bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interferon-γ Attenuates the Survival Activity of G-CSF through PI3K/Akt Signaling Pathway in Mouse Multipotent Progenitor Cells.
Authors: Liu Hong , Mihara Keichiro , Tanaka Hideo , Kimura Akiro 
Published: April 2020

Abstract
Aplastic anemia (AA) is characterized by cytopenias caused by loss of stem cell and immunological relevance. We found out that impaired stem cell could be caused by excess of interferon-gamma (IFN-γ) in the serum or hypersensitivity to IFN-γ in patients with AA, notably severe AA. However, it is not clear how IFN-γ works in terms of the etiology of AA, as it exhibits diverse biological effects on cells. Thus, we investigated the essential role and the mechanism for the apoptotic function of IFN-γ against hematopoietic stem and/or progenitor cells. Although G-CSF augmented the surviving, proliferative, and differentiating activity in 32D cells, mouse multipotent progenitor cells, those effects of G-CSF were abolished by IFN-γ and they were susceptible to apoptosis with IFN-γ treatment. We focused on Akt, which was associated with the control of stem cell fate. IFN-γ did not affect the expression of Akt in 32D cells treated with G-CSF. Intriguingly, although Akt was strongly phosphorylated at Ser473 with G-CSF, IFN-γ attenuated phospho-Akt expression in dose- and time-dependent manner. As Akt is known to be phosphorylated through phosphatidylinositol-3-kinase (PI3K) pathway, we analyzed whether wortmannin, a specific inhibitor of PI3K, might have any synergistic effect on Akt phosphorylation in conjunction with IFN-γ. Wortmannin enhanced the inhibitory effect on Akt phosphorylated by G-CSF in collaboration with IFN-γ, suggesting that the activity of IFN-γ might converge on PI3K pathway. Next, to explore the downstream molecule of Akt, we examined the expression of BAD and NF-kB, which work downstream of Akt. IFN-γ increased the BAD protein reduced by G-CSF. Regardless of addition of IFN-γ, there was no alteration of NF-kB expression. IFN-γ exerted apoptosis against 32D cells through the caspase pathway. Taken together, these results suggest that IFN-γ could exert the inhibitory action on stem cells and/or progenitors by the interference of the PI3K / Akt signaling pathway in AA patients. Our findings may provide a suggestion of the treatment potential for AA such as anti-IFN-γ antibody and contribute the further understanding of AA in terms of immunological aberration.
Defective Ribosomal RNA Maturation in Patients with Diamond Blackfan Anemia.
Authors: Quarello Paola , Aspesi Anna , Flygare Johan , Karlsson Stefan , Ellis Steven R. 
Published: April 2020

Abstract
Diamond Blackfan Anemia (DBA) is a rare congenital red cell aplasia due to a selective defect of erythroid progenitor cell maturation. DBA patients exhibit a wide array of developmental abnormalities and can be predisposed to cancer. Heterozygous mutations in the gene encoding Rps19, a ribosomal protein of the small subunit, have been identified in 25% of DBA cases. By using human cell lines (TF-1) containing small interfering RNA (siRNA) designed to silence RPS19 we have shown that Rps19 deficient cells present an altered ribosomal profile which conveys a decrease in the 40S subunit population. This decrease is due to faulty maturation of the 21S precursor to functional 18S rRNA as illustrated by pulse chase and northern blot analysis of total RNA extracts from the cell lines. Analysis of these intermediates in CD34− cells from the bone marrow of DBA patients harboring mutations in RPS19 revealed a pre-rRNA processing defect similar to that observed in TF-1 cells where RPS19 expression was reduced. This defect was observed to a lesser extent in CD34+ cells from DBA patients with mutations in RPS19. Thus, our data reveal that rRNA processing is disrupted in cells from DBA patients with mutated RPS19.
Ex Vivo Proliferation and Differentiation of CD34+ Cells into Myeloid Progenitor Cells: Cord Blood Versus Mobilized Peripheral Blood.
Authors: De Bruyn Cecile , Delforge Alain , Ngirabacu Marie-Christine , Bron Dominique 
Published: April 2020

Abstract
The purpose of our study was to evaluate the capacities of cord blood (CB) CD34+ cells to proliferate and differentiate ex vivo into myeloid lineage in response to cytokines and to compare them with mobilized peripheral blood (MPB) cells. Briefly, 2.5 × 104 CD34+ cells, isolated from CB (n=10) and MPB (n= 9), were cultured in 5 ml MacoBiotech HP01 (Macopharma) with SCF, Flt3-L, IL-3 and G-CSF. At day 9, 106 cultured cells were replated for 5 additional days. Cells were counted and evaluated for their CD34, CD13 and CD15 expression. Differentiation into myeloid compartment was assessed by CD11b and CD16 coexpression on CD15+ cells. We observed that
leucocyte expansion was significantly higher in CB than in MPB at day 9 (24.3±3.8 vs 15.2±1.9) and at day 14 (224.7±54.2 vs 72.9±20.0). A similar difference was observed for CD34+ cell expansion (8.7±1.4 vs 3.4±0.5 at day 9 and 31.3±4.6 vs 7.6±2.4 at day 14). at day 9, despite superior CB leucocyte expansion, CD13+ and CD15+ cell number produced per CD34+ cell seeded at day 0 were similar in CB and in MPB (18.5±2.4 vs 14.4±2.5 for CD13+ and 7.1±1.3 vs 6.0±1.2 for CD15+). Increasing the culture period led to higher numbers of CD13+ and CD15+ cells in CB than in MPB. This increase was due to a total leucocyte expansion rather than to high CD13+ and CD15+ cell percentage. The distribution of CD11b−CD16−, CD11b+CD16− and CD11b+CD16+ subpopulations in CD15+ cells was comparable in CB and in MPB after 9 days of culture, with a majority of relatively immature CD11b−CD16− myeloid progenitor cells.
However, after 5 additionnal days of culture, MPB CD15+ cells expressed a more mature phenotype than CB CD15+ cells, with a dramatic increase of CD11b+CD16− cells (promyelocytes and myelocytes). In conclusion, our study suggests that, despite the high CB cell capacity of expansion in our culture conditions, CB CD34+ cell differentiation process into myeloid lineage appears to be slower. This difficulty of CB cells to reach maturation in vitro is likely to be related with the longer delay of neutrophil recovery after CB transplantation.
Productive Infection of Human Cord Blood Stem Cells and Lineage Progenitors by Porcine Endogenous Retrovirus (PERV).
Authors: Morgan Doris A. , Fitzsimmons John , Argaw Takele , Wilson Carolyn 
Published: April 2020

Abstract
Endogenous retroviruses were once considered “junk DNA” and merely residual evolutionary genetic material. In the human system, recent reports have suggested a link between activation of human endogenous retroviruses (HERV) and certain diseases. Activation may be restricted to an up-regulation of viral specific transcripts or may involve gene translation with the assembly of virus-like particles (Morgan, Exp.Hem., 2004). Against this background of a possible link between activated endogenous retroviruses and disease, the potential risks of porcine to human transmission in the context of xenotransplantation becomes more plausible. The principal focus of this report was to determine the susceptibility of human primary cells to porcine endogenous retroviruses (PERV). Expression of a receptor for PERV has been described to be widespread in human tissues (Ericsson,PNAS,2003) and was expressed in hematopoietic stem cells and progenitors of our study. We exposed human umbilical cord blood (UCB) stem cells and cytokine-induced erythroid, myeloid and megakaryocytic progenitors to retroviral pseudotypes composed of replication competent wildtype PERV-NIH virions that also carry an MLV-based retroviral vector genome encoding β-galactosidase (β-gal) (Wilson, J. Vir., 2000; Harrison, J. Vir., 2004). Three days after exposure to PERV, cells were washed in order to remove unbound virus and assayed for viral RNA and DNA using quantitative PCR and RT-PCR (Argaw, J. Gen. Vir. 2002). Cell pellets from stem cells as well as lineage-induced cells were consistently positive for viral DNA and RNA and persisted during a kinetic study 3,5,6,and 7 days post-exposure to PERV. Control cells that were not exposed to PERV were negative in all parameters. There were no detectable adverse effects of PERV infection on cell proliferation or on terminal maturation of the progenitors of all three lineages. PERV infection does not require lineage commitment and provides a mechanism by which self-renewing stem cells may serve as an in vivo reservoir of virus in human bone marrow. Of note, the natural course of disease (anemia) of a related retrovirus, feline leukemia virus, also relies upon infection of hematopoietic progenitors in the bone marrow.
A Gene Dosage Requirement for Transcription Factor Gfi1 in the Regulation of Myelopoiesis and Myeloproliferative Disorders.
Authors: Grimes H. Leighton , Horman Shane R. 
Published: April 2020

Abstract
The generation of mature myeloid lineage cells from hematopoietic stem cells (HSCs) requires a precise synergy between cytokine signaling and lineage-specific transcription factors. Gfi1 (Growth Factor Independence 1) is a zinc finger transcription factor that is necessary for normal myelopoiesis. Gfi1 knockout mice display an abnormal ratio of phenotypic common myeloid progenitors (CMP) and granulocyte/monocyte progenitors (GMP), and such mice completely lack mature neutrophils. In contrast, the Gfi1 heterozygote mouse presents no obvious phenotype. Here we show a gene dosage requirement for Gfi1 in the differentiation of mature myeloid cells. While bone marrow from wild type littermates generates granulocytic, monocytic and mixed methylcellulose colonies, Gfi1 knockout bone marrow cells yield mainly monocytic colonies. In comparison to wild type littermates, Gfi1+/− bone marrow cells generate lower numbers of granulocytic colonies and higher numbers of monocytic colonies. Interestingly, methylcellulose colonies from both Gfi1 knockout mice and heterozygotes display increased serial replating capacity in comparison to wild type littermates. These data suggest that Gfi1 gene dosage may control self renewal and may relate to oncogenic transformation. Activating mutations in K-Ras are common genetic abnormalities in human acute myeloid leukemia and myeloproliferative disease (MPD). However, expression of activated Ras from endogenous regulatory sequences (knock-in) results in an MPD of varying lethality. The severity of the K-Ras-induced MPD depends on unknown genetic modifiers, as lethality is increased in a Balb/c genetic background. Given the effect of Gfi1 gene dosage on serial replating, we have analyzed the effect of Gfi1 gene dosage on the activated Ras knock-in mouse model of MPD. Preliminary data indicate that lowering Gfi1 gene dosage modifies the phenotype of activated Ras MPD by dramatically increasing the number of immature circulating myeloid progenitors. These results reveal that Gfi1 is a modifier of Ras induced disease pathogenesis.
Cell Cycle Influence of Stem Cell Differentiation in Culture.
Authors: Zomorodian Toska J. , Abedi Mehrdad , Dooner Gerri , Greer Debbie , Johnson Kevin , Demers Delia , Quesenberry Peter J. 
Published: April 2020

Abstract
Hierarchical models of hematopoiesis suppose an ordered system in which stem cells and progenitors with specific fixed differentiation potentials exist. We show that the potential of marrow stem cells to differentiate changes reversibly with cytokine-induced cell cycle transit. To address whether the cell cycle plays a role in the differentiation of stem cells, we co-cultured murine bone marrow Lin- Sca-1+ cells, at different points in their cycle, with the OP9-DL1 system. OP9-DL1 stromal cell layer has been transduced to allow T-cell differentiation in culture. We first induced cell cycle synchrony by exposing the isolated cells to a cytokine cocktail of TPO, Flt-3 and Stem Cell Factor. The cells were exposed to this primary culture for 0, 6, 24, 32 and 40 hours and were subsequently cultured on an OP9-DL1 stromal cell layer grown in 6-well plates. Cells were co-cultured for 8 days and 21 days, in the presence of IL-7 and Flt-3. Cultured cells were evaluated for CD4, CD8, B220, CD19, NK1.1, and Mac-1 surface markers, using flow cytometry. On Day 8, we found a significant hotspot at 32-hours (early-S phase) for B220+ cells (34.3 %), while Mac-1 positive cells demonstrated a 24-hour hotspot (18.1 %). As expected, terminal T and B-cell differentiation (CD 4, CD8, and CD19) was undetectable at 8 days. Three separate short-term (8 day) experiments have confirmed these data. Cells in culture for 21 days similarly show variation in differentiation outcome. CD4 cells demonstrate a peak at the 40 hour time point (mid-S phase) (69.9%), while CD8 positive cells were significantly increased at the 32 hour time point (34.4%). These data indicate both B and T cells show reversible differentiation fluxes linked to cell cycle. This work supports previous evidence that marrow hematopoiesis at the stem cell level is regulated on a continuum and that stem cells have reversible, cycle-related differentiation capacity.
MiRNA Transcription Is Regulated by the PML-RARα Oncoprotein in Acute Promyelocytic Leukemia.
Authors: Nigten Jeannet , Knops Ruth , Nikoloski Gorica , de Witte Theo M. , van der Reijden Bert A. , Jansen Joop H. 
Published: April 2020

Abstract
The discovery of the microRNA (miRNA) molecules has led to new insights into the regulation of gene expression. They are able to bind specific mRNA sequences, and due to inhibition of translation or mRNA degradation, miRNAs cause downregulation of their target genes. To date, several hundreds of unique human miRNAs have been described. So far, for only few of them the target mRNAs have been experimentally confirmed. MiRNAs have been linked to several important biological processes as early stage development, cell growth, cell differentiation and apoptosis. In addition, impaired miRNA expression has been implicated in tumorigenesis. Leukemia is often associated with mutated transcription factors and, as a consequence, deregulated gene expression and impaired proliferation and differentiation. Acute Promyelocytic Leukemia (APL), is characterised by the expression of the mutated transcription factor PML-RARα, which may interfere with the normal function of the retinoic acid receptor α (RARα), a nuclear hormone receptor that acts as a ligand-dependent transcription factor. APL is uniquely sensitive to treatment with the RARα ligand, all-trans retinoic acid (ATRA), which results in the expression of genes that induce terminal granulocytic differentiation of the leukemic blasts. To investigate whether miRNA expression is regulated by ATRA in APL, we performed Taqman miRNA assays for 157 different mature miRNAs in the APL cell line NB4 before and after treatment with ATRA. We found that ATRA induced a more than 10 fold upregulation of 18 miRNAs and a more than 10 fold downregulation of 2 miRNAs. These expression patterns were confirmed in primary APL patient cells before and after treatment with ATRA. To study whether the miRNA expression pattern was dependent on the PML-RARα fusion protein, we used U937 cells stably transfected with a zinc-inducible PML-RARα expression cassette (U937PR9, a kind gift of Dr Pelicci). Upon ATRA treatment, we found that several miRNAs were only induced in the presence of PML-RARα, suggesting that PML-RARα is implicated in the expression of these miRNAs. To investigate whether the PML-RARα fusion protein binds to the endogenous miRNA genes chromatin immunoprecipitation assays were performed with PML-RARα transfected 293 cells. We demonstrated the presence of PML-RARα protein on the miRNA genes. This indicates that the oncoprotein PML-RARα directly influences the expression of these miRNAs. The function of the PML-RARα targeted miRNAs in APL cell differentiation is currently being studied using retroviral expression vectors.
AML1/Runx1 Negatively Regulates the Number of Quiescent Hematopoietic Stem Cells in Adult Hematopoiesis.
Authors: Ichikawa Motoshi , Asai Takashi , Nakagawa Masahiro , Kawazu Masahito , Goyama Susumu , Takeshita Masataka , Chiba Shigeru , Ogawa Seishi , Kurokawa Mineo 
Published: April 2020

Abstract
Transcription factor AML1 (also called Runx1), which was initially isolated from the t(8;21) chromosomal translocation frequently found in the acute myelogenous leukemia FAB M2 subtype, is essential for the development of multilineage hematopoiesis in mouse embryos. By analyzing conditional AML1 knockout mice, we have previously shown that AML1 negatively regulates the number of immature hematopoietic cells defined as lineage-negative, CD34− Sca-1+ c-Kit+ (34KSL) cells in adult hematopoiesis, while it is required for megakaryocytic maturation and lymphocytic development. The former is a significant observation because an increase in hematopoietic stem/progenitor cells due to defective AML1 function may be closely related to the development of human leukemia. In support of this is the fact that mice in which leukemic chimeric protein AML1/ETO is expressed in hematopoietic cells are subject to myeloproliferative disease and develop leukemia after additional mutation. However, it has remained yet to be determined how AML1 contributes to homeostasis of hematopoietic stem cells (HSCs). To address this issue, we analyzed in detail HSC function in the absence of AML1. Notably, cells in the Hoechst 33342 side population (SP) fraction are increased in number in AML1-deficient bone marrow, which suggests enrichment of quiescent HSCs. We quantitatively evaluated HSCs by bone marrow transplantation assays using limiting dilution and found a significant increase in HSC number within the AML1-deficient bone marrow. These results indicate that the number of quiescent HSCs is negatively regulated by AML1, loss of which may result in accumulation of leukemic stem cell pool in AML1-related leukemia.
The Effects of Glycosaminoglycans on Thrombopoietin-Induced Megakaryocytopoiesis.
Authors: Takahashi Kenji , Kashiwakura Ikuo , Toki Tsutomu , Takagaki Keiichi 
Published: April 2020

Abstract
The extracellular matrix (ECM) plays an essential role in normal hematopoiesis. Proteoglycans and glycosaminoglycans (GAGs) are major components of the ECM. In this study, the effects of various GAGs on the proliferation and differentiation of CD34+ megakaryocytic progenitor cells (CFU-Meg) were evaluated in vitro. CD34+ cells were highly purified from steady-state human peripheral blood. The GAGs tested were hyaluronic acid (from human [HA-h], pig and rooster), keratan sulfate, heparan sulfate, chondroitin sulfate (from whale [CS-A], shark or squid cartilage) and dermatan sulfate (DS). When used alone, none of the GAGs supported the clonal growth of CFU-Meg; however, in cultures stimulated by recombinant human thrombopoietin (TPO), HA-h, CS-A and DS significantly enhanced such growth. In particular, the addition of DS resulted in increases of about 1.3-fold, 1.6-fold and 2.0-fold in the numbers of total cells, megakaryocytes and CFU-Meg, respectively, compared with the control culture stimulated by TPO alone after 9–12 days of serum-free liquid culture. Furthermore, DS induced the generation of hyperploid megakaryocytes and promoted pro-platelet formation. Chemical fragmentation and desulfation of DS showed that a chain of at least 12 saccharides is required for colony-promoting activity and that the sulfate groups play an essential role. DS acts on an immature population of CD34+ cells, stimulates the proliferation of CFU-Meg, and enhances the terminal maturation of megakaryocytes and thrombopoiesis. These results suggest that DS has a wide spectrum of action in promoting megakaryocytopoiesis and thrombopoiesis.
PBI-1402: A Low Molecular Weight Synthetic Hematopoietic Growth Stimulant.
Authors: Grouix Brigitte , Julien Nathalie , Lagraoui Mouna , Morin Marie-Josée , Krosl Gorazd , Roy Denis-Claude , Penney Christopher , Gagnon Lyne 
Published: April 2020

Abstract
PBI-1402 is a non-toxic, well-defined low molecular weight synthetic hematopoietic growth stimulant. PBI-1402 promotes the proliferation and maturation of hematopoietic progenitors (myeloid and erythroid populations) yielding a biological efficacy comparable to G-CSF, GM-CSF and EPO in in vitro human bone marrow cell proliferation and colony formation assays. An additive effect is observed when PBI-1402 is combined with G-CSF, GM-CSF and EPO. In human bone marrow colony assay, PBI-1402 enhances the differentiation of pluripotent stem cells: CFU-GEMM, CFU-GM with a predominant effect on BFU-E. Furthermore, PBI-1402 exerts its activity via a different mechanism of action than EPO and stem cell factor (SCF) and at an earlier stage on more immature hematopoietic progenitors. PBI-1402 is targeted as an adjunct to cancer chemo/radiotherapy, bone marrow transplantation and diseases involving neutropenia and anemia.
In GM-CSF Restrain Mitochondria Pathway Apoptosis NF-kB Is Not the Only Way.
Authors: Jiang Bo , Zhang Ri , Wu De Pei 
Published: April 2020

Abstract
After stimulated by GM-CSF, Akt and IkB kinase (IKK) can be phosphorylated by activated PIK3-kinase. Activated IKK phosphorylate IkB, making NF-kB released and translocate to nucleus. Granulocyte-macrophage colony-stimulating factor (GM-CSF) restrain TF-1 cell mitochondria pathway apoptosis through PI3K pathway, so we asked if NF-κB is the only way in this process.
All group were plused TF-1 cells 2.5X105/250μl, group A plused hGM-CSF (100ng/ml ) 50μl; group B plused hGM-CSF (100ng/ml) 50 μl and SN50 (50 μmol/L) 20μl; group C plused hGM-CSF 50μl and SN50 200μl. After cultured for 48 hours, all groups were dyed with Annexin and PI, analyzed in FCM. At the same time all groups were dyed by diluted MitoCapture to analyze mitochondria membrane electrical potential. Drop experimental group B 10μl on slips, so as group C, then observe it under Fluorescence microscopes FL1 channel.
Group A cells that have normal mitochondria membrane electrical potential can form poly-MitoCapture in it, and inspired red fluorescence. Group C cells that have monomer MitoCapture was inspired green fluorescence. The red fluorescence intensity of control, group A, B, C means is 368.93, 1137.59, 1033.61 and 618.37 respectively. Group A and B retained a statistically significant difference with control (P&lt;0.01). Group C retained a statistically significant difference with control (P&lt;0.01), group A and group B (P&lt;0.01). This investigation proves that GM-CSF restrain TF-1 cells can be incompletely blocked by SN50, which is the specificity inhibitor of NF-kB. So we get the conclusion that NF-kB is an important but not the only pathway in the process of GM-CSF restrain TF-1 cells apoptosis. It also suggests that there maybe some other pathway in this process.