ALBERT

Description: During megakaryocyte differentiation, the promegakaryoblast (immature megakaryocyte) increases its ploidy to a 2x DNA content by a poorly understood process called endomitosis. This leads to the formation of a giant cell, the megakaryocyte (MK), which subsequently gives rise to platelets. In this report, we show that endomitotis of human MKs is due to abortive mitosis. Human MKs were obtained by a two-step purification of CD34+ blood or marrow precursors followed by in vitro culture in the presence of MK growth factors. Microcoscopic examination shows that a large number of centrosomes (up to 32) and centrioles are present in polyploid MKs. After nocodazole treatment, more than 20% of the MK are blocked in a typical pseudo-metaphase. Both spontaneous and nocodazole-induced endomitosis are associated with a breakdown of the nuclear envelope and possess a complex mitotic spindle composed of several asters. Spindle microtubules radiate from each aster, creating a spherical structure. At metaphase, expression of the kinetochore phosphoepitope recognized by the 3F3/2 antibody is lost, and the sister chromatides segregate moving toward the spindle poles. After limited segregation, the chromosomes decondense and the nuclear envelope reforms in the absence of cytokinesis, isolating all chromosomes in a single nucleus. It has been proposed that endomitosis could be due to an abnormal CDK1 activity or an absence of cyclin B1. Our results show that cyclin B1 can be detected in all MKs, including those with a ploidy of 8N or more. The cyclin B1 staining colocalizes with the mitotic spindle. Using flow cytometry, the level of cyclin B1 increased until 8N, but remained identical in 16N and 32N MKs. Cell sorting was used to separate the MKs into a 2N/4N and 〉4N population. Both cyclin B1 and CDK1 could be detected in the endomitotic polyploid MKs using Western blot analysis, and a histone H1 kinase activity was associated with immunoprecipitated cyclin B1. We conclude that endomitosis of human MKs is due to abortive mitosis, possibly due to alterations in the regulation of mitotic exit.

Description: The late stages of megakaryocytopoiesis, consisting of the terminal processes of cytoplasmic maturation and platelet shedding, remain poorly understood. A simple liquid culture system using CD34+ cells in serum-free medium has been developed to study the regulation of platelet production in vitro. Platelets produced in vitro were enumerated by flow cytometry. A truncated form of human Mpl-Ligand conjugated to polyethylene glycol (PEG-rHuMGDF) played a crucial role in both proplatelet formation and platelet production. A combination of stem cell factor (SCF), interleukin-3 (IL-3), and IL-6 was as potent as PEG-rHuMGDF for the growth of megakaryocytes (MKs). However, the number of proplatelet-displaying MKs and platelets was increased 10-fold when PEG-rHuMGDF was used. Peripheral blood mobilized CD34+ cells gave rise to a threefold augmentation of platelets compared with marrow CD34+ cells. This finding was related to the higher proliferative capacity of the former population because the proportion of proplatelet-displaying MKs was similar for both types of CD34+ cells. The production of platelets per MK from CD34+ cells was low, perhaps because of the low ploidy of the cultured MKs. This defect in polyploidization correlated with the degree of proliferation of MK progenitors induced by cytokines. In contrast, ploidy development closer to that observed in marrow MKs was observed in MKs derived from the low proliferative CD34+CD41+ progenitors and was associated with a twofold to threefold increment in platelet production per MK. As shown using this CD34+ CD41+ cell population, PEG-rHuMGDF was required throughout the culture period to potently promote platelet production, but was not involved directly in the process of platelet shedding. IL-3, SCF, and IL-6 alone had a very weak effect on proplatelet formation and platelet shedding. Surprisingly, when used in combination, these cytokines elicited a degree of platelet production which was decreased only 2.4-fold in comparison with PEG-rHuMGDF. This suggests that proplatelet formation may be inhibited by non-MK cells which contaminate the cultures when the entire CD34+ cell population is used. Cultured platelets derived from PEG-rHuMGDF– or cytokine combination-stimulated cultures had similar ultrastructural features and a nearly similar response to activation by thrombin. The data show that this culture system may be useful to study the effects of cytokines and the role of polyploidization on platelet production and function.

Description: Abstract 1758 During the last several years, iPS cells have been described as a new powerful tool for disease modeling and drug screening. It became possible to reprogram adult patients cells into specific pluripotent cell lines harboring inherited and/or acquired genetic abnormalities. We have focused our work on modeling Jak2V617F positive MPN patients in iPS cells. By retroviral infection (OCT4, SOX2, KLF4, c-Myc), several iPS cell lines were generated from CD34+ cells of a healthy donor (control), a heterozygous JAK2 V617F patient and a homozygous JAK2 V617F patient. All cell lines expressed pluripotent surface markers (TRA1–81, SSEA4), pluripotent genes (NANOG, OCT4, SOX2) and were capable to induce teratomas in immunodeficient mice. Moreover, they were silenced for the expression of the 4 exogenous transgenes and showed no new clonal karyotypic abnormalities. All the iPS cell lines derived from the JAK2V617F heterozygous and homozygous patients were also heterozygous and homozygous for JAK2V617F. No JAK2 wild type iPS could be obtained from the patients CD34+ cells. Interestingly all the homozygous JAK2V617Fhad no 20q deletion by cytogenetic and CGH analysis demonstrating that this deletion was a secondary event in this MPN. JAK2V617F mutation did not elicit a significant increase in hematopoiesis in comparison to the control iPS. However, marked differences in cytokine sensitivity were found. Homozygous iPS-derived erythroid progenitors had spontaneous growth, hypersensitivity to EPO and gave rise to slightly larger erythroid colonies in methylcellulose assays as compared to the control, whereas erythroid progenitors from JAK2V617F heterozygous iPS has similar Epo-response than those from the control. Interestingly, Megakaryocytes (MK) progenitors from JAK2V617F heterozygous iPS had hypersensitivity to TPO whereas those from homozygous iPS showed a complete TPO independence. Those cytokine-response profiles recapitulate the disease's primary features and validate the iPS as a good tool for JAK2V617F MPN modeling. MK differentiation showed a maturation defect with an excess of immature MKs only in the JAK2 V617Fhomozygous lines, who was derived with a post-PV myelofibrosis, suggesting the existence of intrinsic abnormalities of the MK lineage. Finally, we tested several JAK2, PI3K and ERK inhibitors on the JAK2V617F-mediated erythroid growth and showed that only JAK2 and PI3K inhibitors were able to block the erythroid spontaneous growth. iPS technology recapitulates the cytokine hypersensitivity of JAK2V617F MPNs with marked differences between heterozygous and homozygous mutations, enabling us to screen various pharmaceutical inhibitors. In an addition it can also be used to study the clonal hierarchy in a MPN and in the future to study the synergistic effects of different mutations. Disclosures: No relevant conflicts of interest to declare.

Description: The early emergence of macrophages and their large pattern of tissue distribution during development suggest that they may play a critical role in the initial steps of embryogenesis. In the present study, we show that monocytic cells derived from human embryonic stem cells (hESCs) and from fetal liver follow a differentiation pathway different to that of adult cells, leading to specific functions. Embryonic and fetal monocytic cells differentiated from a CD14lowCD16− precursor to form CD14highCD16+ cells without producing the CD14highCD16− cell population that predominates in adult peripheral blood. Both demonstrated an enhanced expression of genes encoding tissue-degrading enzymes, chemokines, and scavenger receptors, as was previously reported for M2 macrophages. Compared with adult blood monocytes, embryonic and fetal monocytic cells secreted high amounts of proteins acting on tissue remodeling and angiogenesis, and most of them expressed the Tie2 receptor. Furthermore, they promoted vascular remodeling in xenotransplanted human tumors. These findings suggest that the regulation of human fetal and embryonic monocytic cell differentiation leads to the generation of cells endowed mainly with anti-inflammatory and remodeling functions. Trophic and immunosuppressive functions of M2-polarized macrophages link fetus and tumor development, and hESCs offer a valuable experimental model for in vitro studies of mechanisms sustaining these processes.

Description: The megakaryocytic (MK) and erythroid lineages are tightly associated during differentiation and are generated from a bipotent megakaryocyte-erythroid progenitor (MEP). In the mouse, a primitive MEP has been demonstrated in the yolk sac. In human, it is not known whether the primitive MK and erythroid lineages are generated from a common progenitor or independently. Using hematopoietic differentiation of human embryonic stem cells on the OP9 cell line, we identified a primitive MEP in a subset of cells coexpressing glycophorin A (GPA) and CD41 from day 9 to day 12 of coculturing. This MEP differentiates into primitive erythroid (GPA+CD41−) and MK (GPA−CD41+) lineages. In contrast to erythropoietin (EPO)–dependent definitive hematopoiesis, KIT was not detected during erythroid differentiation. A molecular signature for the commitment and differentiation toward both the erythroid and MK lineages was detected by assessing expression of transcription factors, thrombopoietin receptor (MPL) and erythropoietin receptor (EPOR). We showed an inverse correlation between FLI1 and both KLF1 and EPOR during primitive erythroid and MK differentiation, similar to definitive hematopoiesis. This novel MEP differentiation system may allow an in-depth exploration of the molecular bases of erythroid and MK commitment and differentiation.

Description: Early recommendations on prophylactic transfusion of thrombocytopenic patients involved a standard platelet dose of about 0.5 × 1011/10 kg body weight. Given the lack of data supporting this dose, we prospectively studied the dose response to platelet transfusions in adults and children with hematologic malignancies. Each patient received, in similar clinical conditions, a medium, high, and very high dose of fresh (〈 24 hours old) ABO-compatible platelets, in the form of apheresis platelet concentrates (APC). For the adults, the medium dose was defined as APC containing between 4 and 6 × 1011 platelets, the high dose between 6 and 8 × 1011, and the very high dose 〉 8 × 1011; for the children, the three doses corresponded to 2 to 4, 4 to 6, and 〉 6 × 1011 platelets. The end points were the platelet increment, platelet recovery, and the transfusion interval, and the results were compared with a paired t-test. Sixty-nine adults and 13 children could be assessed. Recoveries in the adults were similar with the three doses (from 28% to 30%), but the high and very high doses led to a significantly better platelet increment (52 and 61 × 109/L, respectively) than the medium dose (33 × 109/L, P 〈 .01). The main difference was in the transfusion interval, which increased with the dose of platelets transfused, from 2.6 days with the medium dose to 3.3 and 4.1 days with the high and very high doses, respectively (P〈 .01). The positive effect of the high dose was observed regardless of pretransfusional clinical status, but was more marked in patients with no clinical factors known to impair platelet recovery. In these patients, a platelet dose of 0.07 × 1011 per kg of body weight led to a transfusion interval of more than 2 days in 95% of cases. In patients with clinical factors favoring platelet consumption, the proportion of transfusions yielding an optimal platelet increment and transfusion interval increased with the dose of platelets.The platelet dose-effect was also significant in the children, in whom the high and very high doses led to 1.5-fold to twofold higher posttransfusion platelet counts and transfusion intervals. We conclude that transfusion of high platelet doses can reduce the number of platelet concentrates required by thrombocytopenic patients and significantly reduce donor exposure. © 1998 by The American Society of Hematology.

Description: Background T cell replete (TCR) HLA haploidentical hematopoietic stem cell transplantation (SCT) using high dose cyclophosphamide post-transplant (HDCy) to prevent graft versus host disease (GvHD) and graft’s rejection is a promising approach which extends the possibility of grafting patients who lack a conventional donor. Notably, non relapse mortality (NRM) is greatly decreased compared to T-cell depleted haploidentical SCT, giving a low rate of lethal infections or severe GvHD and allowing the possibility to reduce doses of conditioning regiment without impairing the engrafting of the TCR bone marrow. Nevertheless, HDCy post transplant might increase the risk of cardiac toxicity. Methods From March 2012 to July 2013, 11 consecutive adult patients received a TCR haplo-SCT for high risk hematological malignancies: de novo or secondary acute myeloid leukemia (AML) with unfavorable cytogenetics in CR1 (n=5), refractory AML (n=2), chronic myeloid leukemia (n=2), and refractory Hodgkin disease (n=2). Patients with myeloid disease received a myeloablative conditioning (MAC) (Cy 30mg/Kg/d at day -4 and -3 + TBI 12Gy or Busulfan 3.2mg/Kg/d for 4 days + Cy 14.5mg/Kg at day-3 and day-2 + Fludarabine 90mg/m2) or a reduced intensity conditioning (RIC) with Melphalan 140 mg/m2 + Thiotepa 10mg/m2 + Fludarabine 160mg/m2. Two patients with Hodgkin disease and 1 patient who rejected a previous MAC-double cord blood received a RIC with Cy 14.5mg/Kg at day-6 + TBI 2gy + Fludarabine 150mg/m2. Ten patients received HDCy 50 mg/kg/day on days +3 and +4, plus tacrolimus and mycophenolate mophetil for GvHD prophylaxis. In one patient, HDCy was reduced to 50mg/Kg at day+3. All patients but one received a bone marrow graft. Median age was 39.1 (range 22-62). Donors were related for 10, and unrelated for 1 patient with a median of 5.4/10 allele matches (range 5-8). Cardiac evaluation pre-SCT was performed for all patients. Cardiac ultrasound was normal for all (median left ventricular ejection fraction was 62%; range 53-67%). None of the patients had cardiovascular risk factors such as hypertension, diabetes, obesity or history of valvular or ischemic cardiopathy. Median Sorror score was 0.63 (range 0-2). The rate of early deaths (within the 1st month post transplant) related to cardiac toxicity was compared to that of the whole cohort of 875 patients who received an allogeneic HSCT in our center from May 1984 to July 2013 without HDCy post transplantation. Results Among the 11 patients who received HDCy post SCT, 3 patients developed a compressive pericardial effusion at day 4, day 14 and day 41. An extensive microbiological research found no infectious etiology of this cardiac complication and toxicity of HDCy post transplant was the most probable. One patient had received a MAC (TBI 12Gy –Cy) and the 2 other patients had received the RIC Mel-Thio-Flu regimen. Evolution was reversible for 2 patients after the administration of angiotensin-converting enzyme inhibitors and/or pericardial drainage. One patient died at day+16 of a refractory cardiogenic shock. In the control cohort, early death correlated to cardiac toxicity was reported in 6 patients out of 875 (0.69%), as compared to 1/11 (9%) in the haploidentical cohort. In the control cohort, median age of patients who died from cardiac toxicity was 40.1 (range 30-51). All but 1 received a MAC including HDCy pre-transplantation. No HDCy was administered after SCT. The death occurred at a median time of 14.6 days post-transplant. No early death correlated to cardiac toxicity was reported after 1 month post SCT. Conclusion Although limited by the small number of patients, the rate of cardiac toxicity seems to be increased after HDCy post TCR haplo-SCT. This toxicity is probably multi-factorial and HLA mismatches syndrom may contribute to the severity of symptoms. Nevertheless, such severe and early cardiac complication is an unusal observation in patients without any particular cardiovascular risk factor or other co-morbidities at the time of transplant. Caution should be taken during the days following the administration of HDCy in collaboration with intensive care specialists. Disclosures: Leblond: Roche : Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Mundipharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau.

Description: Treatment of high-risk leukemia with transplantation of allogenic bone marrow or stem cells from peripheral blood is limited by the scarcity of HLA-matched related donors; only 30% of patients were eligible. Unrelated cord blood transplantation (UCBT) have become emerging therapies for adult patients without matched donors, We previously demonstrated that NK cells generated after haplo-mismatched hematopoietic stem cells transplantation are blocked at an immature state characterized by specific phenotypic features and impaired functioning, having negative impact for clinical relapse. In the present work, we studied NK cells after UCBT. This study included 27 adults’ patients (median age: 42 years), with high-risk hematologic malignancies, following a non-myeloablatif regimen. Fourthy one percent (11/27) of patients experienced initial engraftment, whereas 16 subjects (59%) experienced either primary or secondary graft failure. Neutrophil engraftment occurred at a median of 20.5 days. The incidence of the relapse was 26% (7/27) and was a major cause of death in 5 subjects. The cumulative incidence of grades II, and III acute GVHD was 33.3%. Infection occurred only in 2 patients (7.4%). The survival rate was 73% at 6 months. Analysis of DNA polymorphisms in the peripheral blood revealed full (median 99.7) donor-type chimerism. Peripheral blood samples were collected during the first year following UCBT in order to realize an extensive phenotypic study of NK cells. This analysis was realized on the cytotoxic NK CD56dim cell subset, which reaches a count number at one-month post graft (M1) close to healthy volunteer. Our results shown that NK cells phenotype post-UCBT was closed to adult controls, contrasting with NK cells generated after haplo-mismatch allograft. Indeed, excepted for the receptors NKG2A and KIR2DL1/DS1, which are respectively increased (M1: p

Description: Abstract 4009 The ex vivo culture of Hematopoietic Stem Cells (HSC) with various combinations of cytokines can increase the number of mature hematopoietic cells that are theoretically capable of rapidly release neutrophils and platelet and reduce recovery duration post transplantation. In patients, the infusion of such cells has been reported, but the short-term effect was not clear. In a randomized, double blind study, we used expanded cells from 4×106/kg peripheral blood hematopoietic selected CD34+cells in comparison to a non manipulated graft containing the same number of CD34+ cells; we designed an ex vivo expansion protocol based on a cocktail of early or late acting cytokines with different culture duration in order to obtain progenitors at various stages of differentiation 1) primitive progenitors obtained from selected CD34+cells cultured for 8 days in presence of fetal liver tyrosine kinase 3 ligand (FLT3-ligand), stem cell factor (SCF), interleukin-3 (IL-3) and thrombopoietin (TPO), 50 ng/ml, each 2) committed megakaryocyte progenitors (Mks) obtained after culture for 10 days in presence of TPO et SCF (50 ng/ml, each) or 3) committed granulocytes and megakaryocyte (GMks) progenitors obtained after culture for 10 days in presence of TPO, SCF and G-CSF (100 ng/ml). Eighteen Non Hodgkin Lymphoma patients submitted to autologous transplantation after a myeloablative regimen consisting of AraC: Day (D)-6 to D-3: 200mg/m2/12h - VP16: 100mg/m2/12h: D-6 to D-3 - BCNU: 300mg/M2: D-6- Melphalan: 140mg/M2: D-2 could be evaluated. Patients in the Expansion Group received graft with 1×106/kg non manipulated cells combined with primitive progenitors issues from 2×106/kg CD34+ cells and Mks progenitors (Mks Expansion Group) or GMks progenitors (GMks Expansion Group) coming from 1×106/kg CD34+cells; unmanipulated cells were used as a source of immunocompetent cells. In the mean, patients of control group (n=10) received 1.3±0.9×108Total Nucleated Cells (TNC)/Kg, 2.7±1.2×106 CD34+/kg, 49±17×104/kg CFU-GM and 17.7±.7104CFU-Mks/kg; in the Mks (n=4) and GMks (n=4) Expansion group, they received respectively in the mean: 61.5±18.5 and 90.1±21.3×108TNC /Kg, 26.5±10.7 et 31± 11.2 x106CD34+/kg, 451± 188 et 557±216×104/kg CFU-GM, 358±212 et 39±18.1 x104/kg CFU-Mk. No cytokines were administered after transplantation. No toxicity was observed after cell infusion. The mean times to reach white blood cell (WBC) recovery (WBC 〉1x 109/l) was significantly shorter after administration of expanded cells, 14 (10–16), 12 (11–14) and 9 (9–10) days respectively in control, Mks and GMks Expansion Group (p=0.01). Median profound neutropenia (neutrophils

Description: Abstract 3416 The relationship between monocytes and mesenchymal stromal cells remains a controversial issue. During embryonic development, the two cell types emerge early and share a large pattern of tissue expression. Using human embryonic stem cells (hES), we recently reported that embryonic monocytes/macrophages were endowed mainly with anti-inflammatory and remodeling functions. Here, we show that a subset of embryonic monocytic cells can give rise to stromal cells. Mesoderm and hematopoietic specification of hES were achieved from embryoid bodies in Iscove's Modified Dulbecco's medium (IMDM) supplemented with 15% fetal bovine serum (FBS) in presence of Bone Morphogenetic Protein 4 (BMP-4, 10 ng/ml) and Vascular Endothelial Growth Factor (VEGF, 5 ng/ml), followed by fetal liver tyrosine kinase 3 ligand (FLT3-ligand, 10 ng/ml), stem cell factor (SCF, 50 ng/ml), interleukin-3 (IL-3, 100 U/ml) and thrombopoietin (TPO, 10 ng/ml). Between day 14–21 of culture, CD45+14+ cells were sorted, cultured for 4 days in presence of Monocyte-Colony Stimulating Factor (M-CSF, 50 ng/ml), Granulocyte-Macrophage CSF (GM-CSF, 20 ng/ml) and IL-3, and subsequently seeded on fibronectin. After culture in Endothelial Cell Growth Medium supplemented with Endothelial GF (EGF), VEGF (25 ng/ml) and bFibroblast GF (bFGF) (1 ng/ml) for 14 days, clones of adherent cells with typical fibroblast-like morphology emerged at a frequency of 2/104 plated embryonic monocytic cells. In order to eliminate contaminating stromal cells before seeding on fibronectin, CD34low43+ hematopoietic cells were sorted at day 10 and CD45+14+ cells were sorted 7 days later. These cells were cultured with the previously described growth factors, but in serum free medium that does not support stromal cell proliferation. In these conditions, we observed that stromal cell developed from CD45+CD14+ embryonic monocytes. These EM-SCs shared several phenotypic and functional characteristics with adult mesenchymal stem cells (MSCs). They could be expanded in vitro in complete alpha–modified Eagle's medium (MEMa) supplemented with 10% FBS, by successive cycles of dissociation. At a density of 500 and 1000 per cm2, EM-SCs formed small colonies of CFU-F. EM-SCs did not express the hematopoietic surface markers CD14 and CD45, nor the endothelial markers CD31 and KDR, and strongly expressed CD105, CD73, CD13 and CD90. In contrast with adult MSCs, they expressed CD133 and low levels of CD34. EM-SCs could not elicit a proliferative response in the presence of allogeneic lymphocytes, and exhibited a suppressive effect on T-cell proliferation in mixed lymphocyte reaction. Under appropriate conditions, EM-SCs displayed osteogenic, chondrogenic and adipogenic differentiation. They could also adopt a smooth muscle cell but not an endothelial or a cardiac phenotype. Compared to adult MSCs, EM-SCs did not expressed telomerase reverse transcriptase, but demonstrated longer telomeres and enhanced expression of genes encoding growth factors, adhesion proteins, tissue degrading enzymes, and anti-inflammatory chemokines. EM-SCs also secreted high amounts of proteins involved in tissue remodeling and angiogenesis. Thus, a rare subset of embryonic monocytic cells can give rise to a population of stromal cells with high immunosuppressive and remodeling functions. A large body of evidence shows that macrophages and stromal cells are involved in tumor development. It remains to be explored whether stromal cells with remodelling potential could derive from tumor-infiltrating macrophages as it can derive from embryonic macrophages. hES cells offer a valuable experimental model for in vitro studies of these differentiation pathways. Disclosures: No relevant conflicts of interest to declare.