bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ineffective Hematopoiesis in Megaloblastic Anemia Is Associated with Higher Frequency of Apoptosis in Each Lineage Immature and Mature Cells.
Authors: Oyake Tatsuo , Ito Shigeki , Kowata Shugo , Murai Kazunori , Ishida Yoji 
Published: April 2020

Abstract
A deficiency of Vitamin B12 is a major cause of megaloblastic anemia (MBA). Ineffective hematopoiesis is observed in MBA, characterized by cytopenia, bone marrow cells with dysplastic change and normal to hypercellularity. We reported that excessive apoptosis of each lineage CD34(+) cells was observed in myelodysplastic syndrome (MDS) in the last ASH meeting. In this study, we investigated the hypothesis that excessive apoptosis induced the ineffective hematopoiesis in MBA. We performed the three color flow cytometric analysis of bone marrow mononuclear cells in 12 MBA patients using PE labeled Annexin V, PerCP labeled anti-CD34 antibody and FITC labeled anti-each lineage antibody (anti-glycophorin A (GPA) antibody, anti-CD33 antibody and anti-CD41 antibody). The frequency of apoptosis in subpopulations of immature (CD34(+)) and each lineage (+) cells or those of mature (CD34(−)) and each lineage (+) cells were calculated as the ratio (%) of (cell number with Annexin V(+)) divided by (cell number in the subpopulation). The subpopulations include CD34(+)GPA(+) (immature erythroid), CD34(+)CD33(+) (immature myeloid), CD34(+)CD41(+) (immature megakaryocytic), CD34(−)GPA(+) (mature erythroid), CD34(−)CD33(+) (mature myeloid) and CD34(−)CD41(+) (mature megakaryocytic) cells. Much higher frequency of apoptosis was observed in each lineage CD34(+) cells in MBA (median: 23.8% (range: 10.8–43.6%) in erythroid, 43.5% (12.7–67.3%) in myeloid, 50.1% (21.0–64.1%) in megakaryocytic lineages, P&lt; 0.05, respectively, n=12), compared to those in normal controls (8.5% (1.5–9.9%) in erythroid, 8.5% (2.2–8.8%) in myeloid, 7.7% (4.4–9.3%) in megakaryocytic lineages, respectively, n=10). While, the relatively higher frequency of apoptosis was observed in each lineage CD34(−) cells in MBA patients (median: 15.9% (range: 5.1–20.6%) in erythroid, 16.4% (5.6–23.2%) in myeloid, 16.1% (10.2–24.8%) in megakaryocytic lineages, P&lt; 0.05, respectively, n=12), compared to those in normal controls (4.8% (1.3–6.6%) in erythroid, 2.2% (0.6–4.4%) in myeloid, 3.3% (1.5–7.1%) in megakaryocytic lineages, respectively, n=10).
These results suggest that the excessive apoptosis occurs not only in CD34(+) but also in CD34(−) cells, which induces ineffective hematopoiesis in MBA.
Figure 1. The frequency of apoptosis in CD34+ BM cells Figure 1. The frequency of apoptosis in CD34+ BM cells Figure 2. The frequency of apoptosis in CD34− BM cells Figure 2. The frequency of apoptosis in CD34− BM cells
Functional Distinctions for Smad1 and Smad5 during Hematopoiesis Revealed by the Zebrafish Model System.
Authors: McReynolds Lisa J. , Mullins Mary , Evans Todd 
Published: April 2020

Abstract
Smad1 and Smad5 proteins are intracellular mediators of BMP signaling, which has been implicated in regulating hemato-vascular development. Both Smad 1 and 5 are expressed widely in the developing hematopoietic mesoderm, and mutation of either mouse gene is embryonic lethal, limiting progress in defining their function in hematopoiesis. To compare the functions of these two closely related regulatory proteins, we used specific Smad5 mutants and Smad1 morpholinos in the zebrafish model. In the Smad5 mutant, piggytail (pgy), homozygous embryos are dorsalized and live until day 3–5, but have little to no blood and no circulation. Key hematopoietic regulatory genes are expressed in the pgy/pgy mutant fish, but the pattern of expression is altered in early embryos. Gata1, Gata2, Lmo2, draculin and Scl expression patterns are expanded at the five somite stage toward the more caudal region of the mutant embryos. In contrast, the expression pattern of the general mesoderm marker ‘no tail’ is not affected by loss of Smad5. The cells in the most caudal region of the mutant embryos express Gata1 precociously, indicating that they are specified along the erythroid lineage but fail to differentiate. However, Gata1 expression is lost by 48 hpf, as demonstrated in embryos carrying a gata1:gfp transgene on the pgy/pgy background. As in the pgy embryos, injection of a Smad1 morpholino at the one-cell stage leads to a dorzalized phenotype. However, in contrast to pgy embryos, Gata1 expression is maintained in the Smad1 morphants and Gata1 positive cells are found to circulate in the morphant embryos through at least 72 hpf. Our data support an essential role for Smad5 and not Smad1 in embryonic hematopoiesis. To analyze Smad5 function in the adult hematopoietic system, we established adult pgy fish that carry the gata1:gfp transgene, for analysis of blood cell profiles using flow cytometry. Blood cell populations were monitored under normal conditions and following phenylhydrazine induced hemolytic anemia in wildtype and pgy/+ fish. The pgy/+ adult fish are able to mount an erythropoietic response to hemolytic anemia. However, pgy/+ fish at two days post-treatment accumulate 30% of their erythroid progenitor population at the polychromatophilic stage, compared to 10% in wildtype. The pgy/+ fish are thus unable to reconstitute the erythrocyte population in the kidney at a rate comparable to wildtype fish. In summary, our data indicate that Smad5 has a role in the kinetics of embryonic stage erythropoiesis, distinct from the function of Smad1, and in adult erythropoiesis specifically at the transition from the proeyrthroblast to the polychromatophilic erythroblast stage.
A Role for fbxo5 in Zebrafish Myelopoiesis.
Authors: Rhodes Jennifer , Amsterdam Adam , Ho Karen , Kanki John P. , Hopkins Nancy , Look A. Thomas 
Published: April 2020

Abstract
The zebrafish is a powerful model system for investigating embryonic vertebrate hematopoiesis that allows for the critical in vivo analysis of cell development and lineage determination. Conservation of the variety of blood cell types and key regulatory factors between mammals and zebrafish suggests that the regulatory mechanisms directing lineage specification are similarly conserved. To identify novel genes necessary for normal myeloid cell development, we have analyzed a panel of zebrafish insertional mutants, provided by Nancy Hopkins at Massachusetts Institute of Technology (MIT), and identified mutants with deficient or abnormal distribution of myeloperoxidase (mpo), a gene specifically expressed in zebrafish granulocytes. One of the mutants identified in this screen is disrupted in fbxo5, a gene not previously identified as having a role in hematopoiesis. Using whole mount RNA in situ analysis on mutant and morpholino-injected embryos (morphants) we have determined that decreased levels of fbxo5 result in severely diminished numbers of granulocytes, although erythropoiesis appears normal. Interestingly, fbxo5 morphants have normal levels of pu.1 and l-plastin expression. Since we have previously shown that knockdown of pu.1 results in the loss of mpo-expressing cells, our data suggest that fbxo5 may function downstream or in a parallel pathway to pu.1 during granulopoiesis. To determine if the decreased number of granulocytes was due to programmed cell death, we examined the level of apoptosis in morphant versus control embryos. Whole mount acridine orange staining of apoptotic cells indicated that fbxo5 morphants had increased apoptosis, which can be mostly rescued by co-injection with p53 morpholino. However, the fbxo5/p53 double morphants continued to display a severe decrease in granulocytes, suggesting that the loss of granulocytes is not due to p53-mediated apoptosis. Fbxo5 has been examined previously in Xenopus oocytes, where the protein was found to regulate cell cycle progression by inhibiting APC. Interestingly, we have identified multiple mutants in genes that normally regulate the G2/M checkpoint and mitosis during the cell cycle all which display decreased numbers of granulocytes, suggesting a role for the regulation of the cell cycle in granulopoiesis. While further analysis is needed to determine where and how fbxo5 functions during myeloid cell development, as well as each of the genes disrupted in our additional cell cycle mutants, we have identified that fbxo5 is necessary specifically for the normal development of granulocytes.
A Novel Culture System of Human ES Cells for High Efficient Hematopoietic and Endothelial Differentiation.
Authors: Wang Zack Z. , Chen Tong , Au Patrick , Shao Ying , Jain Rakesh , Scadden David T. 
Published: April 2020

Abstract
Human embryonic stem cell (hESC) lines provide not only an unique system for studying human embryonic development, but also have enormous potential as a source of therapeutic tissues, including those of vascular and hematopoietic lineages. However, it is difficult to generate large-scale of relatively pure population of specific lineage cells derived from hESCs. Here, we describe a 2-dimensional (2-D) culture system for hESC differentiation to generate hematopoietic and endothelial cells in vitro without embryoid body (EB) formation (3-D). To initial differentiation, hESCs (H1 and H9 cell lines) were placed in serum-containing media for 9–10 days without supplement of additional growth factors. Gene expression analysis of 2-D differentiation indicated that Oct-4, a pluripotent gene, was decreased, while genes for GATA-2 and CD31, were increased, suggesting that differentiated hESCs contain hematopoietic and endothelial progenitors. Comparing with 3-D EB formation, 2-D differentiation was able to generate robust differentiated cells (5 to 10-fold higher). To generate a reasonable pure population of hematopoietic and endothelial precursors, CD34+ cells prior to hematopoietic commitment were isolated from 2-D differentiation by immuno-magnetic beads and cultured in the presence of hematopoietic growth factors and endothelial growth factors. After 7–10 days culture of CD34+ cells, both suspension and adherent cells emerged. The number of suspension and adherent cells was dependent on the presence of hematopoietic growth factors or/and endothelial growth factors. The suspension cells expressed hematopoietic marker, CD45, and gave rise to multi-lineage hematopoietic colonies on methylcellulose cultures. The adherent cells expressed endothelial markers, such as CD31 and Ve-cadherin, and took up Dill-labeled ac-LDL. The adherent cells rapidly formed capillary-like structures on Matrigel cultures, while the suspension cells would not. While the adherent cells were implanted into mice, they formed functional blood vessels in vivo. Our culture system paves the road for further large-scale manufacture of hematopoietic and endothelial cells derived from human ES cells. Our studies demonstrated also that CD34+ cells derived from human ES cells contain hematopoietic and endothelial progenitors.
Derivation of Lymphoid Lineage Cells from Human Embryonic Stem Cells on Co-Culture in OP9 and OP9DL1 Stromal Cells.
Authors: Mojica-Henshaw Mariluz P. , Huang Xiaosong , Spangrude Gerald J. 
Published: April 2020

Abstract
We have been able to direct the differentiation of CD34pos cells derived from H9 and H7 hESCs toward lymphoid lineages using OP9 or OP9DL1 feeder layers. OP9 and OP9DL1 stromal cell lines are consistently better than S17 or OP9-jagged in inducing hESC-derived CD34pos cells at different time points in culture. By day 14 in culture, about 8.7% and 7.9% of differentiated H9 hES cells harvested from co-cultures in OP9 or OP9DL1 respectively were CD34pos. The CD34pos cells in these cultures were enriched for by magnetic sorting. The clonogenic potential of differentiated hES cell suspensions and of sorted CD34pos cells were determined using methylcellulose colony assays. The highest clonogenic potential was noted after 18 days in co-culture when we obtained 142 +/− 31 and 218 +/− 38 colonies per million cells plated from OP9 and OP9DL1. Selection for CD34pos cells enriched the clonogenic activity 27-fold. Morphologic analyses of representative colonies from the methylcellulose cultures showed cells of the erythroid, myeloid, lymphoid and megakaryocytic lineages were generated. Directed differentiation of CD34pos cells towards lymphoid lineages was initiated by replating the sorted CD34pos cells onto irradiated OP9 or OP9DL1 stromal cells supplemented with stem cell factor (SCF), IL3, IL7 and Flt3L. Under these conditions, B lineage (CD19pos) and myeloid lineage (CD14pos or CD15pos) cells were observed when CD34pos cells were grown in OP9, and T lineage cells were observed when CD34pos cells were grown in OP9DL1. CD4posCD8pos cells were evident after 12 days of secondary culture in OP9DL1 and were prominent by day 20. Only a few of the CD4pos, CD8pos and CD4posCD8pos cells, however, were noted to be CD3pos. Addition of SCF + IL3, SCF + Flt3L, or SCF + Flt3L + IL7 at initiation of hESC differentiation increased the percentage of CD34pos cells and/ or increased the hematopoietic (clonogenic) potential of sorted CD34pos cells. CD34pos cells derived from hESCs grown in OP9DL1 for 14 days and replated in OP9DL1 supplemented with Flt3L + IL7 +/− SCF generated the highest percentages of CD4posCD8pos cells. Additional phenotypic analyses and functional studies of sorted CD4pos, CD8pos and CD4posCD8pos cells generated from these cultures are needed to further characterize their lymphoid properties.
The Role of Alpha Hemoglobin Stabilizing Protein (AHSP) in the Formation of Hemoglobin A.
Authors: Zhou Suiping , Weiss Mitchell J. , Gow Andrew J. 
Published: April 2020

Abstract
Alpha hemoglobin stabilizing protein (AHSP) binds α hemoglobin (Hb) to stabilize its structure and limit its chemical reactivity in vitro and in vivo. Addition of β Hb to α Hb-AHSP displaces AHSP to form HbA (α2β2) tetramer. Recently, we determined that binding of AHSP to free α Hb generates a novel structure in which the helix F of α Hb is disordered and the heme iron is reduced to the ferrous form and coordinated by the distal histidine (ferrous α Hb-AHSP). Over time, the heme iron within this complex oxidizes to the ferric form (ferric α Hb-AHSP) and the F helix reorders, generating a bis-histidyl heme iron that exhibits decreased capacity to catalyze the formation of reactive oxygen species. At room temperature in ambient oxygen, the conversion of ferrous to ferric α Hb-AHSP occurs over several hours. We proposed that in vivo, these complexes could serve as transient intermediates to maintain α Hb in a stable state until β Hb is available to form tetrameric HbA. The current study addresses two questions related to this hypothesis:
Does bis-histidyl ferric α Hb-AHSP form more readily under physiologic conditions? Can ferrous and ferric α Hb-AHSP complexes form functional tetrameric HbA upon reaction with β Hb?
The conversion of ferrous to ferric α Hb-AHSP is temperature-dependent with complete conversion occurring in 40 minutes at 37°C, as opposed to 5 hours at 22°C. Furthermore, the conversion is accelerated by partial oxygen saturation, increasing the likelihood that ferric-AHSP Hb will form in vivo. Using cellulose acetate gel electrophoresis, we demonstrated that β Hb displaces AHSP to form tetrameric HbA from both α Hb-AHSP complexes. The resulting tetramer from ferrous α Hb-AHSP is similar to HbA, both spectroscopically and in terms of oxygen binding. In contrast, the Hb tetramer formed from ferric α Hb-AHSP and β Hb is similar to HbY (αFeIII2βFeII2) spectroscopically. However, the α globin heme iron within this tetrameric complex appears to be occluded, as the rate of cyanide binding is reduced. Furthermore, it is resistant to reduction by sodium dithionite or methemoglobin reductase, suggesting that the α Hb retains its bis-histidyl nature. Together, these studies show that both α Hb-AHSP complexes are likely to form under physiological conditions. Upon interaction with β Hb, ferrous α Hb-AHSP forms functional Hb A tetramer. In contrast, ferric α Hb-AHSP is more chemically inert, but may not be able to form viable HbA.
Structure of the Calcium-Rich Signature Domain of Human Thrombospondin-2.
Authors: Britt C. , Bernstein Douglas A. , Annis Douglas S. , Misenheimer Tina M. , Hannah Blue-Leaf A. , Mosher Deane F. , Keck James L. 
Published: April 2020

Abstract
There are five members of the thrombospondin (TSP) family in vertebrates. This family of proteins comprises two separate subsets, group A and group B. Group A includes TSP-1 and TSP-2. TSP-1 is a major platelet α-granule component and contains a polymorphism that has been linked to coronary artery disease (CAD). TSP-2, although not found in platelets, is important for megakaryopoiesis. TSPs contain a signature domain that is found with high fidelity throughout the TSP family and consists of tandem epidermal growth factor (EGF)-like modules, 13 aspartate-rich calcium-binding repeats and a C-terminal lectin-like module. The signature domains of TSP-1 and TSP-2, which have three EGF-like modules, share 77% identity, without any insertions or deletions. In this study, we describe the 2.6 angstrom resolution crystal structure of the glycosylated signature domain of human TSP-2. Components of the domain interact extensively to form three striking structural features: a stalk that consists of the two N-terminal EGF-like modules, a wire that consists of the asparate-rich repeats, and a globe that consists of the lectin-like module. The third EGF-like module serves as a clasp to tie together the globe and the ends of the wire. The TSP-2 signature domain is stabilized by 30 bound Ca2+ ions and 18 disulfide bonds. The structure reveals functions of the residues at the position of the CAD-linked polymorphism in TSP-1 and many of the positions of the over 100 skeletal dysplasia-linked mutations in TSP-5. Finally, the structure allows for a better understanding of interactions between TSPs and a number of binding partners, including integrins, CD47, vonWillebrand factor, type V collagen, fibroblast growth factor, cathepsin-G and neutrophil elastase.
Heterogeneity of Endothelial Sheet Migration: Role in Angiogenic Plasticity.
Authors: Ofori-Acquah Solomon F. , Stevens Troy 
Published: April 2020

Abstract
The vascular system is a complex network of conduit and microvascular vessels exposed to different microenvironments that imprints unique phenotypic traits on individual endothelial cell populations. Endothelial cells in fully differentiated blood vessels in adult tissues have a quiescent phenotype characterized by an increased resistance to proliferate, migrate or undergo apoptosis. The intrinsic capacity of endothelial cells to switch from a quiescent to angiogenic phenotype however plays an important role in wound healing, several vascular proliferative disorders and tumor angiogenesis. Endothelial cells revert to an angiogenic phenotype as isolated cells in culture nonetheless they are contact inhibited at confluence reflecting their in vivo phenotype in the endothelium. In this study, we used time-lapse video microscopy to study early events in endothelial sheet migration in confluent monolayers of primary endothelial cells derived from conduit (PAECs) and microvascular (PMVECs) blood vessels. Recordings were made in a live cell chamber and were restricted to six hours to minimize the effect of proliferation on sheet migration. PMVECs at the wound edge were significantly highly spread and squamous in appearance compared to PAECs, which had a distinctly more cuboidal morphology. Majority (&gt;95%) of PMVECs at the wound edge produced extensive lamellipodia based on morphology and dynamics that measured 25 μm ± 4 μm. By contrast, PAECs formed significantly smaller lamelipodia which extended by 8 μm ± 4 μm. On average PMVEC sheets migrated at a speed of 12.5 μm per hour covering a total distance of 75 μm ± 15 μm (n=6). Sheet migration rate in PMVECs was 3-fold faster than in PAECs (3.8 μm per hour), which covered a total distance of 23 μm ± 10 μm in the same time period. To unravel the molecular basis for this functional diversity, gene micro array analysis was performed. We identified unique transcriptional profiles for cell-cell adhesion molecules, integrins and disintegrin-metalloproteases each with a distinct role in collective cell migration. In particular, integrin alpha 7, which is a major regulator of lamellipodia formation was found to be 20-fold more abundant in PMVECs than in PAECs. This study provides molecular and functional evidence for heterogeneity of endothelial sheet migration. This central finding highlights variability in angiogenic plasticity in fully differentiated endothelial cells, which may have important ramifications for anti-angiogenesis therapy.
Hypomethylation Therapy of Decitabine in Patients with Myelodysplastic Syndromes (MDS) Induces Apoptosis and Reduces Proliferation.
Authors: Jilani I. , Kantarjian H. , Gorre M. , Issa J.-P. , Bennett J. , Raza A. , Ravandi F. , Saba H. , Albitar M. 
Published: April 2020

Abstract
Background: MDS is characterized by cytopenias believed to be the direct result of increased apoptosis of the hematopoietic cells in the bone marrow despite an increase in proliferation. Aberrant methylation resulting in leukemogenesis is frequent in MDS and is a potential target for pharmacologic therapy. Decitabine (Dacogen™; DAC), which has been shown to be effective in treating patients with MDS (Saba et al, Kantarjian et al), indirectly depletes methylcytosine, resulting in hypomethylation of target genes. However, it is not known whether this correction of methylation leads to reducing the apoptosis, allowing normal cells to grow, or by increasing the apoptosis killing off the tumor cells.
Methods: We studied apoptosis and proliferation in patients with MDS treated on a randomized protocol to receive either DAC or best supportive care (SC). Apoptosis as measured by annexin V and mitochondrial potential was studied in various subpopulations of cells using multiparameter flow cytometry. Proliferation was also measured in a similar fashion using BrdU incorporation. Bone marrow (BM) and peripheral blood (PB) samples were collected from patients at baseline and at various times on therapy.
Results: At baseline, the DAC group showed no significant difference in apoptosis, proliferation or percent of CD34+ cells from the SC group in BM (n = 39 and 33 patients, respectively) or PB (n = 51 and 50, respectively). After three months on treatment a significant increase in apoptosis (annexin V) in CD34+ cells was noted in the DAC arm but not in the SC arm (Wilcoxon test, P=0.01). Similar results were obtained when disturbance in mitochondrial potential was measured in the blast population (P=0.02). Interestingly, a similar increase in apoptosis was observed in CD8+ cells in the DAC arm (P=0.02). The increase in apoptosis was augmented with additional courses of DAC therapy. A greater reduction in proliferation (BrdU incorporation) in the CD34+ cells in the decitabine arm compared to SC (P=0.01) was also observed. Those DAC-treated patients who had a higher proliferation rate (BrdU incorporation) at diagnosis were more likely to achieve a CR than those with low level of proliferation (P=0.03).
Conclusions: Decitabine therapy in patients with MDS leads to hypomethylation, but the net effects include high levels of apoptosis and the death of the neoplastic cells and a complimentary reduction of proliferation of the leukemic cells.