Vincent Praloran

Publications of Vincent Praloran

• Combination of low O(2) concentration and mesenchymal stromal cells during culture of cord blood CD34(+) cells improves the maintenance and proliferative capacity of hematopoietic stem cells.

  Authors: Mohammad Hammoud, Marija Vlaski, Pascale Duchez, Jean Chevaleyre, Xavier Lafarge, Jean-Michel Boiron, Vincent Praloran, Philippe Brunet de la Grange, Zoran Ivanovic

  Journal of cellular physiology. 09/2011; 227(6):2750-8.

  The physiological approach suggests that an environment associating the mesenchymal stromal cells (MSC) and low O(2) concentration would be most favorable for the maintenance of hematopoietic stemThe physiological approach suggests that an environment associating the mesenchymal stromal cells (MSC) and low O(2) concentration would be most favorable for the maintenance of hematopoietic stem cells (HSCs) in course of ex vivo expansion of hematopoietic grafts. To test this hypothesis, we performed a co-culture of cord blood CD34(+) cells with or without MSC in presence of cytokines for 10 days at 20%, 5%, and 1.5% O(2) and assessed the impact on total cells, CD34(+) cells, committed progenitors (colony-forming cells-CFC) and stem cells activity (pre-CFC and Scid repopulating cells-SRC). Not surprisingly, the expansion of total cells, CD34(+) cells, and CFC was higher in co-culture and at 20% O(2) compared to simple culture and low O(2) concentrations, respectively. However, co-culture at low O(2) concentrations provided CD34(+) cell and CFC amplification similar to classical culture at 20% O(2) . Interestingly, low O(2) concentrations ensured a better pre-CFC and SRC preservation/expansion in co-culture. Indeed, SRC activity in co-culture at 1.5% O(2) was higher than in freshly isolated CD34(+) cells. Interleukin-6 production by MSC at physiologically low O(2) concentrations might be one of the factors mediating this effect. Our data demonstrate that association of co-culture and low O(2) concentration not only induces sufficient expansion of committed progenitors (with respect to the classical culture), but also ensures a better maintenance/expansion of hematopoietic stem cells (HSCs), pointing to the oxygenation as a physiological regulatory factor but also as a cell engineering tool. J. Cell. Physiol. 227: 2750-2758, 2012. © 2011 Wiley Periodicals, Inc.

• Slow-cycling/quiescence balance of hematopoietic stem cells is related to physiological gradient of oxygen.

  Authors: Amélie V Guitart, Mohammad Hammoud, Persio Dello Sbarba, Zoran Ivanovic, Vincent Praloran

  Experimental hematology. 10/2010; 38(10):847-51.

  Regulation of hematopoiesis depends on cytokines, cellular interactions, transcription, and metabolic factors. Among the latter, O(2) has been neglected for a long time. Recently, an increasingRegulation of hematopoiesis depends on cytokines, cellular interactions, transcription, and metabolic factors. Among the latter, O(2) has been neglected for a long time. Recently, an increasing number of publications evidenced the regulatory role of physiological low O(2) concentrations (0.1-5%; similar to those in bone marrow) on the in vitro behavior of hematopoietic stem cells. This brief review utilizes the article of Eliasson and colleagues in this Journal to summarize the major results and questions about the relationships between O(2) and hematopoiesis. In order to be concise and interesting for readers unfamiliar with this field, we selected only the most significant data that either reinforce or contradict the conclusions of Eliasson et al., but we also provide references of reviews with a more detailed bibliography. A critical analysis of some key publications provides partial answers to three important questions: is the term hypoxia appropriate to describe physiological low O(2) concentrations? Is a very low O(2) level sufficient to control the quiescence/slow cycling balance of hematopoietic stem cells? Is the O(2) concentration able to modify the effect of cytokines on hematopoietic stem cells? We propose to use in situ normoxia instead of the confusing term hypoxia when working with normal cells at physiological low O(2) concentrations. We suggest that a very low O(2) concentration is necessary but not sufficient to induce hematopoietic stem cell quiescence. We review some articles showing that O(2) variations modify the effect of cytokines.

• Obtaining of CD34+ cells from healthy blood donors: development of a rapid and efficient procedure using leukoreduction filters.

  Authors: Yann Peytour, Amélie Guitart, Arnaud Villacreces, Jean Chevaleyre, Francis Lacombe, Zoran Ivanovic, Vincent Praloran

  Transfusion. 05/2010; 50(10):2152-7.

  Human CD34+ cells are mandatory to study many aspects of human hematopoiesis. Their low frequency in blood or marrow and ethical reasons limit their obtainment in large quantities. LeukoreductionHuman CD34+ cells are mandatory to study many aspects of human hematopoiesis. Their low frequency in blood or marrow and ethical reasons limit their obtainment in large quantities. Leukoreduction filters (LRFs) are discarded after preparation of red blood cells. The CD34+ cell concentration in healthy donor blood is low (1×10(3) -4×10(3) /mL), but their number trapped in one LRF after filtration of 400 to 450mL of blood is high (0.4×10(6) -1.6×10(6) ). To develop a procedure allowing obtainment of purified CD34+ cells from LRFs with a good yield, white blood cell (WBC) recoveries after a 500-mL continuous or after sequential elution (50- or 20-mL fractions) were compared. Different WBC and mononuclear cell (MNC) centrifugation methods were tested to minimize their PLT contamination before the CD34+ cell immunomagnetic selection. Cell functionality was finally analyzed under various culture conditions. The 20-mL back-flushing of LRFs allowed the most efficient WBC recovery. The next steps (110×g centrifugation, MNC separation on Ficoll, and washes) resulted in a cell suspension in which the lymphocyte recovery was approximately 76±10% and the PLT contamination below 1.6%. After immunomagnetic selection, 4×10(5) to 6×10(5) cells containing approximately 85% of functional CD34+ cells were obtained. This procedure allows the easy, rapid (<5hr), and efficient preparation of large quantities of CD34+ cells having functional activities similar to those of CD34+ cells from other sources. Therefore, easily available and virally safe, LRFs represent an important and regular WBC source to work with human CD34+ cells, but also with other WBC types.

• CD34+ cells obtained from "good mobilizers" are more activated and exhibit lower ex vivo expansion efficiency than their counterparts from "poor mobilizers"

  Authors: Zoran Ivanovic, Milica Kovacevic-Filipovic, Michel Jeanne, Leslie Ardilouze, Anne Bertot, Milène Szyporta, Francis Hermitte, Xavier Lafarge, Pascale Duchez, Marija Vlaski, Noel Milpied, Mirjana Pavlovic, Vincent Praloran, Jean-Michel Boiron

  Transfusion. 10/2009;

  BACKGROUND: The classification of patients into "good" or "poor" mobilizers is based on CD34+ cell count in their peripheral blood (PB) after granulocyte-colony-stimulating factor (G-CSF) injection.BACKGROUND: The classification of patients into "good" or "poor" mobilizers is based on CD34+ cell count in their peripheral blood (PB) after granulocyte-colony-stimulating factor (G-CSF) injection. We hypothesized that, apart from their mobilization from marrow to the blood, the response to G-CSF of CD34+ cells also includes activation of proliferation, metabolic activity, and proliferative capacity. STUDY DESIGN AND METHODS: Mobilized PB CD34+ cells purified from samples obtained by cytapheresis of multiple myeloma or non-Hodgkin's lymphoma patients of both good (>50 CD34+ cells/microL) and poor (</=50 CD34+ cells/microL) mobilizers were studied. The initial cell cycle state of CD34+ cells after selection and their kinetics of activation (exit from G(0) phase) during ex vivo culture were analyzed. Their proliferative capacity was estimated on the basis of ex vivo generation of total cells, CD34+ cells, and colony-forming cells (CFCs), in a standardized expansion culture. Indirect insight in metabolic activity was obtained on the basis of their survival (viability and apoptosis follow-up) during the 7-day-long conservation in hypothermia (4 degrees C) in the air or in atmosphere containing 3% O(2)/6% CO(2). RESULTS: CD34+ cells obtained from good mobilizers were in lower proportion in the G(0) phase, their activation in a cytokine-stimulated culture was accelerated, and they exhibited a lower ex vivo expansion efficiency than those from poor mobilizers. The resistance to hypothermia of good immobilizers' CD34+ cells is impaired. CONCLUSION: A good response to G-CSF mobilization treatment is associated with a higher degree of proliferative and metabolic activation of mobilized CD34+ cells with a decrease in their expansion capacity.

• Low-oxygen and high-carbon-dioxide atmosphere improves the conservation of hematopoietic progenitors in hypothermia.

  Authors: Michel Jeanne, Milica Kovacevic-Filipovic, Milène Szyporta, Marija Vlaski, Francis Hermitte, Xavier Lafarge, Pascale Duchez, Jean-Michel Boiron, Vincent Praloran, Zoran Ivanovic

  Transfusion. 05/2009;

  BACKGROUND: During short-term storage of hematopoietic cells (HCs) at 4 degrees C a substantial decline in number and in functional capacity of progenitors occurs after 3 days. We hypothesized thatBACKGROUND: During short-term storage of hematopoietic cells (HCs) at 4 degrees C a substantial decline in number and in functional capacity of progenitors occurs after 3 days. We hypothesized that physiologic O(2) and CO(2) concentrations of hematopoietic tissue microenvironment (approx. 3% O(2) and approx. 6% CO(2)) could improve cell viability and functionality during storage at 4 degrees C. STUDY DESIGN AND METHODS: Mobilized peripheral blood (PB) CD34+ cells from multiple myeloma or non-Hodgkin's lymphoma patients were stored in flasks containing air (approx. 20% O(2) and approx. 0.05% CO(2)) or 3% O(2)/6% CO(2) atmosphere, for 3, 5, and 7 days at 4 degrees C. The total number of cells, the number of cells in G0 or G1 phase of cell cycle, and the apoptosis rate were determined. The functional capacity of stored cells was assessed by the capacity of progenitors to form colonies in methylcellulose (colony-forming cells [CFCs]) and of stem cells to repopulate the bone marrow (BM) of immunodeficient mice (SCID-repopulating cell [SRC] assay). RESULTS: The total number of viable cells and cells in G1 phase as well as the number of total CFCs were significantly higher at 3% O(2)/6% CO(2) than in air at all time points. Cells in G0 phase and SRC were equally preserved in both conditions. CONCLUSION: Atmosphere with low O(2) and high CO(2) concentration (3% O(2)/6% CO(2)) in hypothermia (+4 degrees C) during 7 days of storage prevents cell damage and preserves a high number of functional HSCs and progenitors mobilized in PB by granulocyte-colony-stimulating factor.

• Low O 2 concentrations enhance the positive effect of IL-17 on the maintenance of erythroid progenitors during co-culture of CD34+ and mesenchymal stem cells.

  Authors: Aleksandra Krstić, Marija Vlaski, Mohammad Hammoud, Jean Chevaleyre, Pascale Duchez, Gordana Jovčić, Diana Bugarski, Pavle Milenković, Philippe Bourin, Jean-Michel Boiron, Vincent Praloran, Zoran Ivanović

  European cytokine network. 02/2008; 20(1):10-16.

  Co-culture of haematopoietic cells with a stromal cell layer does not mimic the physiological, micro-environmental niche, whose major feature is a low oxygen (O(2)) concentration. Thus, in order toCo-culture of haematopoietic cells with a stromal cell layer does not mimic the physiological, micro-environmental niche, whose major feature is a low oxygen (O(2)) concentration. Thus, in order to study the effects of IL-17 in a context which better approximates the physiological state, we investigated its effects on cell expansion, colony-forming ability, and the phenotypical profile of normal, human blood CD34+ cells co-cultured for five days with MSC layers at various O(2) concentrations (20%, 12.5% and 3% O(2)). We demonstrated that IL-17 enhances CD34+ and total CFC production during the five days of MSC/CD34+ co-culture. This effect depends upon the O(2) concentration, reaching its maximum at 3% O(2), and is more pronounced on erythroid progenitors (BFU-E). In addition, the stimulation of IL-6 production by IL-17 in MSC cultures and co-cultures is enhanced by low O(2) concentration. The expression of some differentiation markers (CD34, CD13 and CD41) on haematopoietic cells in co-cultures also depends upon the oxygen concentration. Our results strengthen the concept that physiological levels of O(2) (mistakenly called hypoxia), should be considered as an important environmental factor that significantly influences cytokine activity.

• Interleukin-6 (IL-6) and low O(2) concentration (1%) synergize to improve the maintenance of hematopoietic stem cells (pre-CFC).

  Authors: Milica Kovacević-Filipović, Marijana Petakov, Francis Hermitte, Christelle Debeissat, Aleksandra Krstić, Gordana Jovcić, Dijana Bugarski, Xavier Lafarge, Pavle Milenković, Vincent Praloran, Zoran Ivanović

  Journal of cellular physiology. 08/2007; 212(1):68-75.

  Low O(2) concentration (1%) favors the self-renewal of hematopoietic stem cells and inhibits committed progenitors (CFC). Since IL-6 influences both stem cells and committed progenitors at 20% O(2),Low O(2) concentration (1%) favors the self-renewal of hematopoietic stem cells and inhibits committed progenitors (CFC). Since IL-6 influences both stem cells and committed progenitors at 20% O(2), we studied its effects in cultures at 1% O(2). The pre-CFC activity in Lin- population of mouse bone marrow was analyzed following 10 days of serum-free culture in medium (LC1) supplemented with IL-3 with and without IL-6, at 20 and 1% O(2) and phenotypic differentiation and proliferative history monitored. The IL-6 receptor expression and initiation of VEGF-A synthesis were also investigated. At 20% O(2), the effects of IL-6 on pre-CFC were negligible but effects on CFC were apparent; conversely, at 1% O(2), the IL-6 enhances activity of pre-CFC but not of CFC. Unlike at 20% O(2), at 1% O(2) a subpopulation of cells remained Lin- in spite of extensive proliferation. However, the absolute number of Lin- cells, did not correlate with pre-CFC activity. A relative increase in VEGF transcripts at 1% O(2) in presence of IL-3 alone was enhanced by the addition of IL-6. IL-6 enhanced pre-CFC activity at 1% O(2) and this was correlated to the induction of VEGF. These data reinforce the concept that physiologically low oxygenation of bone marrow is a regulator of stem cell maintenance. Since the 20% O(2) does not exist in tissues in vivo, further studies in vitro at lower O(2) concentrations should revise our knowledge relating to cytokine effects on stem and progenitor cells.

• Imatinib and nilotinib induce apoptosis of chronic myeloid leukemia cells through a Bim-dependant pathway modulated by cytokines.

  Authors: Francis Belloc, François Moreau-Gaudry, Maialen Uhalde, Laurie Cazalis, Marie Jeanneteau, Francis Lacombe, Vincent Praloran, François-Xavier Mahon

  Cancer biology & therapy. 07/2007; 6(6):912-9.

  It is an important challenge to better understand the mechanisms of tyrosine kinase inhibitors-induced apoptosis in CML cells. Thus, we have investigated how this apoptosis can be modulated byIt is an important challenge to better understand the mechanisms of tyrosine kinase inhibitors-induced apoptosis in CML cells. Thus, we have investigated how this apoptosis can be modulated by extracellular factors. Apoptosis induced by imatinib and nilotinib was determined in BCR-ABL expressing cell lines and primary CML CD34+ cells. Both molecules induced apoptosis of BCR-ABL expressing cells. This apoptosis was inhibited by protein synthesis inhibition in both K562 and CML CD34+ cells. In K562, 80% inhibition of the BCR-ABL auto-phosphorylation by either imatinib or nilotinib induced a two fold increase in Bim-EL expression and induction of apoptosis in 48 h. Bim accumulation preceded apoptosis induction which was completely abolished by depletion in Bim using shRNA. However, the anti-proliferative effect of imatinib was preserved in Bim-depleted cells. When K562 cells were cultured in a cytokine containing medium, the pro-apoptotic effect of nilotinib was decreased by 68% and this was related to a decrease in Bim-EL dephosphorylation and accumulation. Similarly, the presence of a combination of cytokines inhibited 88% of NIL- and 39% of IMA-induced apoptosis in primary CML CD34+ cells. In conclusion, both nilotinib and imatinib induce apoptosis through Bim accumulation independently of cell cycle arrest. However, the pro-apoptotic effect of both molecules can be attenuated by the presence of cytokines and growth factors, particularly concerning nilotinib. Thus BCR-ABL inhibition restores the cytokine dependence but is not sufficient to induce apoptosis when other signaling pathways are activated.

• Proteasome inhibition specifically sensitizes leukemic cells to anthracyclin-induced apoptosis through the accumulation of Bim and Bax pro-apoptotic proteins.

  Authors: Arnaud Pigneux, François-Xavier Mahon, François Moreau-Gaudry, Maialene Uhalde, Hubert de Verneuil, Francis Lacombe, Josy Reiffers, Noel Milpied, Vincent Praloran, Francis Belloc

  Cancer biology & therapy. 05/2007; 6(4):603-11.

  Proteasome inhibitors are a novel class of compounds that might increase sensitivity to chemotherapy for acute myeloid leukemia (AML). We quantified apoptosis in THP-1 cells incubated with idarubicinProteasome inhibitors are a novel class of compounds that might increase sensitivity to chemotherapy for acute myeloid leukemia (AML). We quantified apoptosis in THP-1 cells incubated with idarubicin (IDA) alone or together with a low concentration of MG132 or bortezomib. The combination of both drugs yielded a percentage of apoptotic cells that was significantly higher than the additive effect of both drugs administered separately (p < 0.01). Isobologram analysis showed that both MG132 and bortezomib interacted synergistically with IDA to induce apoptosis of THP1 cells. Western blot analysis of Bax and Bim show an acumulation of these pro-apoptotic proteins in THP1 treated cells. This increase in Bim preceded the induction of apoptosis and participated in idarubicin-induced apoptosis. Proteasome inhibition also potentiated IDA-induced apoptosis in primary blast cells from 22 AML patients while no such effect was found on normal lymphocytes, PHA-stimulated lymphocytes, normal cord blood CD34+ cells or bone marrow normal myeloid cells. These data show that MG132 and bortezomib specifically sensitize leukemic cells to IDA through an increase in BIM and Bax pro-apoptotic Bcl-2 family proteins.

• JAK2V617F detection and dosage of serum erythropoietin: first steps of the diagnostic work-up for patients consulting for elevated hematocrit.

  Authors: François Girodon, Eric Lippert, Pascal Mossuz, Irène Dobo, Nathalie Boiret-Dupré, Jean-François Lesesve, Sylvie Hermouet, Vincent Praloran

  Haematologica. 04/2007; 92(3):431-2.

  The predictive values of common biological criteria for the diagnosis of polycythemia vera were studied in a cohort of patients with high hematocrit. We found JAK2V617F and erythropoietin assays wereThe predictive values of common biological criteria for the diagnosis of polycythemia vera were studied in a cohort of patients with high hematocrit. We found JAK2V617F and erythropoietin assays were the most relevant first tests. Classification of patients according to their JAK2V617F status and erythropoietin levels facilitated the choice of further diagnostic investigations.

• Latent myeloproliferative disorder revealed by the JAK2-V617F mutation and endogenous megakaryocytic colonies in patients with splanchnic vein thrombosis.

  Authors: Marjorie Boissinot, Eric Lippert, Francois Girodon, Irene Dobo, Marc Fouassier, Claude Masliah, Vincent Praloran, Sylvie Hermouet

  Blood. 12/2006; 108(9):3223-4.

• Expression of HMGA2 in PB leukocytes and purified CD34+ cells from controls and patients with Myelofibrosis and myeloid metaplasia.

  Authors: Joris Andrieux, Chrystele Bilhou-Nabera, Eric Lippert, Marie-Caroline Le Bousse-Kerdiles, Brigitte Dupriez, Nathalie Grardel, Olivier Pierre-Louis, Christophe Desterke, Vincent Praloran, Jean-Luc Laï, Jean-Loup Demory

  Leukemia & lymphoma. 10/2006; 47(9):1956-9.

• The JAK2-V617F mutation is frequently present at diagnosis in patients with essential thrombocythemia and polycythemia vera.

  Authors: Eric Lippert, Marjorie Boissinot, Robert Kralovics, François Girodon, Irène Dobo, Vincent Praloran, Nathalie Boiret-Dupré, Radek C Skoda, Sylvie Hermouet

  Blood. 10/2006; 108(6):1865-7.

  We determined the allelic frequency of the JAK2-V617F mutation in DNA and assessed the expression levels of the mutant and wild-type JAK2 mRNA in granulocytes from 60 patients with essentialWe determined the allelic frequency of the JAK2-V617F mutation in DNA and assessed the expression levels of the mutant and wild-type JAK2 mRNA in granulocytes from 60 patients with essential thrombocythemia (ET) and 62 patients with polycythemia vera (PV) at the time of diagnosis. Using allele-specific quantitative polymerase chain reaction (qPCR), we detected JAK2-V617F in 75% of ET and 97% of PV at diagnosis. The total JAK2 mRNA levels were elevated in ET, PV, and secondary and idiopathic erythrocytosis, suggesting that hyperactive hematopoiesis alters JAK2 expression. The expression levels of JAK2-V617F mRNA were variable but strongly correlated with the allelic ratio of JAK2-V617F determined in DNA. Thus, differences in JAK2-V617F expression, markedly lower in ET than in PV, reflected different percentages of granulocytes carrying the mutation. Moreover, allelic ratios higher than 50% JAK2-V617F, indicating the presence of granulocytes homozygous for JAK2-V617F, were found in 70% of PV at diagnosis but never in ET.

• Whole-blood leuko-depletion filters as a source of CD 34+ progenitors potentially usable in cell therapy.

  Authors: Zoran Ivanovic, Pascale Duchez, Doris A Morgan, Francis Hermitte, Xavier Lafarge, Jean Chevaleyre, Vincent Praloran, Bernard Dazey, Gérard Vezon, Jean-Michel Boiron

  Transfusion. 02/2006; 46(1):118-25.

  BACKGROUND: Used leuko-depletion filters (LDFs), containing billions of white blood cells (WBCs), are discarded. Because the steady-state blood contains low quantities of stem and progenitor cellsBACKGROUND: Used leuko-depletion filters (LDFs), containing billions of white blood cells (WBCs), are discarded. Because the steady-state blood contains low quantities of stem and progenitor cells that are retained in LDFs, the viability and the functional properties of mononuclear cells (MNCs) and CD 34+ cells recovered from LDFs were investigated. STUDY DESIGN AND METHODS: WBCs were recovered from LDFs by use of a closed system. MNCs and CD 34+ cells were isolated from freshly LDF-recovered WBCs or after their overnight incubation. The CD 34+ cells were enumerated, as well as the number of colony-forming unit (CFU)-granulocyte-macrophage, burst-forming unit-erythroid, and CFU-Mixed. The expansion in clinical-scale volume cultures (serum-free medium plus stem cell factor, granulocyte-colony-stimulating factor, and megakaryocyte growth and development factor) was performed starting from MNCs, freshly isolated CD 34+ cells, and CD 34+ cells isolated after overnight incubation of WBCs. The erythroid, megakaryocytic, eosinophilic, and monocyte-myelocytic lineage differentiation of LDF-recovered CD 34+ cells was challenged in liquid cultures by adding relevant cytokines. RESULTS: Nearly 450 x 10(3) viable CD 34+ cells were recovered per LDF. These cells exhibit unimpaired colony-forming ability. It is possible to expand these cells ex vivo, but their response to cytokines is different compared to mobilized peripheral blood and cord blood CD 34+ cells. Thus, further work is necessary to optimize their ex vivo expansion. These cells give rise to the mature cells and precursors of erythroid, megakaryocytic, eosinophilic, and monomyelocytic lineage in liquid cultures. CONCLUSION: MNCs and CD 34+ cells recovered from the LDFs exhibit unimpaired functional capacities. Recent development of ex vivo technologies for expansion, retro-differentiation, and differentiation reinforces the value in cell therapy of these LDG-recovered peripheral blood progenitor cells that are routinely discarded.

• A clinical-scale expansion of mobilized CD 34+ hematopoietic stem and progenitor cells by use of a new serum-free medium.

  Authors: Zoran Ivanovic, Pascale Duchez, Bernard Dazey, Francis Hermitte, Isabelle Lamrissi-Garcia, Frédéric Mazurier, Vincent Praloran, Josy Reiffers, Gérard Vezon, Jean-Michel Boiron

  Transfusion. 02/2006; 46(1):126-31.

  BACKGROUND: The autologous transplantation of CD 34+ cells expanded ex vivo in serum-free conditions dramatically reduces post-myeloablative neutropenia in myeloma patients. In our cell therapy unit,BACKGROUND: The autologous transplantation of CD 34+ cells expanded ex vivo in serum-free conditions dramatically reduces post-myeloablative neutropenia in myeloma patients. In our cell therapy unit, cells for this clinical assay have been expanded under GMP with serum-free Irvine Scientific (IS) medium with stem cell factor (SCF), granulocyte-colony-stimulating factor (G-CSF), and megakaryocyte growth and development factor (MGDF; 100 ng/mL, respectively). Because this clinical-grade IS medium is no longer available, a new serum-free medium, Maco Biotech HP 01 (Macopharma), was evaluated. STUDY DESIGN AND METHODS: Purified CD 34+ cells (Isolex 300i, Baxter) from mobilized peripheral blood samples of myeloma patients were thawed, washed, and cultured, as for previous clinical assays. Twenty million CD 34+ cells were resuspended per 1 L of SCF-, G-CSF-, and MGDF-supplemented medium (HP 01 or IS), introduced into 3-L culture bags (AFC), and cultured for 10 days in 5 percent CO(2), at 37 degrees C, and at 100 percent humidity. RESULTS: A higher amplification of total nucleated cells (NCs) and colony-forming cells (CFCs) was obtained with HP 01 medium than with IS medium (42+/-16.6-fold vs. 20.5+/-5.9-fold for NCs and 26.7+/-7.4-fold vs. 15.5+/-2.5-fold for CFCs, respectively), whereas an increase in CD 34+ cells (3.5+/- 1.2-fold for HP 01 vs. 2.7+/- 1.5-fold for IS) was not significant. IS medium partially maintained SCID-repopulating cells (SRC), whereas the culture in HP 01 medium fully maintained the stem cell activity for 10 days. A higher frequency of CD 41+ cells after expansion in HP 01 than in IS medium was also observed. CONCLUSION: Maco Biotech HP 01 medium is suitable for clinical-scale expansion of CD 34+ cells with the SCF, G-CSF, and MGDF cytokine cocktail, permitting an intensive amplification of CFCs and maintenance of SRCs.

• Molecular remission in chronic myeloid leukemia patients with sustained complete cytogenetic remission after imatinib mesylate treatment.

  Authors: Marie Colombat, Marie-Pierre Fort, Claudine Chollet, Gerald Marit, Catherine Roche, Claude Preudhomme, Josy Reiffers, Vincent Praloran, François-Xavier Mahon

  Haematologica. 02/2006; 91(2):162-8.

  BACKGROUND AND OBJECTIVES: Imatinib mesylate induces a complete cytogenetic response (CCR) in many patients with chronic myeloid leukemia (CML). However, the ultimate goal of therapy for CML isBACKGROUND AND OBJECTIVES: Imatinib mesylate induces a complete cytogenetic response (CCR) in many patients with chronic myeloid leukemia (CML). However, the ultimate goal of therapy for CML is complete elimination of Philadelphia chromosome positive cells or BCR-ABL rearrangements. We studied molecular responses in CML patients in CCR after imatinib treatment. DESIGN AND METHODS: Real-time quantitative reverse transcriptase polymerase chain reaction analysis were used to monitor BCR-ABL levels in 59 CCR patients. Negative results were confirmed by two different techniques performed in two different laboratories. Patients were considered in complete molecular remission if they had four undetectable analyses from two separate samples taken three months apart. RESULTS: The median follow-up was 41 months (17-53). The median BCR-ABL/ABL ratio at the time of CCR was 0.3 % (0-9.88). Patients were split into two groups: group A (n=43) comprised patients with a detectable BCR-ABL/ABL ratio throughout the follow-up and group B (n=16) included those with an undetectable level of BCR-ABL/ABL (< 10(-5)) i.e. in complete molecular remission. No relapses were observed in group B, while 13 group A patients lost their CCR. The probability of losing CCR in this group was 33.2 % >+/-18.0. By Cox regression analysis the best factor for predicting the probability of achieving molecular remission was having a CCR at 6 months (p=0.038) or at 3 months (p=0.024). INTERPRETATION AND CONCLUSIONS: Molecular remission after imatinib treatment, i.e. BCR-ABL/ABL< 10-5 in peripheral blood, is not a rare event, particularly in patients achieving CCR at 6 months.

• Oxygen concentration influences mRNA processing and expression of the cd34 gene.

  Authors: Philippe Brunet de la Grange, Christophe Barthe, Eric Lippert, Francis Hermitte, Francis Belloc, Francis Lacombe, Zoran Ivanovic, Vincent Praloran

  Journal of cellular biochemistry. 02/2006; 97(1):135-44.

  CD34 is a cell surface glycoprotein expressed on hematopoietic stem and progenitor cells that disappears with their maturation. This gene is transcribed in two alternatively spliced mRNAs that encodeCD34 is a cell surface glycoprotein expressed on hematopoietic stem and progenitor cells that disappears with their maturation. This gene is transcribed in two alternatively spliced mRNAs that encode full length and truncated form of CD34 cell surface antigen. Some publications suggested that CD34 full length plays a role in the maintenance of their self renewal capacity. An examination of CD34 regulation by a low O2 concentration that ensures a better maintenance of stem cells may provide important insights into the molecular control of hematopoiesis. Using human cord blood CD34+ cells, we first compared the effect of short term (24 h) culture in hypoxia (1% O2) and normoxia (20% O2) on the expression of full length and truncated form of cd34 transcripts and on the expression of the CD34 antigen. Hypoxia maintained a larger quantity of cd34 full length transcripts and a higher cd34 full length/cd34 truncated form ratio than normoxia. After 72 h of culture at 1% and 20% O2, sorted CD34low sub-population from 1% O2 primary culture still contained more cd34 full length mRNAs than those from 20% O2, maintained better CD34 antigen expression during secondary culture at 20% O2 and contained more undifferentiated cells. This work provides the first evidence of the regulation of the cd34 gene by hypoxia resulting in a delayed higher and longer antigen expression by cord blood cells. We suggest that this phenomenon is related to the better maintenance of primitive stem cells in hypoxia.

• Very low O2 concentration (0.1%) favors G0 return of dividing CD34+ cells.

  Authors: Francis Hermitte, Philippe Brunet de la Grange, Francis Belloc, Vincent Praloran, Zoran Ivanovic

  Stem cells (Dayton, Ohio). 02/2006; 24(1):65-73.

  Physiological bone marrow oxygen concentrations are everywhere lower than 4% and almost null in some areas. We compared the effects of 20%, 3%, and 0.1% O2 concentrations on cord blood CD34+ cellPhysiological bone marrow oxygen concentrations are everywhere lower than 4% and almost null in some areas. We compared the effects of 20%, 3%, and 0.1% O2 concentrations on cord blood CD34+ cell survival, cycle, and functionality in serum-free cultures for 72 hours with or without interleukin-3 (IL-3). As from 24 hours, IL-3 improved cell survival and proliferation in all conditions. After 72 hours, cells were 1.5 and 2.5 times more in quiescence (G0) at 3% and 0.1% O2, respectively, than at 20%; transforming growth factor-beta signaling seemed not to be involved. To explore cell cycle further, fresh CD34+ cells were stained with PKH26 and cultured for 72 hours, and then undivided and divided cells were sorted. At 0.1% O2, 46.5%+/-19.1% of divided cells returned to G0 compared with 7.9%+/-0.3% at 20%. Colony formation and nonobese diabetic/severe combined immunodeficient mice engraftment efficiency were similar after 3 days at 20% and 0.1% O2 concentrations but lower than at T0. In conclusion, a low O2 concentration, close to those found in bone marrow stem cell niches, induces the G0 return of CD34+ cells without impairing their functional capacity.

• Serum erythropoietin measured by chemiluminescent immunometric assay: an accurate diagnostic test for absolute erythrocytosis.

  Authors: Pascal Mossuz, François Girodon, Sylvie Hermouet, Irène Dobo, Eric Lippert, Magali Donnard, Veronique Latger-Cannard, Nathalie Boiret, Vincent Praloran, Jean-Claude Lecron

  Clinical chemistry. 07/2005; 51(6):1018-21.

• Overproduction of BCR-ABL induces apoptosis in imatinib mesylate-resistant cell lines.

  Authors: Vanessa Desplat, Francis Belloc, Valérie Lagarde, Catherine Boyer, Junia V Melo, Josy Reiffers, Vincent Praloran, François-Xavier Mahon

  Cancer. 01/2005; 103(1):102-10.

  BACKGROUND: Imatinib mesylate, a BCR-ABL tyrosine kinase inhibitor, induces apoptosis in chronic myeloid leukemia cells. Resistance to imatinib is currently the most important concern of thisBACKGROUND: Imatinib mesylate, a BCR-ABL tyrosine kinase inhibitor, induces apoptosis in chronic myeloid leukemia cells. Resistance to imatinib is currently the most important concern of this treatment. One of the main mechanisms of this resistance is overexpression of BCR-ABL. METHODS: In the current study, the authors investigated the correlation between BCR-ABL overexpression and apoptosis in BaF/BCR-ABL and LAMA84 cell lines resistant to imatinib suddenly deprived of the inhibitor, and compared with their sensitive counterpart. RESULTS: Removal of imatinib from culture medium led to a decrease in Bcr-Abl protein expression by Day 5, which was sustained for > or = 3 weeks of imatinib deprivation. Apoptosis was observed after 3 days of imatinib deprivation in resistant lines accompanied by caspase activation, loss of membrane asymmetry (annexin V staining), and alteration of mitochondrial potential (dihexyloxacarbocyanine iodide [DiOC6]). Transient activation of the STAT5/Bcl-xL pathway and Akt kinase activity preceded these responses. CONCLUSIONS: Thus, imatinib removal led to apoptosis of BCR-ABL-overexpressing leukemic cells, a phenomenon that could be exploited to sensitize imatinib-resistant cells to the cytotoxic effect of other drugs.