bluuebunny/crossref_metadata_2025_split huggingface dataset:
Application of the Cone and Plate(let) Analyzer as a Point-of-Care Method for Monitoring Anti-Platelet Therapy.
Authors: Spectre Galia , Brill Alexander , Gural Alexander , Shenkman Boris , Turetzky Natalia , Savion Naphtali , Varon David 
Published: April 2020

Abstract
Platelet anti-aggregants play a major role in the treatment of cardiovascular disorders. Recently it has been suggested that a significant proportion of patients fail to respond to these agents according to different testing methods. We have developed a new method for evaluating the response to platelet anti-aggregants, the modified Cone and Plate(let) Analyzer (CPA) test. The method is based on the observation that pre-activation of platelets by an agonist will result in reduced platelet deposition on a polystyrene surface under arterial flow conditions (the phenomenon of platelet adhesion refractoriness). In vitro studies demonstrated that the basic platelet adhesion (surface coverage, SC 12.3±6.8%) was significantly reduced in response to pre-incubation of the sample (for 1 min) with arachidonic acid (AA, SC 2.1±1.5%), adenine diphosphate (ADP, SC 1.3±0.6%) and epinephrine (EPI, 2.9±0.9%). This effect was selectively inhibited by aspirin (for the response to AA, SC 8.1±3.8%), and by 2-Methylthio-AMP triethylammonium (2-MeSAMP), a selective inhibitor of P2Y12 (for the response to ADP, SC 4.8±2.0%), p&lt;0.001. The potential application of this method for monitoring the effect of aspirin therapy (daily doses of 100, 300 and 500 mg), among 20 healthy volunteers was investigated in comparison to turbidimetric aggregometer. All volunteers responded to aspirin in the three doses according to the aggregometer test (reduced max. aggregation from 88±8% to 12.4±7%), with no evidence for a dose response manner in this dose range. Basic platelet adhesion as tested by the CPA (SC 9.8±2.2%), was unaffected by aspirin in the three tested doses. However, the response to pre-incubation with AA was significantly inhibited by aspirin in all patients (SC of 0.6±0.3% before aspirin therapy and 3.5±1.3%, 4.4±1.7%, and 4.1±2.0% following therapy with 100, 200 and 500 mg aspirin per day, respectively, p&lt;0.001 for all doses), again with no dose response effect. This effect was specific for AA, since no difference in SC was observed when ADP was used as an agonist. A significant correlation between the effect of aspirin as tested by the modified CPA and by the turbidimetric aggregometer using AA as an agonist (R2=0.55) was observed. In conclusion, the CPA was found useful point-of-care method for testing the response of platelets to aggregating agonists as well as for monitoring the effect of anti-platelet drugs.
Direct and Indirect Effects of Oxymetholone on Growth and Apoptosis of Hematopoietic Progenitor Cells In Vitro.
Authors: Jo Deog-Yeon , Kim Seong-Woo , Hwang Jin-Hee , Yun Hwan-Jung , Kim Samyong 
Published: April 2020

Abstract
Despite a tarnished reputation, androgens remain a common treatment for aplastic anemia, especially in the Orient. Increased apoptosis of hematopoietic cells is characteristic of aplastic anemia; however, it has not been established whether androgens affect apoptosis of hematopoietic progenitor cells. In most previous studies regarding the in vitro effects of androgens on hematopoietic cells, whole bone marrow cells were used, rather than purified hematopoietic progenitor cells such as CD34+ cells. With these questions in mind, we investigated the direct and indirect effects of oxymetholone and other androgens on apoptosis and growth of normal hematopoietic progenitor cells (HPCs) in vitro. Oxymetholone did not rescue normal BM CD34+ cells and colony-forming cells (CFCs), other than mature erythroid CFCs, from apoptosis induced by growth factor deprivation. Unexpectedly, both testosterone and 5-dihydrotestosterone (5-DHT) at a concentration of 10−5 M, but not oxymetholone, increased the percentage of annexin-positive apoptotic cells (62.2 ± 5.9%, P &lt; 0.05; 61.7 ± 6.4%, P &lt; 0.05, respectively) compared with the controls (52.6 ± 5.6%). The addition of either stromal cell-derived factor-1 (SDF-1) or stem cell factor (SCF) partially relieved the increase in apoptosis induced by 5-DHT, and the addition of both SDF-1 and SCF completely reversed it. Oxymetholone did not rescue CFCs from interferon-gamma (IFN-g)-induced inhibition of clonal growth of BM CD34+ cells in methylcellulose cultures. Furthermore, oxymetholone did not mitigate IFN-g-induced suppression of CD34+ cell survival in the presence of growth factors. In a methylcellulose clonogenic assay, oxymetholone stimulated the clonal growth of colony-forming unit-erythroid at low concentrations, while not affecting colony-forming unit-granulocyte/macrophage or burst-forming unit-erythroid. Oxymetholone did not reverse the IFN-g-induced inhibition of colony formation by CD34+ cells. Interestingly, oxymetholone stimulated the production of SCF and thrombopoietin in normal human bone marrow stromal cells (BMSCs) through transcriptional regulation while inhibiting the production of interleukin-6. In agreement with this, oxymetholone-treated BMSCs better supported the survival and growth of HPCs. These results suggest that oxymetholone exerts most of its myelostimulatory effects via the regulation of cytokine production in BMSCs, rather than by direct action on hematopoietic progenitor cells.
Quantitative and Qualitative Protein Expression Mapping of Highly Enriched G-CSF Mobilized CD34+ Stem Cells from Peripheral Blood.
Authors: Lannert Heinrich , Franz Thomas , Eckstein Volker , Hofmann Reiner , Lenze Angela , Horsch Kerstin , Miesala Katrin , Ho Anthony D. 
Published: April 2020

Abstract
Background: Proteome analysis is a direct measurement of proteins in terms of their presence and relative abundance in a defined system. The overall aim of a proteomic study is characterization of the complex network of cell regulation. Different states of a cell can be compared and specific qualitative and quantitative protein changes can be identified. We focused our first proteom-investigations on G-CSF mobilized CD34+ stem cells from peripheral blood (PB).
Methods: Mononuclear cells from healthy donors were isolated by a standard Ficoll-Hypaque gradient separation method after leucapheresis from PB. An Auto-MACS (Miltenyi) and FACS Vantage SE cell sorter (Becton Dickinson) was used to highly enrich (&gt;99%) CD34+ cells fractions. Sample preparation, determination of protein concentrations, 2D-gel-electrophoresis with a 1D-separation isoelectric focusing (IEF) with immobilised pH gradient (IPG) strips (17cm), 2D-separation with SDS-PAGE were performed and described in Proteome Works System (BioRad), pH range from 3 until 10 and molecular weight from 5 until 200kDa. Sypro ruby stained gels were used for protein identification by peptide mass fingerprint analysis with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS).
Results: A.) The pattern of the G-CSF mobilized CD34+ fraction from PB cells is complex and shows &gt;1000 protein spots (dependent on protein concentration) in silver stain using PDQuest.
B.) The most dominant 150 protein spots were characterized by MALDI-TOF analyses. A sample of identified spots and their functional groups are specified:
1. Cytosketeletal proteins: tubulin, actin, profilin, tropomyosin. 2. Signaling proteins: enolase, rho GDP dissociation inhibitor 2, glutathione S transferase, nucleophosmin. 3. Metabolism: phosphoglycerate kinase I, glyceraldehyde-3P-dehydrogenase. 4. Protein folding: heat shock proteins (HSP 60, HSP70c), molecular chaperones (GRP78).
Conclusions: Proteomics is a highly effective method to describe the gen-expression and the cell biology of stem cells on real protein transcription. Highly purified CD34+ cells from PB demonstrate a complex proteom. Our preliminary results show that cell cycle determining chaperones (folding of other protein and responsible for their functional activation or deactivation) are dominantly expressed. Thus cytoskeletal proteins dominate the protein pattern of CB stem cells. Further Proteom-comparison with other subsets of stem cells from different origins (e.g. fetal liver, cord blood, bone marrow) are currently achieved.
Figure Figure
Bonr Marrow Reconstitution from Mouse Embryonic Stem Cell-Derived Hematopoietic Cells.
Authors: Yoshimoto Momoko , Hsi Chang , Umeda Katsutsugu , Iida Midori , Heike Toshio , Nakahata Tatsutoshi 
Published: April 2020

Abstract
Differentiation of embryonic stem (ES) cells in vitro yield different kinds of hematopoietic progenitors including primitive and definitive hematopoietic cells. It has been reported that HOXB4 induction enable york sac (YS) cells and embryoid body-derived cells to engraft in the irradiated adult mice, however, since the characteristic of ES-derived transplantable cells is not clear, generating hematopoietic stem cells (HSCs) in vitro still remains to be resolved. We previously reported the generation of definitive HSCs from both early YS and intraembryonic paraaortic splanchnopleures (P-Sp) on AGM-S3 stromal cells derived from the aorta-gonad-mesonephros (AGM) region at 10.5 days post coitum (Matsuoka, et al; Blood 2001) Co-cultureing on AGM-S3, these YS cells and PS-Sp cells acquired the reconstituting potential of adult irradiated mice. Here we intended to make HSCs using this stromal cells. We differentiated ES cells labeled with GFP on OP9 stromal cell, which is supportive for Hematopoietic differentiation. After 4 days, we sorted Flk1+ cells, which is considered as a marker of hemangilblasts, and transferred them onto A-9, subline of AGM-S3, or OP9 stromal cell layer with cytokines. After several days incubation, we examined the emergence of CD34+ c-kit+ cells and colony forming ability of CD34+ or CD34− cells. CD34+ cells contained more CFU-Mix than CD34− cells. When compared on A-9 or OP9, cultured cells on OP9 contained more CFU activity than on A-9. We sorted and cultured CD34+ c-kit+ cells on OP9 for 7–10 days, and confirmed Ter119+, Gr-1+, or Mac-1+ cells differentiated from CD34+ c-kit+ cells by FACS analysis. Next, we cultured Flk1+ cells on A-9 or OP9 for 7–15 days and transplanted all the collected cells into 2.4Gy irradiated NOD-SCID mice. After 3 months after transplantation, FACS analysis showed no GFP+ cells in the recipient BM. However, PCR analysis detected donor derived DNAs in BM when Flk1+ cells were cocultured on A-9. We next transplanted 1×104 of CD34+ CD45+ or CD34+ CD45− cells from Flk1+ cells cocultured on A-9 or OP9 into 2.4 Gy irradiated NOD-SCID mice. PCR analysis revealed donor derived DNAs in mice transplanted with CD34+ CD45+ cells on A-9 and with CD34+ CD45− cell on OP9. These data suggested that CD34+ cells differentiated from Flk1+ cells have powerful hematipoietic activity and showed different potential cultured between on A-9 and on OP9.
A Novel hTERC Deletion Manifesting with Features of Dyskeratosis Congenita and Genetic Anticipation.
Authors: Benke Stan , Houston D. S. , Dokal Inderjeet , Vulliamy Tom 
Published: April 2020

Abstract
The gene encoding the RNA component of human telomerase (hTERC) is mutated in families with the autosomal dominant form of dyskeratosis congenita (DC). The phenomenon of genetic anticipation has recently been reported to accompany this form of DC, with disease severity increasing in offspring of affected individuals. It has been postulated that anticipation in these families relates to the adverse impact of hTERC mutations on inherited telomere length, with progressive telomere shortening seen in succeeding generations (Nat Gen2004; 36:447). We describe here a novel hTERC mutation, with affected individuals presenting in adulthood with mild mucocutaneous abnormalities, bone marrow failure and a pattern of penetrance supporting the presence of disease anticipation. The proband in the family studied presented at age 49 with squamous cell carcinoma of the tongue and a history of oral leukoplakia which he had developed at age 30. Peripheral blood on presentation was remarkable only for a mild macrocytic anemia. During treatment of his malignancy, severe and irreversible bone marrow hypoplasia was precipitated by a single cycle of cisplatinum chemotherapy. The patient’s brother at age 25 had been previously diagnosed with severe aplastic anemia; this was refractory to standard immunosuppression with cyclosporine and antithymocyte globulin. No somatic abnormailites were identified in this patient. Testing for Fanconi anemia in both siblings was negative. Direct sequencing analysis of hTERC in these patients revealed both to be heterozygous for a novel hTERC mutation (79 deletion C). Further studies among family members documented heterozygosity for the mutation in the mother of these two siblings. At age 77, she displayed none of the mucocutaneous signs associated with DC, while the only abnormality seen in her peripheral blood was an elevated mean corpuscular volume. The hTERC mutation seen in this family most likely exerts its effects through disruption of the pseudoknot domain. The findings of an individual with normal longevity, minimal phenotypic expression and affected offspring are further evidence of genetic anticipation being an important feature of autosomal dominant DC. Correlation with determination of telomere length has been initiated.
Parathyroid Hormone (1–34) Treatment Induces Hematopoietic Precursor Cell Expansion in Murine Long-Term Bone Marrow Culture.
Authors: Drize Nina , Svinareva Daria , Nifontova Irina , Chertkov Joseph 
Published: April 2020

Abstract
Parathyroid hormone (PTH) induces activation and increases the number of osteoblasts. An increase the stem cells number (Lin-Sca1+cKit+) was observed in mice after 4 weeks of PTH treatment, that suggest osteoblasts participation in regulation of hematopoiesis (Calvi, et al., 2003, Nature, 425, 841). Long-term bone marrow culture (LTBMC) was used for the study of PTH influence on hematopoietic progenitors of different stages of maturation. Rat PTH (1–34) (final concentration, 10−7M) was supported during whole culture period. Cell number, colony-forming units in culture (CFU-GM, CFU-C-21 days) and cobblestone area forming cells (CAFC) were measured during 10 weeks. The number of mature cells in culture did not changed during PTH treatment. The number of studied progenitors did not changed significantly during 3 and 6 weeks of PTH treatment. The number of CFU-GM, CFU-C-21 days and CAFC 14 days, CAFC 21 days, CAFC 28 days increased 7 –10 fold in suspension fraction of LTBMC after 10 weeks of PTH treatment.
Table 1. Number of different precursor cells in suspension fraction of LTBMC after PTH treatment.
To evaluate the possibility of stem cells expansion by using of PTH treated adherent cell layers (ACL) of LTBMC, the PTH-treated for 4 and 8 weeks cultures, were irradiated with 40 Gy and seeded with 2 × 106 bone marrow cells depleted of adherent cells. Following 24 hours the number of survived CAFC 8 – 28 was analyzed. On PTH-treated ACL the number of CAFC 21 increased 2,7 ± 0,4 fold as compared with fresh bone marrow or culture with non-treated ACLs. The number of CAFC 28, which characterizes the number of marrow repopulating cells, increased 2 fold only after cultivation on ACL treated with PTH for 8 weeks.
Table 2. CAFC number in bone marrow cultivated 24 hours on PTH treated ACL of LTBMC
The data suggest possibility to increase stem cell expansion ex vivo on pharmacologically manipulated microenvironment.
Time of cultivation, weeks Specificity of progenitor Number of precursors, per 100000 cells Control Control PTH treatment 3 CFU-GM 50 ± 5.2 77 ± 5.6 6 CFU-GM 33 ± 4 31 ± 5.9 10 CFU-GM 20 ± 4.8 147.5 ± 3.5 3 CFU-C-21 55 ± 5.9 76.6 ± 11.2 10 CFU-C-21 11.5 ± 1.7 102.0 ± 2.0 3 CAFC-7 21.6 20.3 6 CAFC-7 25.9 26.9 10 CAFC-7 0.9 3.0 3 CAFC-21 1.01 0.69 6 CAFC-21 0.43 0.48 10 CAFC-21 0.28 3.11 3 CAFC-28 0.32 0.55 6 CAFC-28 0.12 0.14 10 CAFC-28 0.1 0.82 Age of CAFC (days) Number of CAFC per 2 x 10^6 bone marrow cells not treated PTH (4 weeks) PTH (8 weeks) CAFC-7 424.1 804.5 647.5 CAFC-14 262.5 529.8 295.2 CAFC-21 93.5 272.7 226.2 CAFC-28 38.3 30.1 79.2
Human Marrow Megakaryocyte Differentiation: Multiparameter Correlative Analysis Identifies Von Willebrand Factor as a Sensitive and Distinctive Marker for Early (2N and 4N) Megakaryocytes.
Authors: Tomer Aaron 
Published: April 2020

Abstract
Human megakaryocyte differentiation and maturation were studied in fresh marrow aspirates using multiparameter flow cytometric correlative analysis. The expression of glycoprotein (GP)IIb/IIIa, GPIIIa, GPIb and CD36 correlated directly with cell size and ploidy (r ≥ 0.97) however, GPIb acquisition was relatively slow. von Willebrand factor (vWF) is robustly expressed by early (2N and 4N) megakaryocytes, enabling their complete resolution from the other marrow cells at a level superior to that achieved with GPIIb/IIIa. Expression of myeloid CD45 and IgG-FcγRII (CDw32) increased with megakaryocyte maturation and contrasted with the declining expression of HLA-DR (negative in platelets). Interleukin-6 receptor expression in megakaryocytes was higher than in other marrow cells. Using the time-of-flight technique, the diameter of the megakaryocyte population was 37±4μm (mean±1SD) compared to 14±2μm for the total marrow cells, ranging from 21±4μm for 2N cells to 56±8μm for 64N cells. Cell size directly correlated with cell DNA (r=0.98). Receptor density of GPIIb/IIIa and GPIb decreased with the transition from 2N to 4N cells, then reached maximum at 32N-cells. We conclude that the present methods are useful for studying in vivo human megakaryocytopiesis in normal and altered states. The expression of vWF is a sensitive and distinctive marker for the identification of young marrow megakaryocytes.
Oncogenic Tyrosine Kinases Regulate Proliferative and Survival Signals through Activation of Id1.
Authors: Gu Ting-Lei , Tam Winnie F. , Chen Jing , Lee Benjamin H. , Wang Andrew Z. , Cooper Michael R. , Monti Stefano , Golub Todd , Gilliland Gary D. 
Published: April 2020

Abstract
The oncogenic tyrosine kinases, such as BCR-ABL, TEL-ABL, TEL-PDGFbR and FLT3-ITD, play a major role in the development of hematopoietic malignancy. They activate many of the same signal transduction pathways, including the RAS/MAPK pathway, STAT/JAK pathway and PI3-K/AKT pathway. Although activation of signal transduction pathway has been extensively studied, few bone fide target genes have been identified. To identify the critical target genes that are required for transformation in hematopoietic cells, specific tyrosine kinase inhibitors were applied to 32Dcl3 cells expressing various activated tyrosine kinases. Using Affymetrix oligonucleotide arrays, we have identified inhibitor of DNA binding 1 (Id1), which is involved in development, cell cycle and tumorigenesis, as a common target gene of oncogenic tyrosine kinases. To characterize the biological consequence of Id1 expression, we inhibited the expression of Id1 in two human leukemia cell lines (Molm-14 and K562) by an antisense Id1 construct. In both cell lines, we observed a growth inhibition associated with release of inhibition of p27Kip. In addition, inactivation of Id1 sensitized K562 cells to Trail-induced apoptosis. Our findings suggest that Id1 is an important target of constitutively activated tyrosine kinases, and may be a therapeutic target for leukemias involving oncogenic tyrosine kinases.
Transcription Factors Analysis in Neuronal-Like Cells Derived from Adult Human Bone Marrow.
Authors: Bulvik Shlomo , Ben-Zur Tali , Levy Yossef S. , Burshtein Alex , Melamed Eldad , Offen Daniel 
Published: April 2020

Abstract
Stem cells are immature cells with self-renewal capacity and ability to differentiate into multiple cell types including bone, cartilage, fat, tendon and muscle. Transplantation of stem cells or their derivatives to the adult brain have been proposed as future therapy for neurodegenerative diseases. Parkinson’s disease (PD) is a progressive neurodegenerative disease involving the deterioration of dopaminergic neurons of the substantia nigra, a part of the midbrain. Most of the currently used drugs are based on dopamine replacement which, although it reduces symptoms is limited owing to severe side effects. Alternatively, strategies of cell therapy for the treatment of PD focused on replacing damaged neurons with cells that may improve the functioning of the damaged cell population. In this study, we demonstrate that bone marrow stromal cells isolated from human iliac crest can be induced to differentiate into neural-like cells. Human bone marrow stromal cells differentiation was induced with N2 supplements, retinoic acid, butylated hydroxyanisole, isobutylmethylxanthine and dibutyryl-cyclic-AMP. A few hours after differentiation the cells changed their phenotype to neural-like cells, spindle shaped, with cell bodies and long branches. Moreover, immunostaining demonstrated the expression of specific neuronal markers such as nestin, neurofilaments and neuron specific enolase. To define the transcription factors associated with the differentiation, we used the TranSignal Protein/DNA kit (Panomics Inc). This new array-based technology allows functional analysis of dozens of eukaryotic transcription factors. Nuclear proteins were purified and incubated with mix labeled DNA binding oligonucleotides corresponding to the consensus sequences of 96 transcription factors. The DNA probes were then dissociated from the complexes and used to hybridize the TranSignal Array. We found that the differentiated human bone marrow stromal cells induced the expression of several neuron-specific transcription factors such as GAG (glycosaminoglycan), FKHR (human forkhead in rhabdomyosarcoma) and DR-2. The activation of these transcription factors in a time course manner was evident also by electrophoretic mobility shift assay. In conclusion, we show that the induced differentiation of neuronal like cells from bone marrow is associated with activation of specific neuronal transcription factors. Our study advances the possible use of autologous transplantation as a new strategy for cell therapy in neurodegenerative diseases.
Transdifferentiation of Bone Marrow-Derived Cells Co-Cultured with Fetal Liver Cells into Hepatic-Like Cells without Fusion.
Authors: Yamada Yasuhiro , Yonemura Yuji , Nishimoto Eishi , Mitsuya Hiroaki 
Published: April 2020

Abstract
Several research groups have reported that bone marrow cells (BMCs) transdifferentiate into hepatocytes in rodents. However, it is yet to be studied what factors effectively trigger and sustain the transdifferentiation of BMCs to hepatocytes. In the present study, we investigated whether murine BMCs in the presence of fetal liver cells (FLCs) could differentiate into hepatic-like cells in vitro without fusion. Fractionated BMCs from C57Bl/6-TgN(ACTbEGFP)10bs mice and FLCs from B6.129S7-Gt(ROSA)26Sor mice were co-cultured at 1x105 cells and 1x106 cells in 10% FCS-containing medium supplemented with hepatocyte growth factor on laminin-coated dishes. Hepatocyte-specific markers among BMCs were detected as assessed by immunocytochemistry for albumin and reverse transcription-polymerase chain reaction (RT-PCR) for alpha-fetoprotein (AFP), albumin, and cytokeratin-19 mRNAs. We also found that Sca-1+ BMCs containing both hematopoietic stem cells and AFP-expressing cells could differentiate into hepatic-like cells and such cells were seen adherent to dish together with FLCs in the early phase of culture. Moreover, the AFP-expressing cells were found in a Sca-1+ cKit- cell fraction, which also differentiated into CD45− GFP+ albumin+ cells and proved to be positive for GFP but negative for LacZ as assessed by RT-PCR and immunocytochemistry. These results suggest that albumin+ cells developed through transdifferentiation from BMCs but not through spontaneous cell fusion between BMCs and FLCs.