maximal rise of γH2AX was detected at 2 h in G1 cells and the cell cycle phase specificity was much less apparent. A linear relationship between the drug concentration and increase of γH2AX immunofluorescence was seen only up to 200 nM TPT; a decline in γH2AX was apparent at a concentration range between 0.4 and 1.6 μM TPT. Thus, the intensity of γH2AX immunofluorescence, as a marker of cell survival following TPT treatment, can be used only within a limited range of drug concentration. Following treatment with TPT, CPT or MTX the peak of H2AX phosphorylation preceded caspase-3 activation and the appearance of apoptosis-associated DNA fragmentation, both selective to S-phase cells. Progression of apoptosis was paralleled by a decrease in γH2AX immunofluorescence. On the basis of our laboratory results, the present clinical study is evaluating ex vivo the feasibility of assessing DNA damage induced by treatment with topoisomerase inhibitors in patients with acute leukemias.