bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of EVI1 in Cell Cycle Regulation: Relevance of Specific Target Genes.
Authors: Perkins Archibald S. , Xiao Ying-Yi , Lezon-Geyda Kimberly , Lin Sharon , Camargo Fernando , Tuck David , Yatsula Bogdan
Published: April 2020

Mds1-Evi1 is a complex locus with two distinct transcriptional start sites, four possible polyadenylation sites, and nineteen exons spread over half a megabase that encodes at least six protein isoforms containing variable numbers of DNA-binding zinc finger motifs. In mouse and some other species, the locus is a common site of proviral insertion in myeloid leukemia, while in human it is a site of nonrandom chromosomal rearrangement in myelodysplasia, AML, and CML blast crisis. One such translocation is t(3;21), encoding the AML1-MDS1-EVI1 (AME) fusion protein. We have characterized the RNA transcripts and proteins generated by Mds1-Evi1 in murine leukemic cells bearing Evi1 proviral insertion using three different antisera specific for the N-terminal and C-terminal domains of EVI1, as well as MDS1, and find consistent overexpression of three protein isoforms of 135, 123, and 103 kDa (EVI1A, B, and C, respectively) while other isoforms, including MDS1-EVI1, are not normally expressed. However, by knocking lacZ into the Mds1 first exon we showed that it is normally expressed in hematopoietic progenitors, similar to Evi1. The N-terminal zinc finger domain present in the EVI1A and B isoforms binds to the GACAAGATA motif with high affinity, and single missense mutations in zinc finger six abrogate high affinity binding. Using two different bioassays, we have demonstrated that cellular transformation by either the EVI1A isoform or by AME is dependent on DNA binding via this N-terminal domain, thus indicating that transformation is dependent on the action of this domain on specific target genes. To determine the biological effect of Evi1 on leukemic cell growth, short hairpin RNAs were used to suppress Evi1, and this resulted in slowing of the cell cycle and prolongation of the G2/M phase. RNA expression profiling revealed significant changes in gene expression accompanied Evi1 suppression, including some involved in cell cycle control. Upon long term Evi1 suppression, numerous markers of myeloid differentiation were induced, indicating the acquisition of a differentiation-like phenotype. These data indicate an effect of EVI1 on both cell cycle and differentiation, and suggest that the effect on cell cycle is direct. To gain further insight into the nature of EVI1 target genes, we utilized tet-regulated expression of Evi1A, and identified a set of putative targets of the A isoform that includes genes directly involved in cell cycle. However, the fold changes are relatively minor, and although the kinetics of activation suggests these are direct targets, this has not been conclusively demonstrated. We predict on the basis of these data that EVI1 expression modulates several key regulators of cell cycle and this results in an acceleration of cell cycle. With high level EVI1 expression, block to differentiation is induced as well.
Hyperhomocysteinemia in Relation to Total and Cardiovascular Death after Coronary Artery Bypass Grafting. A Prospective Study.
Authors: Girelli Domenico , Martinelli Nicola , Olivieri Oliviero , Trabetti Elisabetta , Pizzolo Francesca , Friso Simonetta , Bozzini Claudia , Tenuti Ilaria , Faccini Giovanni , Lotto Valentina , Pignatti Pier Franco , Mazzucco Alessandro , Corrocher Roberto
Published: April 2020

Background: Hyperhomocysteinemia (HHCY), a known risk factor for thrombosis, is frequently found in patients with chronic ischemic heart disease, but its independent prognostic impact is still controversial.

Aim: To evaluate the relationship between HHCY and mortality in patients after coronary artery bypass grafting (CABG) surgery.

Methods: We prospectively followed 353 patients (mean age 60.1 ± 9.0; 83.3% males) who underwent elective CABG between May 1996 and May 1999.

Results: after a median follow-up of 58 months, 36 patients (10.1%) had died, 28 because of cardiovascular events (Figure 1). Baseline homocysteine levels >90th of distribution (i.e. >25.2 μmol/L) were independently associated to total mortality: OR = 2.39, 95% CIs 1.02–5.59, P=0.035, after adjustment for age, sex, baseline renal function (glomerular filtration rate estimated by MDRD equation), and high-sensitivity C Reactive Protein, by logistic regression. HHCY was also independently associated with mortality due to cardiovascular events, adjusted as above including the presence or absence of myocardial infarction prior to CABG (OR = 2.66, 95% CIs 1.02–6.96, P=0.045).

Conclusions: Moderate (>25.2 μmol/L) HHCY is an independent predictor of total and cardiovascular mortality after CABG. At least in this specific subgroup of cardiovascular patients, low-cost strategies to lower homocysteine (i.e. supplementation with folate and B vitamins) should be implemented.

Figure Figure
A New Role for FcγRIIA in the Potentiation of Human Platelet Activation Induced by Weak Stimulation.
Authors: Canobbio Ilaria , Stefanini Lucia , Guidetti Gianni F. , Balduini Cesare , Torti Mauro
Published: April 2020

The low affinity receptor for immunoglobulin G, FcγRIIA, is expressed in human platelets, mediates heparin-associated thrombocytopenia, and participates in platelet activation induced by von Willebrand factor. Activation of FcγRIIA occurs upon clustering of the receptor induced by immunocomplexes, and consists in the phosphorylation of two tyrosine residues within the ITAM, typically promoted by an associated Src kinase. The phosphorylated receptor acts as a docking site for SH2 domain-containing signaling proteins, including the tyrosine kinase Syk. This event initiates an intracellular tyrosine kinase-based signaling cascade that eventually leads to phosphorylation and activation of phospholipase C (PLC) γ2, and elicits cellular responses. To date, very little is known on the possible involvement of FcγRIIA in platelet activation induced by soluble agonists. We have found that stimulation of platelets with agonists acting on G-protein-coupled receptors resulted in Src-kinase-mediated tyrosine phosphorylation of FcγRIIA. Treatment of platelets with the blocking monoclonal antibody IV.3 against FcγRIIA, but not with control IgG, inhibited platelet aggregation induced by TRAP1, TRAP4, the thromboxane A2 analogue U46619, and low concentrations of thrombin. By contrast, platelet aggregation induced by high doses of thrombin was unaffected by blockade of FcγRIIA. We also found that the anti-FcγRIIA monoclonal antibody IV.3 inhibited pleckstrin phosphorylation and calcium mobilization induced by low, but not high, concentrations of thrombin. Thrombin- and U46619-induced tyrosine phosphorylation of Syk and PLCγ2, which represent substrates typically involved in FcγRIIA-mediated signaling, was clearly reduced by incubation with anti-FcγRIIA antibody IV.3. Morever, we were able to demonstrated that platelet stimulation by thrombin induced the association of FcγRIIA with Syk. Signaling through immunoreceptor typically takes places in characteristic membrane microdomains called lipid rafts. Upon stimulation with thrombin, FcγRIIA relocated in lipid rafts, and thrombin-induced tyrosine phosphorylation of FcγRIIA occurred within these membrane domains. Controlled disruption of lipid rafts by depleting membrane cholesterol prevented tyrosine phosphorylation of FcγRIIA, and impaired platelet aggregation induced by U46619 or by low, but not high, concentrations of thrombin. These results indicate that FcγRIIA can be activated in human platelets downstream G-protein-coupled receptors, and initiates a tyrosine kinase-based signaling pathway that significantly contributes to platelet activation and aggregation in response to weak stimulation.
Detection and Mechanisms of GPIIb-IIIa Antagonist Induced Thrombocytopenia: Role for Antibody Induced Platelet Activation.
Authors: Linden Matthew D. , Frelinger Andrew L. , Furman Mark I. , Krueger Lori A. , Li Youfu , Fox Marsha L. , Hughes Annette , Lown John , Barnard Marc R. , Przyklenk Karin , Michelson Alan D.
Published: April 2020

After the administration of GPIIb-IIIa antagonists, 1 – 3% of patients become thrombocytopenic, and 0.5 – 1% of patients develop profound thrombocytopenia. This GPIIb-IIIa antagonist-induced thrombocytopenia may be antibody mediated. In this study we demonstrated that GPIIb-specific monoclonal antibody 5B12 augmented platelet activation, as determined by platelet surface P-selectin and leukocyte-platelet aggregation) in the presence of therapeutic concentrations of abciximab, but not epitifibatide or tirofiban. Furthermore, the GPIIIa-specific monoclonal antibody Y2/51 augmented platelet activation in the presence of therapeutic concentrations of eptifibatide or tirofiban, but not abciximab. This activation was abolished by an FcγRII receptor (CD32)-specific antibody and was absent when Fab fragments 5B12 or Y2/51 were used. These results suggest that (i) GPIIb-IIIa antagonists cause conformational changes in GPIIb-IIIa; resulting in (ii) antibody binding to GPIIb-IIIa; and, consequently (iii) platelet activation via the FcγRII receptor. To determine whether this mechanism is operative in vivo, plasma from patients who developed abciximab-induced thrombocytopenia (n=2), eptifibatide-induced thrombocytopenia (n=5) and normal donors (n=7) was incubated with ABO Rh matched donor whole blood with and without therapeutic concentrations of GPIIb-IIIa antagonists. Platelet activation was assessed by flow cytometric measurement of platelet surface P-selectin. Abciximab, but not eptifibatide or tirofiban, increased platelet surface P-selectin in patients who developed abciximab-induced thrombocytopenia. Eptifibatide, but not abciximab or tirofiban, increased platelet surface P-selectin in patients who developed eptifibatide-induced thrombocytopenia. Platelet surface P-selectin in normal donors was not changed by abciximab, tirofiban or eptifibatide. These data (i) provide novel insight into the mechanisms of GPIIb-IIIa antagonist induced thrombocytopenia; and (ii) suggest a possible assay for the clinical diagnosis and prediction of this phenomenon.

Platelet surface P-selectin after addition of a GPIIb-IIIa antagonist (% baseline fluorescence). *p<0.05 Abciximab Eptifibatide Tirofiban Normal donors 103 105 99 Eptifibatide-induced thrombocytopenia 103 170 * 104 Abciximab-induced thrombocytopenia 1571 * 108 105
The 3-Dimensional Architecture of Platelets in the Native State by Electron Cryotomography.
Authors: Marsh Michael P. , Chang Juan , Chen Junmei , Lopez Jose , Chiu Wah
Published: April 2020

Conventional electron microscopy requires that samples be chemically fixed, embedded in plastic and stained prior to examination. This technique always raises uncertainty as to whether the observations are faithful to the native cellular organization. These limitations are overcome with the technique of electron cryomicroscopy, in which samples are prepared and observed in the frozen, hydrated state. A modification of this technique, electron cryotomography, is a method capable of recording images from the same specimen area at different tilt angles under low electron dose conditions, revealing the spatial relationships of organelles and molecular assemblies within a cell that has not been subjected to chemical fixation or metal staining.

Here, we used electron cryotomography to examine the 3-dimensional architecture of platelets in the resting state and in various stages of activation. Platelets in citrated plasma isolated from healthy human subjects were allowed to settle onto a perforated carbon grid and frozen immediately in liquid ethane or were allowed to settle for different time periods on a grid coated with fibrillar type 1 collagen before being frozen. The platelets suspended across holes were embedded in a thin layer of vitreous ice. Successive images of these frozen, hydrated platelets were then acquired as the stage was tilted through a range of ±70° using a JEM2010F electron cryomicroscope. Each image was recorded using a 4kx4k CCD camera. The digital images were computationally aligned and merged to reconstruct a 3-dimensional density map with an 4.0 gigapixel volume. Multiple tilt-series were reconstructed and analyzed using segmentation and visualization tools. The platelet reconstructions were annotated in terms of the sizes, shapes and relative locations of the subcellular components, including mitochondria, α and dense granules, dense clusters, the dense tubular system, microtubules, and the surface-connected canalicular system. We observed great variability in the extent of ordered structure in the α granules, and in the filopodia adhering to the grid surface. The tomograms resolve the fibrous actin bundles driving filopod extension and the individual actin cross-links within the bundles. In some platelets, we observed membrane tethers that attached to the surface but were devoid of actin. This represents the first 3-D visualization of human platelets under native conditions. These observations and the ease of generating such data suggest that electron cryotomography can become a useful tool for dissecting the molecular sequence of cytoskeletal rearrangements occurring during platelet activation and granule secretion and for understanding the structural basis of platelet diseases.
Targeted Gene Deletion Demonstrates That Adhesion Molecule ICAM-4 Is Critical for Erythroblastic Island Formation.
Authors: Lee Gloria , Lo Annie , Short Sarah , Mankelow Tosti , Parsons Stephen , Spring Frances , Mohandas Narla , Anstee David , Chasis Joel Anne
Published: April 2020

Erythroid progenitors proliferate, differentiate and enucleate within specialized bone marrow subcompartments, termed erythroblastic islands, which are comprised of developing erythroblasts surrounding a central macrophage. Growing evidence suggests that within erythroblastic islands adhesion events, in concert with cytokines, play critical roles in regulating erythropoiesis and apoptosis. We are exploring the potential function of erythroid ICAM-4, a recently characterized member of the immunoglobulin superfamily, in erythroblastic island formation. We earlier identified α4β1 and αV integrins as ICAM-4 binding partners. Since erythroblasts express α4β1 and ICAM-4 and macrophages exhibit αV, ICAM-4 is an attractive candidate for mediating erythroblast-erythroblast and erythroblast-macrophage attachments. Indeed, two synthetic peptides that block ICAM-4/αV adhesion caused a marked decrease in the percentage of islands formed. To more definitively test whether ICAM-4 attachments are active in erythroblastic islands we generated ICAM-4 knockout mice and compared the capacity of single cell suspensions from freshly harvested ICAM-4 null and wild type bone marrow to form erythroblastic islands in vitro, using a reproducible live cell island reconstitution assay that we have established. Islands and their cellular components were identified and quantitated by three-color immunofluorescent microscopy employing fluoresceinated erythroid-specific TER119 antibody, macrophage-specific F4/80 antibody and a DNA probe. Strikingly, we observed a 47% decrease in the percentage of islands formed from bone marrow of ICAM-4 null mice compared to wild type littermates (n=10 and n=10, respectively). We also studied the ability of ICAM-4 null erythroblasts to form islands in vivo by analyzing intact islands freshly harvested from mouse bone marrow. Similar to the in vitro data we found a marked decrease in the percentage of islands formed in the bone marrow of ICAM-4 null mice compared to wild type littermates. The null mice had 44% fewer islands than wild type mice. Taken together, the results of this phenotypic analysis provide convincing evidence that ICAM-4 is one of the adhesion molecules critical for erythroblastic island formation. We postulate that this newly identified erythroblast receptor may be important not only for adhesive integrity of the island structure but also for initiating intracellular signaling essential for normal erythroid terminal differentiation.
Outcomes in CCG-2961, a Children’s Cancer Group Phase III Trial for Untreated Acute Myeloid Leukemia (AML).
Authors: Lange Beverly J. , Smith Franklin O. , Dinndorf Patricia A. , Arndt Carola A.S. , Barnard Dorothy R. , Dusenbery Kathryn , Feig Steven A. , Feusner James H. , Luna-Fineman Sandra , Neglia Joseph , Seibel Nita L. , Sender Leonard , Shannon Kevin M. , Weiman Margie , Gerbing Robert B. , Alonzo Todd A.
Published: April 2020

CCG-2961 tested an intensively timed induction therapy consisting of cytarabine (AC), etoposide, thioguanine, dexamethasone, idarubicin and daunorubicin. Patients in remission after induction were randomized to a second induction course (Arm A) or a 3-drug combination of fludarabine, AC, and idarubicin (Arm B). Course 3 for patients with related donors was bone marrow transplantation (BMT); for those without donors, high dose AC/l-asparaginase. After Course 3 patients without donors were randomized to 14 infusions of Interleukin 2 (IL2) over 18 days or follow-up. CNS prophylaxis was intrathecal AC. Eligibility included all subtypes of de novo AML except acute promyelocytic leukemia and AML in patients with Down syndrome. CCG-2961 opened in Oct.1996 and closed in Dec. 2002. The DSMC suspended the study between Oct. 1999 and May 2000 while the 2961 Committee developed supportive care policies to reduce treatment-related mortality (TRM). CCG-2961 enrolled 900 de novo patients aged 3 days to 21 years, with 495 and 405 patients accruing pre-and post suspension respectively. Remission induction rate is 88.5%. With median follow-up of 3.6 years (range: 0 – 8.1 years), event-free survival (EFS) at 3 years is 44±3% and survival (OS) 57±3%. Disease-free survival (DFS) following Course 2 Arms A and B are not different, although relapse is significantly higher in Arm A (7.3% .vs. 3.1% P=0.018) and TRM more common in Arm B (7.9% vs.4.2% P=0.059), despite 7 less days of neutropenia in Arm B (P<0.001). DFS is 65±9% for patients with a donor versus 50±5% for patients without a donor (P=0.005); respective OS, 74±8% and 66±5% (P=0.221). However, among 98 patients in CR1 with t(8;21) or inv(16) cytogenetics, outcomes in those without and with a donor were no different: DFS (61±12% vs. 72±18%, P = 0.49) and OS (78±10% vs. 77±17%, l P= 0.85). DFS with and without IL2 is 55±9% and 60±8%(P=0.606). Outcomes improved progressively over time. EFS pre- and post-suspension are 41±4% and 47±5%(P=0.038); OS, 52±5% and 63±5%(P=0.005); TRM is 17±3% pre- and 12±3% post-suspension (P=0.039). Factors predictive of inferior EFS are age >17 years, Afro-American and Hispanic ethnicity, body mass index <10th or >95th percentile for age, absence of related marrow donor, WBC > 50,000/mm3, karyotype with −7/7q, −5/5q- or > cytogenetic 5 abnormalities, FLT3/ITD, >15 % morphologic blasts on day 14 or >0.5% immunologically detectable blasts at the end of induction. CCG-2961 confirms the efficacy and high TRM of intensively timed therapy. Neither fludarabine nor IL2 increases DFS or OS, and availability of a donor does not improve outcomes in those with favorable cytogenetics.
Purified Murine Hematopoietic Stem Cells Contribute to the Generation of Cardiomyocytes Via Cell Fusion.
Authors: Fukata Mitsuhiro , Ishikawa Fumihiko , Shimazu Hideki , Ito Hiroyuki , Shimoda Kazuya , Akashi Koichi , Shultz Leonard D. , Harada Mine
Published: April 2020

Background and aim: It has been reported that donor-maker+ cardiomyocytes appear following the transplantation of total bone marrow (BM) cells or enriched hematopoietic stem/progenitor cells. However, recent reports suggested that highly purified hematopoietic stem cells (HSCs) might not adapt the fate of cardiomyocytes. We aimed to identify the cell populations in BM that possess the capacity to give rise to caridomyocytes. Additionally, we aimed to clarify whether cardiomyogenic potential of the hematopoietic tissue-derived cells require cell fusion with host-derived cardiomyocytes.

Method: To examine the developmental plasticity of hematopoietic tissue-derived cells, we employed the newborn transplantation model, in which age-related decline of regenerative capacity can be restored by environment factors (Conboy et al., Nature 2005). We first separated Lineage antigen−CD45+ (Lin−CD45+) cells and Lineage antigen−CD45− (Lin−CD45−) cells from BM of mice constitutively expressing GFP. Each cell population was intravenously injected into irradiated syngeneic newborn C57BL/6 mice within 48hours after the birth. The cardiac tissue of recipients was analyzed for the presence of donor GFP+ cardiomyocytes at 1 month post-transplantation. 37.0±23.9 GFP+ cardiomyocytes were detected per 40 sections in the myocardium of recipients transplanted with Lin−CD45+ (n=6), while no GFP+ cardiomyocytes were detected in recipients transplanted with Lin−CD45− (n=4). As judged by the expression of CD29 on Lin−CD45− cells and osteogenic and adipogenic potential in vitro, Lin−CD45− likely contained mesenchymal stem cells (MSCs). We further purified c-Kit+Lin−Sca-1+ cells (KLS) HSCs and c-Kit+Lin−Sca-1− progenitors by cell sorting, and transplanted limiting numbers of each population into syngeneic recipients. The number of GFP+ cardiomyocytes was compatible with the injected cell dose both in the transplantation of HSCs and progenitors, suggesting that both HSC and progenitors can give rise to cardiomyocytes in vivo. Finally, to determine the mechanism of cardiomyogenic potential of HSCs, we transplanted GFP+ KLS BM cells into CFP transgenic newborn mouse. Linear unmixing analysis using laser-scanning confocal microscopy revealed that donor-derived GFP+ cardiomyocytes coexpressed CFP. These results indicated that HSCs require cell fusion for generating cardiomyocytes.

Conclusion: Highly purified BM-derived hematopoietic stem/progenitor cells can contribute to the post-natal generation of cardiomyocytes through cell fusion, not through transdifferentiation.
Myogenic Developmental Potential of Hemangioblasts Derived from Mouse Embryonic Stem Cells.
Authors: Burke Michael J. , Kennedy Patrick , Wang Lei , Bhatt Devang , Tse William T.
Published: April 2020

The hemangioblast, the progenitor common to hematopoetic and endothelial lineages, expresses the vascular endothelial growth factor (VEGF) receptor flk-1 on its surface. Based on lineage-tracking studies indicating that skeletal muscle cells are derived from flk-1-expressing precursors (Sato et al. Genesis35:153–159, 2003), we tested the hypothesis that the hemangioblast is also a progenitor to the muscle lineage. We studied the development of skeletal muscle progentiors from mouse embryonic stem cells cultured in suspension to form embryoid bodies (EB). These EB were harvested at different time points, disaggregated into single cells and analyzed by flow cytometry. After 10–18 days of culture, the EB contained abundant cells that expressed α7-integrin, a skeletal muscle-specific cell surface antigen. Immunofluorescence microscopic study of sorted and cytospun α7-integrin-positive cells from EB revealed positive staining with MyoD, sarcomeric α-actinin and desmin antibodies, indicating that they were bona fide muscle cells. To test if these muscle cells were derived from hemangioblasts, green fluorescent protein (GFP)-expressing EB were collected at day 4 of development, disaggregated into single cells and replated in methylcellulose medium with the addition of VEGF, stem cell factor and thrombopoietin. After 5 days in methylcellulose culture, clusters of loosely aggregated cells appeared. These cells have features characteristic of hemangioblasts and exhibit large nuclear-to-cytoplasmic ratios. The hemangioblast colonies were individually picked, diaggregated into single cells and plated with a myoblastic cell line (C2C12 cells) in media containing 10% fetal calf serum. Co-culturing of the hemangioblasts with a myoblastic feeder layer was designed to provide the environmental signals necessary for myogenic development. After 48 hours, the co-culture medium was changed to 2% serum to promote muscle differentiation. Three days later, the plates were fixed and analyzed. A small number of GFP-expressing cells assumed an elongated, multinucleated, myofiber-like appearance and stained positiviely for skeletal muscle markers α7-integrin and desmin. We next tested the myogenic potential of hemangioblasts in vivo. The tibialis anterior muscles of C57BL/6 mice were injected with cardiotoxin, a protein kinase inhibitor that depolarizes muscle fibers and induces muscle regeneration. After two days, hemangioblast colonies were picked from methylcellulose culture and injected into the regenerating muscles. Occasional GFP-expressing fibers that stained positive for desmin were seen when analyzed several weeks post-injection. These results indicate that the hemangioblast may be a progenitor not only to hematopoetic and endothelial lineages, but also to skeletal muscle. Future experiments include investigating the significance and mechanism of the hemangioblast’s development into muscle and explore the role of cell-cell fusion in this phenomenon.
Multi-Potent Adult Progenitor Cells from Swine Bone Marrow.
Authors: Zeng Lepeng , Rahrmann Eric , Hu Qingsong , Wang Xiaohong , Zhang Jianyi , Verfaillie Catherine M.
Published: April 2020

Here, we show that Multi-potent Adult Progenitor Cells (MAPCs) can be derived from adult and fetal swine bone marrow. Swine MAPC (swMAPC) contain initially multiple different progenitors and mature cells; however, cultures are homogenous by 50 population doublings and can grow past 150 population doublings. SwMAPCs are CD44, CD45, and MHC-I, II negative, express Oct3a/4 at levels close to those seen in embryonic stem cells, and have telomerase activity leading to no telomere shortening with expansion. We also have very solid evidence that swMAPCs differentiate into most mesoderm, neuroectoderm, and endoderm lineages as demonstrated by a significant up-regulation of transcription factors and other lineage specific proteins in a time dependent fashion similar to development, measured by Q-RT-PCR as well as immunohistology. In addition, swMAPCs induced to the endothelial lineage form vascular tubes, to the hepatic lineage produce albumin and urea, and to the smooth muscle lineage display significant calcium flux in response to smooth muscle agonists. In addition, we are investigating the swMAPC differentiation into cardiomyocytes. Preliminary data indicates swMAPCs express TBX5, Nkx2.5, and GATA6 by day 7 at significant levels. As we have seen for rodent MAPCs, when swMAPCs are allowed to grow at high density, Oct3a/4 levels drop to undetectable ranges, the cells take on a mesenchymal stem cell (MSC) phenotype, and the differentiation potential is lost. When replated under low density conditions, oct3a/4 expression or differentiation capacity can not be re-induced, and cells remain MSC like. Therefore, we suggest that Oct3a/4 is not an in vitro culture phenomena but is already present in swMAPCs and cannot be recovered once lost with standard culturing techniques.
Human Cord Blood Cells Are Capable of Generating Amylase-Producing Pancreatic Acinar Cells without Cell Fusion.
Authors: Minamiguchi Hitoshi , Ishikawa Fumihiko , Fleming Paul A. , Yang Su , Drake Christopher J. , Ogawa Makio
Published: April 2020

Pancreatic acinar cells and hepatocytes arise from the same cell population located within the embryonic endoderm during embryogenesis. It has been reported that a multi-potent population of liver cells is capable of differentiating into pancreatic cells. Thus, pancreatic acinar cells and hepatocytes may share a common precursor. Recent studies revealed that murine and human hematopoietic cells have the ability to generate hepatocytes in vivo. To determine whether human cord blood cells can generate pancreatic cells, we carried out in vivo xenograft experiments. We transplanted 105 CD34+ cord blood cells into conditioned newborn NOD/SCID/beta-2 microglobulin-null mice via facial vein injection and, 2–4 months later, examined pancreata from recipient mice showing high level (16–52%, n=9) human multilineage hematopoietic engraftment in the bone marrow. RT-PCR and immunohistochemical analyses revealed human amylase mRNA and human amylase protein, respectively, in the pancreata of recipient mice. Using fluorescence in-situ hybridization (FISH), we also identified human alpha-satellite DNA positive cells with a morphology characteristic of pancreatic acinar cells. Recently, a number of investigators have reported that the apparent reconstitution of tissue cells such as hepatocytes by hematopoietic stem cells is the result of spontaneous cell fusion between donor hematopoietic cells and recipient tissue cells. To negate this possibility, we used FISH for human and mouse DNA and examined the pancreas for the evidence of fusion between human hematopoietic cells and recipient pancreatic cells. We performed four-color analysis using FISH with human pan-centromere probe, mouse pan-centromere probe, and species non-specific probes for pancreatic amylase mRNA, combined with Hoechst nuclear counter staining on paraffin-embedded sections of pancreata from recipient mice. We identified cells that were amylase mRNA-positive expressed human-specific alpha-satellite DNA and exhibited morphological characteristics of pancreatic acinar cells but did not express mouse-specific gamma-satellite DNA. These findings establish that human umbilical cord blood cells are capable of generating pancreatic acinar cells via a non-fusion mechanism.
NOD/SCID Repopulating Cells Contribute Only to Short-Term Repopulation in the Baboon.
Authors: Beard Brian C. , Mezquita Pau , Kiem Hans-Peter
Published: April 2020

The nonobese diabetic/severe combined immune deficiency (NOD/SCID) xenotransplantation model is widely used as an assay to study human candidate stem cells. However, in contrast to large animal or human studies, the NOD/SCID model is limited by the short life span of the recipient and the limited proliferative demand placed on transplanted cells and the inability to support differentiation into all hematopoietic lineages. We have previously performed a direct comparison in the repopulation ability of gene modified baboon CD34+ cells in an autologous transplantation versus a xenotransplant model in irradiated NOD/SCID mice. Baboon CD34-selected marrow cells were transduced with a gammaretrovirus vector and infused into irradiated baboons and NOD/SCID mice. Our results showed a significant decline in the percentage of transduced cells in the baboon 2–3 months following transplant, while the levels in the NOD/SCID mice remained high and stable up to 12 weeks. A limited integration site analysis could only detect 2 common retrovirus integration sites (RIS) in the NOD/SCID and monkey. Here we have extended this analysis and performed LAM-PCR and locus-specific PCR on 30 clones recovered from 12-week old NOD/SCID b2M mice reconstituted with transduced baboon CD34+ cells. We cloned the sequences of these RIS and generated specific primers for these sequences. We then performed PCR analysis on samples in the baboon to determine whether the clones were present in the baboon peripheral blood at different time points after transplantation. We analyzed samples early (2 to 6 weeks) and late (6 to 12 months) after transplantation. We were able to identify five common integrants in the baboon early after transplant (2 weeks), two of which could still be detected at 6 weeks. None of the clones present in the NOD/SCID b2M mice, however, was found in the baboon during the long-term follow-up (6 and 12 months post-transplant). These results confirm that repopulating cells in the NOD/SCID mouse model contribute only to short-term repopulation in a clinically relevant large animal model and suggest that distinct hematopoietic stem/progenitor cell populations are responsible for the hematopoietic reconstitution in NOD/SCID mice versus nonhuman primates.
Role of Consolidation Therapy in the Treatment of Patients up to 60 Years with High Risk AML.
Authors: Krauter Jürgen , Heil Gerhard , Hoelzer Dieter , Ottmann Oliver , Martin Hans , Lübbert Michael , Finke Jürgen , Heit Wolf , Fiedler Walter , Kanz Lothar , Brossart Peter , Schlimok Günter , Kirchner Hartmut , Raghavachar Aruna , Brugger Wolfram , Ganser Arnold
Published: April 2020

In the prospective multicenter AML 01/99 trial, 484 patients with acute myeloid leukemia up to 60 years (385 with de novo and 99 with secondary AML) were treated with risk-adapted therapy. Patients with CBF-leukemias [t(8;21) or inv(16)] or normal karyotype and good response (GR) to induction I (≤5% blasts in day 15 BM) were considered standard risk (SR), all other patients as high risk (HR). Patients with t(15;17) were excluded. Induction course I consisted of standard dose araC, idarubicine and etoposide (IVA-I). Patients with GR to IVA-I continued with IVA-II on day 21 as double induction. In patients with bad response (>5% bone marrow blasts on day 15), the second cycle consisted of FlAG/Ida. Double induction was followed by an early consolidation with intermediate dose araC. As late consolidation, HR patients with an HLA-matched sibling were scheduled for a matched related donor (MRD) transplantation whereas patients without a family donor should undergo autologous peripheral blood stem cell transplantation (autoPBSCT). PBSC were mobilized after early consolidation. A pilot study within the 01/99 trial showed superior results for matched unrelated donor (MUD) transplantation compared to patients who had undergone autoPBSCT. Therefore, since 2003 all HR patients without a sibling donor were scheduled for MUD transplantation. 234 patients were classified as HR, of whom 137 (58%) achieved complete remission. 31 (13%) of the HR patients died during induction. After 70 months, overall survival (OS) was significantly worse for HR patients (28%) than for SR patients (51%). For HR patients, there was no difference in OS and relapse-free survival (RFS) between those patients classified as HR because of bad response to IVA-1 (n = 76), unfavourable karyotype (n = 83) or both characteristics (n = 75). Moreover, RFS was neither different between HR patients with de novo or secondary AML nor between HR patients with normal karyotype, complex karyotype or other high risk cytogenetics. Regarding the different regimens for late consolidation, the “as treated” analysis of the HR patients revealed that OS and RFS was significantly better after an MRD transplantation (n=34, OS 68%, RFS 65%) than after autoPBSCT (n=26, OS 23%, RFS 7%). OS and RFS for the 29 patients who received MUD transplantation was also significantly better than after autoPBSCT with 56% and 52% respectively. In conclusion, HR patients as defined in our study do not benefit from an autologous PBSCT even after intensive double induction and early consolidation. Therefore, an allogeneic transplantation from a related or unrelated donor should be performed.
Identification of Candidate Regulators of Hematopoietic Stem Cell Aging Using a Combined QTL Mapping and Microarray Analysis Approach.
Authors: Oakley Erin J. , Van Zant Gary
Published: April 2020

A combination of quantitative trait locus (QTL) mapping and microarray expression profiling was used to identify five novel candidate genes for regulating hematopoietic stem cell aging in two strains of inbred mice, C57BL/6 (B6) and DBA/2 (D2). Despite their ability to self-renew and maintain essentially normal levels of hematopoietic function throughout the lifetime of an organism, it has become increasingly evident that hematopoietic stem cells (HSCs) are subject to qualitative and quantitative changes over time, and that these changes may affect both the rate of aging and longevity of organisms. In mice, the effect of aging on stem cells is highly strain-specific, thus suggesting that genetic regulation plays a role in HSC aging. Our lab has identified a QTL on murine chromosome 2 that is associated with the variation in frequency of HSCs between aged B6 and D2 mice. In B6 mice, the frequency of HSCs increases with age, whereas in D2 mice the population of HSCs decreases as the animals age. Using a congenic mouse model in which the region surrounding the QTL in D2 mice was introgressed onto a B6 background, genome wide gene expression analyses were performed using sorted lineage negative hematopoietic cells, which are enriched for primitive stem and progenitor hematopoietic cells. RNA isolated from lineage negative cells from chromosome 2 congenic and B6 background mice at young and old ages was reverse transcribed, labeled, and hybridized to Affymetrix Murine Genome 430 arrays. Using an overall p-value cut-off of ≤ 0.01, a total of 4,368 transcripts were differentially expressed between background and congenic mice, out of 39,000 transcripts present on the mouse genome chip. Following a ‘QTL to Gene’ strategy, we identified 123 differentially expressed transcripts located in the congenic interval on chromosome 2. We then assessed for hematopoietic tissue specific expression patterns, the presence of single nucleotide polymorphisms (SNPs) in the transcripts, and cis-regulation of transcript expression. Five genes were identified that met these criteria, and thus are considered candidate regulators of hematopoietic stem cell aging. We used real time PCR to validate differential expression of a subset of the identified genes. Interestingly, a majority of the genes are associated with cell cycle control and/or protein targeting and transport processes. Additionally, all five cis-regulated genes were found to be down-regulated in aged congenic mice compared to aged B6 mice, thus suggesting coordinated regulation by a common promoter region(s) located within the chromosome 2 QTL. These findings suggest that stem cell aging is associated with specifc intrinsic cellular events and provide insight into the genetic mechanisms underlying hematopoietic stem cell aging.
An Early Block to Erythro-Megakaryocytic Development Conferred by Loss of Transcription Factor GATA-1.
Authors: Stachura David L. , Chou Stella T. , Weiss Mitchell J.
Published: April 2020

Transcription factor GATA-1 is essential at multiple stages of hematopoiesis. Murine gene targeting and analysis of naturally occurring human mutations demonstrate that GATA-1 is required for the maturation of committed erythroid precursors and megakaryocytes. Prior studies also suggest additional, poorly defined, roles for GATA-1 at earlier stages of erythro-megakaryocytic development. To investigate these functions further, we studied hematopoietic differentiation of Gata1− murine embryonic stem cells on OP9 stroma with the cytokine thrombopoietin (TPO) present. Initially, the Gata1− cultures generated a wave of mutant megakaryocytes, but these were rapidly overgrown by a unique population of TPO-dependent blasts that continued to proliferate for more than 6 months in culture. These immature Gata1− cells arose reproducibly in culture without growth lag or crisis, indicating that they derive directly from loss of GATA-1 and not from random genetic events acquired during cell culture. The cells express transcription factors GATA-2, FOG-1 and PU.1 and exhibit the surface phenotype Lin−, Sca1−, IL7R−, CD41+, cKit+, CD9+, and GPIblow. Importantly, upon restoration of GATA-1 function, these cells undergo both erythroid and megakaryocytic differentiation, as assessed by morphology, ultrastructural analysis and the induction of lineage-specific markers. Clonal analysis shows that individual cells maintain the capacity for erythro-megakaryocytic differentiation. Hence, we term this unique population G1ME for Gata1− -Megakaryocyte-Erythroid. To determine if G1ME cells are present in vivo, we analyzed E13.5 fetal livers of Gata1−/Gata1wild-type chimeric embryos. Flow cytometry analysis demonstrates an expanded population of cells expressing the G1ME surface phenotype. Individual cells within this population also exhibit TPO-dependency, extensive proliferative capacity and GATA-1-dependent biphenotypic erythro-megakaryocytic maturation in vitro. Our findings indicate that the loss of GATA-1 impairs the maturation of a specific megakaryocyte-erythroid progenitor. This defines a new role for GATA-1 at a relatively early stage of hematopoiesis and provides potential insight into recent discoveries that human GATA1 mutations promote acute megakaryoblastic leukemia (AMKL), a clonal malignancy with features of both erythroid and megakaryocyte maturation.
RUNX1 Directly Regulates Band 3 Transcription.
Authors: Xie Jingping , Hiebert Scott W. , Koury Mark J. , Brandt Stephen J.
Published: April 2020

RUNX1 (AML1 or CBFA2) regulates the expression of a number of genes important to hematopoiesis. Gene knockout studies demonstrated that a heterodimeric complex of RUNX1 and its DNA binding partner, core binding factor-beta (CBFbeta), is essential for definitive hematopoiesis. Here, we report that RUNX1 directly represses expression of the Band 3 gene prior to terminal erythroid differentiation. Band 3 is one of four major components of the erythrocyte membrane skeleton and is important for maintenance of cytoskeletal architecture and electroneutral Cl-/HCO3− exchange across the red cell membrane. Band 3 expression, like that of beta-globin, increases dramatically with terminal erythroid differentiation. In a previous study, we identified an upstream region in the mouse Band 3 gene designated as B3URE (for Band 3 upstream regulatory region) bound by multiple transcription factors, including TAL1 (also known as SCL), RUNX1, Ldb1, and GATA1, that acts as an orientation- and position-independent and tissue-specific repressive element. Chromatin immunoprecipitation (ChIP) analysis showed that RUNX1 was associated with the B3URE in intact MEL cells and electrophoretic mobility shift analysis confirmed specific RUNX1 interaction with RUNX1 binding sites in the B3URE. Together with CBFbeta, RUNX1 inhibited reporter activity from a construct linking the B3URE with 1 kb of Band 3 promoter in transiently transfected MEL but not COS cells. DNA affinity precipitation analysis with wild-type and mutant oligos established that RUNX1 and CBFbeta in MEL cell nuclear extracts could interact with the B3URE in vitro and suggested that RUNX1 recruits TAL1 and Ldb1 to DNA. Northern blot and quantitative real-time PCR analysis demonstrated that enforced expression of RUNX1 dramatically inhibited dimethylsulfoxide (DMSO)-induced Band 3 gene expression. Quantitative ChIP analysis showed that histone acetylation in the B3URE increased more than 4-fold, while histone methylation decreased ~50% after 3 days of DMSO-induced differentiation. Over the same time frame, the promoter region underwent significantly less acetylation but more extensive demethylation. Finally, changes in B3URE acetylation and methylation were attenuated and inhibited, respectively, in RUNX1-transfected MEL cells relative to vector controls. In sum, these results demonstrate that the Band 3 gene is a direct target of RUNX1 in erythroid cells and indicate that the B3URE contributes to the tightly regulated expression of this gene in differentiating erythroid progenitors. One mechanism by which RUNX1 regulates Band 3 transcription may be by influencing histone acetylation/methylation in this upstream regulatory region.
Outcomes after HLA-Matched Sibling Transplants or Chemotherapy in Children with Acute Lymphoblastic Leukemia in Second Remission: A Collaborative Study of the Children’s Oncology Group (COG) and the Center for International Blood and Marrow Transplant Research (CIBMTR).
Authors: Eapen Mary , Raetz Elizabeth , Zhang Mei-Jie , Muehlenbein Catherine , Devidas Meenakshi , Abshire Thomas , Billett Amy , Homans Alan , Camitta Bruce , Carroll William , Davies Stella
Published: April 2020

The best treatment for children with B-precursor acute lymphoblastic leukemia (ALL) in second remission after a marrow relapse is controversial. To address this question, we compared outcomes in 192 patients enrolled on COG chemotherapy trials 9110 (n=109), 9310 (n=59) and 9411 (n=29) and 189 HLA-matched sibling transplant recipients reported to the CIBMTR. Patients received treatment between 1991 and 1997. Patients with isolated extra-medullary relapse were excluded. Median follow-up was 103 (range 34–159) and 101 (16–160) months after chemotherapy and transplantation, respectively. Groups were similar with respect to sex, leukemia sub-type, leukocyte count at diagnosis and duration of first remission. Chemotherapy recipients were younger at diagnosis (5 vs. 8 years) and less likely to have combined marrow and extra-medullary site relapse (12% vs. 30%). To adjust for time-to transplant bias, we used left-truncated Cox regression models to examine treatment outcomes. The relative efficacy of chemotherapy and transplantation depended on the duration of first clinical remission and transplant conditioning regimen as shown in the Table below. Comparing children with early relapse (<36 months from diagnosis) the 5-year probabilities of leukemia-free survival (LFS) were 23%, 44% and 8% after chemotherapy alone, transplantation with a total body radiation (TBI)-containing regimen and a non-TBI regimen, respectively. In contrast to these findings, relapse and treatment failure were similar after transplantation with a TBI-containing regimen or chemotherapy after a late relapse (≥36 months from diagnosis). The 5-year probabilities of LFS were 61% and 60% after chemotherapy alone and a TBI-containing regimen, respectively and significantly lower at 30% after a non-TBI regimen. These data support HLA-matched related donor transplantation using a TBI-containing regimen in second remission for patients with ALL and early relapse.

Early relapse (<36 months) Late relapse ≥36 months) ( Outcome N Relative Risk (95% CI) P-value N Relative Risk (95% CI) P-value Leukemia recurrence Chemotherapy 115 1.00 81 1.00 TBI-containing regimen 92 0.50 (0.27–0.68) 0.0004 61 1.02 (0.62–1.68) 0.91 Non-TBI containing regimen 19 1.36 (0.81–2.31) 0.24 14 2.51 (1.23–5.16) 0.01 Treatment failure Chemotherapy 115 1.00 81 1.00 TBI-containing regimen 92 0.53 (0.37–0.75) 0.0003 61 0.93 (0.63–1.37) 0.70 Non-TBI containing regimen 19 1.49 (0.93–2.37) 0.09 14 2.87 (1.60–5.13) 0.0004