bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expansion of Murine Amniotic Fluid c-Kit High Lin- Cells Maintaining Their Hematopoietic Potential.
Authors: Andre-Schmutz Isabelle , Ditadi Andrea , Boudil Amine , Ezine Sophie , Cavazzana-Calvo Marina 
Published: October 2019

Abstract
Abstract 1475
Poster Board I-498
We have recently described the hematopoietic potential of ckit+ Lin- cells from the murine and human amniotic fluid (1). These cells were able to generate all types of blood cells in vitro and as far as mice are concerned, to generate a complete hematopoietic system once transplanted to immunodeficient recipients. This strong hematopoietic potential was accompanied by a molecular signature measured by unicellular RT-PCR, characteristic of fetal hematopoietic progenitors. Indeed coexpression of Gata2, Lmo2 and Aml1 was found in 28% of ckit+Lin- AF cells.
Intriguingly, murine ckit+Lin- AF cells can be subdivided into two fractions depending on the level of ckit expression (low or high). In in vitro assays, we demonstrated that hematopoietic potential was strictly restricted to the ckit high expressing fraction. The expansion of these cells would have great impact even in the clinical field as AF could be seen as a source of transplantable hematopoietic stem cells (HSCs). Many of the early studies that documented some expansion ability of HSCs included fetal calf serum in the protocol. Given the poorly defined combination of factors in serum and the variability between different serum lots, these protocols were often difficult to reproduce. Serum-free media supplied with specific inducers have been shown to bring several advances in driving direct differentiation of embryonic stem cells. Murine AF Lin-ckitlo and ckithi were cultured in serum- and feeder layer-free culture conditions. Lin-ckitlo AF cells died within a few days. Conversely, Lin-ckithi AF cells were maintained for up to 6 weeks, with a proliferation rate of more than 100 during the first three weeks. Their phenotype remained stable, ckithi CD45+ and Lin-. The hematopoietic potential tested in methylcellulose assays showed an increased frequency of mixed CFU-GEMM (from 24% to 84%). In vivo, CFU-S12 composed of erythroid, myeloid and Lin-ckit+Sca1+ progenitor cells were observed after the injection of AF ckithi in lethally irradiated recipients. Gene expression profile analyzed by single cell multiplex RT-PCR analysis correlated with the in vitro and in vivo results of differentiation. LMO2 was coexpressed with Gata2 and Aml1 in 66% of expanded cells, demonstrating the maintenance of an overall pattern of expression.
Collectively, our results indicate that the hematopoietic potential of AF resides in the ckithi fraction and that these cells can be expanded in serum-free condition for prolonged periods of time without reduction of their hematopoietic potential. This strongly supports the idea that AF may be an excellent source of cells for therapeutic applications.
1. Ditadi 2009

Disclosures:
No relevant conflicts of interest to declare.
Geminin Regulates Hematopoietic Stem Cell Proliferation and Differentiation.
Authors: Shinnick Kathryn M. , Barry Kelly A. , Eklund Elizabeth A. , McGarry Thomas J. 
Published: October 2019

Abstract
Abstract 1478
Poster Board I-501
Hematopoietic stem cells supply the circulation with mature blood cells throughout life. Progenitor cell division and differentiation must be carefully balanced in order to supply the proper numbers and proportions of mature cells. The mechanisms that control the choice between continued cell division and terminal differentiation are incompletely understood. The unstable regulatory protein Geminin is thought to maintain cells in an undifferentiated state while they proliferate. Geminin is a bi-functional protein. It limits the extent of DNA replication to one round per cell cycle by binding and inhibiting the essential replication factor Cdt1. Loss of Geminin leads to replication abnormalities that activate the DNA replication checkpoint and the Fanconi Anemia (FA) pathway. Geminin also influences patterns of cell differentiation by interacting with Homeobox (Hox) transcription factors and chromatin remodeling proteins. To examine how Geminin affects the proliferation and differentiation of hematopoietic stem cells, we created a mouse strain in which Geminin is deleted from the proliferating cells of the bone marrow. Geminin deletion has profound effects on all three hematopoietic lineages. The production of mature erythrocytes and leukocytes is drastically reduced and the animals become anemic and neutropenic. In contrast, the population of megakaryocytes is dramatically expanded and the animals develop thrombocytosis. Interestingly, the number of c-Kit+ Sca1+ Lin- (KSL) stem cells is maintained, at least in the short term. Myeloid colony forming cells are also preserved, but the colonies that grow are smaller. We conclude that Geminin deletion causes a maturation arrest in some lineages and directs cells down some differentiation pathways at the expense of others. We are now testing how Geminin loss affects cell cycle checkpoint pathways, whether Geminin regulates hematopoietic transcription factors, and whether Geminin deficient cells give rise to leukemias or lymphomas.

Disclosures:
No relevant conflicts of interest to declare.
Nrf2, a Critical Regulator of Oxidative Stress, Is Required for HSC Function and Cytokine Response.
Authors: Merchant Akil , Singh Anju , Joseph Giselle , Wang Qiuju , Zhang Ping , Brennan Sarah , Biswal Shyam , Matsui William 
Published: October 2019

Abstract
Abstract 1492
Poster Board I-515
Previous studies have established an important role for reactive oxygen species (ROS) in regulating the function and life-span of hematopoietic stem cells (HSC). Nuclear factor erythroid-2–related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates cellular responses to ROS and detoxification pathways implicated in chemoresistance, however, its role in normal stem cells is unknown. We analyzed Nrf2null mice and found increased total bone marrow cellularity, cKit+Sca1+Lin− (KSL) stem-progenitor cells, and long-term quiescent HSC (CD34−KSL) compared to wild type mice (p<0.05). Transplantation of equal numbers of KSL cells from Nrf2wt and Nrf2null resulted in a five-fold decrease in peripheral blood chimerism from Nrf2null derived cells at 16 weeks (15% wild type vs. 3% null, p<0.05). Unlike other models of deficiencies in genes associated with ROS handling, such as ATM or the FoxO family of transcription factors, basal ROS levels were not elevated in Nrf2null HSC. However, Nrf2null bone marrow cells demonstrated increased sensitivity to induced oxidative stress and in vitro treatment with H2O2 resulted in a 2 fold decrease in colony formation in methylcellulose. We also examined the in vivo sensitivity of Nrf2null cells to oxidative stress by irradiating (400 rads) stably chimeric mice 20 weeks following transplantation with either Nrf2wt or Nrf2null HSC. Mice receiving Nrf2null HSC demonstrated a 50% decrease in peripheral blood chimerism at 4 months following radiation compared to no change in Nrf2wt recipients (p<0.05) confirming that loss of Nrf2 leads to increased sensitivity to oxidative stress. Microarray gene expression analysis from Nrf2wt and Nrf2null mice revealed down regulation of the G-CSF cytokine receptor in Nrf2null HSC and suggested that defective cytokine signaling may contribute to the HSC dysfunction seen in Nrf2null bone marrow cells. To test this hypothesis, we attempted to rescue the function of Nrf2null HSC by treating mice with exogenous G-CSF. Nrf2wt and Nrf2null mice were treated with one week of daily G-CSF and then HSC were harvested and transplanted. In contrast to the defects in engraftment of untreated Nrf2null HSC, there was no significant difference in peripheral blood chimerism following transplantation of G-CSF treated Nrf2wt or Nrf2null HSC, thus demonstrating that G-CSF treatment could rescue the HSC defect in mutant mice. In conclusion, the Nrf2 transcription factor appears to be a novel and essential regulator of normal HSC function through the modulation of oxidative stress response and cytokine signaling.

Disclosures:
No relevant conflicts of interest to declare.
NF-Ya Is Essential for Hematopoieic Stem Cell Proliferation.
Authors: Bungartz Gerd , Garrett Russell , Emerson Stephen G. 
Published: October 2019

Abstract
Abstract 1507
Poster Board I-530
Proliferation, self-renewal and differentiation of hematopoietic stem cells (HSCs) must be tightly regulated in order to sustain hematopoiesis over a lifetime and to prevent uncontrolled expansion of cells. Several genes have been implicated in the regulation of HSC behavior, such as HoxB4, Notch1, Lef-1 and others. Previous studies from our and other laboratories have demonstrated that the heterotrimeric transcription factor NF-Y is a potent inducer of many of these regulatory genes by over-expressing NF-Ya, the regulatory subunit of NF-Y. Furthermore, Bhattarchaya et al. showed that the deletion of NF-Ya in mice leads to lethality before day E8.5, highlighting its importance in mouse development. While there is no doubt that NF-Y plays a role in the progression of the cell cycle in vitro, different groups – targeting different subunits for deletion or silencing – have obtained different results. In order to comprehensively investigate the in vivo function of NF-Y in the hematopoietic system, we utilized a conditional knockout mouse model. We found that the bone marrow (BM) cellularity decreases sharply starting as soon as one day after the ablation of NF-Ya. Our data indicate that the cell loss can be attributed to a combination of cell cycle arrest in G2/M-phase of the cell cycle and apoptosis at 24 hours after the gene deletion.
Since NF-Y has been identified as a master regulator of genes involved in HSCs behavior, we focused on the effects of the NF-Ya deletion within the HSC compartment. We found a down regulation of HoxB4, Notch-1, Lef-1 and Bmi-1 following NF-Ya deletion. However, 24h after induction of NF-Ya deletion the HSC population appeared unaffected and their numbers remained stable, likely due to their predominantly quiescent status.
To investigate the capability of stem cells to progress though the cell cycle, we activated HSCs using the interferon inducer poly IC and observed that, once activated, also HSCs accumulate in the G2/M-phase of the cell cycle. Finally, to test whether the deletion of NF-Ya impairs or absolutely abrogates HSC function, we performed long term experiments including competitive BM transplantation and colony formation assays that demonstrate that NF-Y activity is absolutely essential for HSC function.
Altogether, our data identify NF-Y plays a pivotal role in the survival of hematopoietic cells and the progression of cells though the G2/M-phase of the cell cycle in vivo. Additionally, while we found that NF-Y ablation leads to reduced expression of many genes important for HSC behavior, this had no immediate effects on the maintenance of these cells due to their quiescent nature.

Disclosures
No relevant conflicts of interest to declare.
FcγRIIa Rescues Normally-Poor αIIbβ3-Mediated Outside-in Signaling in Mouse Platelets.
Authors: Boylan Brian , McKenzie Steven E. , Newman Debra K. , Newman Peter J. 
Published: October 2019

Abstract
Abstract 153
Outside-in signal transduction is one of several amplification pathways that platelets employ following their adhesion to components of the extracellular matrix or to multivalent ligands like fibrinogen and von Willebrand factor. Previous studies have shown that outside-in signal transduction initiated by the binding of fibrinogen to its receptor, the integrin αIIbβ3, results in the activation of β3-associated Src family kinases (J Cell Biol 157:265, 2002; J Cell Sci 121:1641, 2008) that phosphorylate key tyrosine residues within the cytoplasmic domain of the transmembrane immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor protein, Fcγ;RIIa (Blood 112:2780, 2008). “Activation” of FcγRIIa sets off a cascade of events that facilitate assembly and activation of other key signaling intermediates, including the tyrosine kinase Syk and phospholipase Cγ2. PLCγ2, through its lipase activity, generates lipid products that support a multitude of cellular activation responses, including cytoskeletal rearrangements leading to platelet shape change and spreading, secretion of platelet granules, and activation of additional cell surface integrins. Curiously, mice lack the gene encoding FcγRIIa, and murine platelets are notoriously poor at sending αIIbβ3-mediated outside-in signals into the cell. The purpose of the current investigation, therefore, was to determine whether enforced expression of FcγRIIa in mouse platelets might serve to amplify murine platelet activation downstream of ligand binding to αIIbβ3. We found that the spreading of platelets derived from FcγRIIa transgenic mice was more robust and extensive compared to that of their wild-type counterparts. ADP- and collagen related peptide-induced granule secretion, as measured by P-selectin exposure, was also enhanced, as was the degree of integrin of αIIbβ3 activation, as reported by the binding of the activation-dependent monoclonal antibody, JON/A. Taken together, these data provide further support for importance of FcγRIIa in transmitting αIIbβ3-mediated amplification signals into the cell, and help to explain the often-observed defect in αIIbβ3-mediated signal transduction in murine platelets.

Disclosures:
Newman: New York Blood Center: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy.
Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR1) - a Putative Diagnostic Marker for Chronic Lymphocytic Leukemia (CLL).
Authors: Uhrmacher Sabrina , Hertweck Magdalena , Paesler Julian , Erdfelder Felix , Filipovich Alexandra , Poll-Wolbeck Simon Jonas , Razavi Regina , Gandhirajan Rajesh Kumar , Gehrke Iris , Hallek Michael , Kreuzer Karl Anton 
Published: October 2019

Abstract
Abstract 1587
Poster Board I-613
In chronic lymphocytic leukemia (CLL) WNT signaling is constitutively active and several members of this signaling pathway are uniformely upregulated in these cells. Apart from classical WNT receptors like FZD and LRP6, receptor tyrosine kinase-like orphan receptor 1 (ROR1) has been shown to function as a receptor for WNT proteins, too. Furthermore, it could recently be demonstrated that ROR1 is frequently expressed on the surface of CLL cells and might therefore serve as a therapeutic target in this disease. However, so far only little is known about the expression status of this protein in different patients. Moreover, a diagnostic antibody for flow cytometric investigations is lacking. Thus, the aim of our study was to i) establish a directly labelled anti-ROR1 antibody for flow cytometry, ii) to confirm previous results on ROR1 expression in CLL, iii) to investigate ROR1 expression in different cell compartments and iv) correlate our findings to known markers of risk and disease progression. Peripheral blood of CLL patients as well as healthy volunteers was subjected to flow cytometric analysis. Besides standard determination of leukocyte subpopulations ZAP70 and CD38 status was assessed according to current diagnostic recommendations. In addition, ROR1 surface expression was first detected by flow cytometry using a specific primary antibody directed against ROR1 and a fluorescent labelled secondary antibody. Using this experimental setting we found that ROR1 is expressed on 63.4% of all neoplastic CLL cells and also on 30.5% of T cells in the peripheral CLL blood. In contrast, no ROR1 expression could be detected on NK cells, B cells, CD8+- or CD4+-T cells of healthy individuals. To improve the analytical technique the ROR1 antibody was directly conjugated with Phycoerythrin (PE) and the experiments were repeated. With the conjugated antibody we detected ROR1 expression on 97.1% of neoplastic CLL cells and virtually on no T lymphocytes. ROR1 expression levels correlated neither with the expression of ZAP70 nor with CD38. Again, we could not detect ROR1 expression on peripheral blood cells of our healthy volunteers. Taken together, ROR1 expression appears to be highly restricted to CLL cells. If in addition to CD5 and CD19 ROR1 detection is included into diagnostic flow cytometric panels the specificity and sensitivity of immunophenotypic CLL diagnostics may be greatly enhanced.

Disclosures
Hallek: Roche: Consultancy, Honoraria, Research Funding.
Mutation in the Nucleophosmin Gene (NPM1) Is a Stable Marker for Minimal Residual Disease Monitoring in Acute Myeloid Leukemia Patients with Increased Sensitivity and Specificity Compared to Expression of the Wilms Tumor (WT1) Gene.
Authors: Kristensen Thomas , Preiss Birgitte Strange , Friis Lone , Møller Michael B. 
Published: October 2019

Abstract
Abstract 1602
Poster Board I-628
Mutation in exon 12 of the nucleophosmin (NPM1) gene occurs in approximately 60% of acute myeloid leukemia (AML) patients with normal karyotype. To date, molecular minimal residual disease (MRD) monitoring in this patient group has primarily been based on expression of the Wilms tumor gene (WT1), although expression of WT1 in non-leukemia cells limits the specificity of this marker. Mutation in the NPM1 gene is potentially a superior MRD marker compared to WT1 gene expression by being specific to the malignant clone. The use of NPM1 mutation as a MRD marker would furthermore be in line with the widespread use of leukemia cell specific fusion-genes as MRD markers in AML patients with balanced translocations. In the present study, we therefore evaluated NPM1 mutation as a MRD marker with respect to stability, sensitivity and specificity in direct comparison to WT1 gene expression. A total of 13 relapsed AML patients with normal karyotype that were positive for mutation in NPM1 and WT1 gene expression at the time of diagnosis were included in the study. The NPM1 mutational load and WT1 gene expression was analyzed by real-time qPCR in up to 22 peripheral blood mononuclear cell samples per patient from the time of primary diagnosis to latest follow-up to compare the kinetics of the two markers during periods of morphological remission and relapse events. The 13 patients experienced a total of 18 morphological relapses which were all accompanied by high levels of NPM1 mutation, along with high WT1 mRNA levels, thus demonstrating complete stability of NPM1 mutation during relapse in the present material. During periods of complete morphological remission, the NPM1 mutational load was below detection limit (< 1/1000 cells) in all samples. In contrast, WT1 gene expression was detectable in 70% of these samples, thus demonstrating the limited specificity of this marker. This background WT1 expression in non-leukemia cells reached levels of up to 1% of the levels detected at the time of diagnosis thus limiting the de facto MRD marker sensitivity of WT1. All samples with detectable levels of NPM1 mutation after a period of complete molecular remission were followed by a morphological relapse within weeks. The present study therefore shows that mutation in NPM1 is a stable and more sensitive and specific, and therefore superior, molecular MRD marker than WT1.

Disclosures
No relevant conflicts of interest to declare.
Excellent Prognosis of Children with ETV6-RUNX1 Positive (+) Acute Lymphoblastic Leukemia (ALL) in the FRALLE 2000 Protocol.
Authors: Gandemer Virginie , Auclerc Marie-Françoise , Petit Arnaud , Brethon Benoît , Ballerini Paola , Michel Gerard , Perel Yves , Auvrignon Anne , Mechinaud Francoise , Schmitt Claudine , Schneider Pascale , Vermylen Christiane , Lejars Odile , Leblanc Thierry , Gabert Jean , Macintyre Elizabeth , Cayuela Jean-Michel , Leverger Guy , Baruchel Andre 
Published: October 2019

Abstract
Abstract 1628
Poster Board I-654
Since the cloning of the t(12 ;21) in 1995 the prognosis of children with ETV6-RUNX1(+)ALL seems to further increase in the current era. From december 2000 to july 2008, 1461 children and adolescents aged from 1 to 20 years with B cell lineage ALL have been treated according the FRALLE protocols: one for standard-risk (SR) ALL (age 1-9 y, WBC < 50 G/L, no extra medullary involvement) F2000-A; the other one FRALLE 2000-B for high and very high risk ALL (HR) (all other pts). The two protocols mainly differ by a larger use of dexamethasone and a reduced use of anthracyclins in F2000A and the use of HDMTX and a double delayed intensification in FRALLE 2000B. ETV6-RUNX1 presence has been assessed by RT-PCR in 1392 pts (i.e. 96%) and found positive in 321 pts, i.e. 23%. Initial features include a median age of 4.2 y (1.2-15.6), a sex ratio (M/F) of 1.2, a median WBC of 10 G/L(1-293). Both peripheral blood D8 response to prednisone (PRED) and D21 marrow response to chemotherapy were evaluable in 316 pts: 94% of the pts have a rapid early response at D8 and D21. Only 3% of the pts were qualified as D8 poor PRED responders and 3% slow marrow responders at D21. The D35-42 CR rate is 100%. End of induction (EOI) minimal residual disease (MRD) using Ig-TCR methods is known in 259 out of 321 pts (81%) pts. Only 4 pts (2%) had a very high (≥10-2) EOI-MRD. Five year EFS, DFS and OS are 95±2%, 95±2%, 98±1%, respectively. The 5y EFS is 96±2% for the 252 SR pts and 90±7% for the 69 HR pts, p=0.30. The 5y EFS is significantly better for children with ETV6-RUNX1(+)ALL compared to those with ETV6-RUNX1(-) B-lineage ALL: 95±2% vs 84±2%, respectively (p=.001). Nine relapses have been reported in the bone marrow (4) the testis (4), the CNS (1) after a median time of 44 m (25-68), i.e. significantly longer than in the ETV6-RUNX1(-) cases (28m (1-92), p=.01). More testis relapses are observed in boys with ETV6-RUNX1(+)ALL (4 out 7 relapses vs 2 out of 62 in boys with ETV6-RUNX1(-) ALL). These results represent a significant progress compared to the previous protocol F 93 (191 children) both in terms of EFS and overall survival (5y EFS: 95±2% vs 78±3%, p=.001; 5 y OS: 98±1% vs 92±2%, p=.001).

Conclusion
an excellent prognosis of children with t(12;21)/ETV6-RUNX1 positive acute lymphoblastic leukemia is now observed in the FRALLE 2000 protocol. The question of a cautious de-escalation in this subgroup will be envisaged in the next protocol.

Disclosures
No relevant conflicts of interest to declare.