Search

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 (< or =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. [ABSTRACT FROM AUTHOR]

BACKGROUND: Herein are reported the results obtained in all multiple myeloma patients transplanted with peripheral blood hematopoietic progenitor cells submitted to ex vivo expansion. STUDY DESIGN AND METHODS: Patients had blood progenitor cell mobilization with cyclophosphamide and filgrastim. CD34+ cells were expanded for 10 days in a medium containing granulocyte-colony-stimulating factor (G-CSF), stem cell factor, and megakaryocyte growth and development factor (MGDF). Twenty-seven patients underwent transplantation with expanded and nonexpanded cells and 7 patients underwent transplantation with expanded cells only. RESULTS: The median fold cell expansion was 29.1. The number of colony-forming unit-granulocyte-macrophage (CFU-GM) and CD34+ cells, and the long-term culture-initiating cell (LTC-IC) activity increased with median fold values of 14.7, 2.75, and 2.25, respectively. Postmyeloablative neutropenia was abrogated in 24 of 27 patients transplanted with expanded cells plus nonexpanded cells. The median duration of severe neutropenia was 0 days and correlated with the number of cells and CFU-GM infused. Survival was similar to that of a historical control group. Our LTC-IC and NOD-SCID mice studies showed that the expanded cells are able of sustaining long-term hematopoiesis. Seven other patients received transplantation with expanded cells alone. Absolute neutropenia was abrogated in 6 patients. The median duration of neutropenia was 0 days. Two patients who received the lower number of total cells or CFU-GM had brief secondary neutropenia, which resolved after G-CSF injections. CONCLUSION: CD34+ cells expanded ex vivo can abrogate absolute and severe neutropenia after high-dose therapy. The results of the amplification process are strongly related to the delay of hematopoietic recovery. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 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. [ABSTRACT FROM AUTHOR]

Since, several years the integration of a clinical pharmacist in medical units led to improve the patients' care in France. In the frame of stem cell engraftment, patients belong to a particularly complex population, notably due to pediatric patients or because the age to engraft adult patients is higher. Moreover, because of many reasons, such as numerous medications intake or long-term immunosuppression, these patients are very fragile and at risk of complications. Since the 6th edition, the JACIE standard gives a definition of the role of clinical pharmacist with its competence area and its place in the medical ward. In the aim to standardize the procedures of stem cell transplantation, this 8th congress of the francophone Society of bone marrow transplantation and cellular therapy has proposed a collective proposition of the place and the missions of clinical pharmacists in the transplant units., (Copyright © 2018 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

The evolution of HLA typing and transplantation techniques makes it easier to identify a donor for hematopoietic stem cell (HSC) transplantation. This activity, strongly regulated by regulatory or normative texts, implies in addition biological, medical, para-medical and sometimes psychological evaluations. The benefit/risk discussion is complicated because it must take into account the benefit/risk ratio for the recipient, and the donor risk. No Evidence-Based Medicine data is available and serious events are very rare situations. Biovigilance declarations and their analysis are of fundamental importance. Certain obvious and definite contraindications could be detected very early in the process. It is important to assess whether a risk factor or pathology contributes to increasing the risk associated with collection. In case of recipient risk, the situation should be discussed with the patient team. These recommendations focus on adult peripheral blood HSC donors. They refer to donor information, confidentiality of exchanges, the impact of moral or material pressures, declarations of biovigilance, collegiality and traceability of difficult decisions, desirable experience and training for doctors in charge, use of expert advice informed by an explicit exchange on the possible risks, parsimony of therapeutic interventions and minimization of risks for the donor. We also recommend creation, availability and use by the community of tools and documents (registries, questionnaires, synthetic recommendations, feedback, and collegial qualification meetings) useful for practice., (Copyright © 2017. Published by Elsevier Masson SAS.)

Background: Umbilical cord blood (UCB) has been used as an alternative source of donor hematopoietic stem cells for hematologic transplant setting over the past decade. This study attempted to evaluate potential predictors of cord blood quality., Methods: A total of 750 UCB samples were studied (male, n = 365; female, n = 385). The impact of neonatal sex, weight and stromal cell-derived factor-1α polymorphism on the quality of these UCB samples was investigated., Results: Male neonatal UCB was significantly richer in CD34(+) cells than was female UCB (P < 0.001), whereas female UCB was richer in total nucleated cells (P = 0.01). There was a slight correlation between CD34(+) cells concentration and UCB sample weight (P < 0.01) that could be attributed to the higher weight of male neonates. The use of tetra-polymerase chain reaction to detect stromal cell-derived factor-1α polymorphisms in 180 neonates revealed no differences between A/A, G/G and A/G allelic combinations., Conclusions: These data emphasize the lack of predictive factors for CD34(+) cells and total nucleated cell concentrations in UCB samples before processing., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Artemether (AM) plus azithromycin (AZ) rectal co-formulations were studied to provide pre-referral treatment for children with severe febrile illnesses in malaria-endemic areas. The target profile required that such product should be cheap, easy to administer by non-medically qualified persons, rapidly effective against both malaria and bacterial infections. Analytical and pharmacotechnical development, followed by in vitro and in vivo evaluation, were conducted for various AMAZ coformulations. Of the formulations tested, stability was highest for dry solid forms and bioavailability for hard gelatin capsules; AM release from AMAZ rectodispersible tablet was suboptimal due to a modification of its micro-crystalline structure., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Our ex vivo expansion procedure starting from cord blood (CB) CD34+ cells enabled expansion of committed progenitors (CPs) without a negative impact on hematopoietic stem cells (HSCs) exhibiting both short- and long-term repopulating capacity. Upgraded to clinical scale (Macopharma HP01 in the presence of stem cell factor, FLT3-L [100 ng/mL each], granulocyte-colony-stimulating factor [10 ng/mL], and thrombopoietin [20 ng/mL]), it is being used for an ongoing clinical trial (adult allogeneic context) yielding promising preliminary results. Transplantation of ex vivo expanded CB cells is becoming a reality, while the issue of expanded cells' cryopreservation emerges as an option that allows the conservation of the product for transportation and future use. Here, we investigated whether it is possible to maintain the functional HSC and CP properties after freezing and thawing of expanded cells., Study Design and Methods: We compared cryopreservation efficiency of the ex vivo expanded CB cells using the standard protocol (freezing solution human serum albumin (HSA)-dimethyl sulfoxide [DMSO]) with the newly designed protocol based on an enriched freezing solution (HP01-DMSO) with respect to the viability index, number of CD34+ and total cells, and recovery of CPs (colony-forming units) and HSCs (NOG/Scid/gamma-null mice engraftment)., Results: Cryopreservation and thawing of expanded CB cells using the "standard" procedure (HSA-DMSO) reduced recovery of the CPs (40%) and HSCs (drastically decreasing engraftment capacity). HP01-based protocol resulted in improvement of preservation of both CPs (>60%) and HSCs (nonaltered engraftment capacities)., Conclusion: Functional maintenance of the expanded graft by cryopreservation is feasible in conditions compatible with human cell therapy requirements., (© 2012 American Association of Blood Banks.)

This report brings the first experimental evidence for the presence of long-term (LT) repopulating hematopoietic stem cells (HSCs) and Side Population (SP) cells within human steady state peripheral blood CD34(+) cells. Ex vivo culture, which reveals the LT-HSC, also increases short-term (ST) HSC engraftment capacity and SP cell number (as well as the SP subpopulations defined on the basis of CD38, CD90 and CD133 expression) which are very low in freshly isolated cells. Thus, ex vivo incubation either allows the expansion of the small fraction of HSCs or reveals "Scid Repopulating Cells - SRC" that are present in the initial CD34(+) cell population but unable to engraft. In addition, among these CD34(+) cells, we confirm the presence of committed progenitors at frequencies similar to those found in cord blood CD34(+) cells. These cells, obtained from leukoreduction filters (LRFs) and rejected in the course of the preparation of red blood cell concentrates, are an abundant and reliable material for obtaining committed progenitors, short- and long-term HSCs of therapeutic interest, especially after the ex vivo expansion phase. Our results open a perspective to set up new therapeutic protocols using expanded LRFs-recovered CD34(+) cells as a source of HSCs for autologous or allogeneic transplantation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Pharmaceutical development and manufacturing process optimization work was undertaken in order to propose a potential paediatric rectal formulation of azithromycin as an alternative to existing oral or injectable formulations. The target product profile was to be easy-to-use, cheap and stable in tropical conditions, with bioavailability comparable to oral forms, rapidly achieving and maintaining bactericidal concentrations. PEG solid solution suppositories were characterized in vitro using visual, HPLC, DSC, FTIR and XRD analyses. In vitro drug release and in vivo bioavailability were assessed; a study in rabbits compared the bioavailability of the optimized solid solution suppository to rectal solution and intra-venous product (as reference) and to the previous, non-optimized formulation (suspended azithromycin suppository). The bioavailability of azithromycin administered as solid solution suppositories relative to intra-venous was 43%, which compared well to the target of 38% (oral product in humans). The results of 3-month preliminary stability and feasibility studies were consistent with industrial production scale-up. This product has potential both as a classical antibiotic and as a product for use in severely ill children in rural areas. Industrial partners for further development are being sought., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Our previously published ex vivo expansion procedure starting from cord blood CD34+ cells enables a massive expansion of total and CD34+ cells and committed progenitors without negative impact on stem cells exhibiting both short- and long-term repopulating capacity. It was upgraded to clinical scale [Macopharma HP01(®) medium in presence of SCF, FLT3-L (100 ng/ml each), G-SCF (10 ng/ml), and TPO (20 ng/ml)] and is in use for an ongoing clinical trial (adult allogeneic context), yielding encouraging results. In order to test the possibility to use the expanded cells in distant transplantation centers, we studied the functional stability at +4°C (usual temperature of transportation) of hematopoietic progenitors and stem cells 48 h after expansion. If the cells were washed and resuspended in 4% albumin solution (actual procedure for immediate injection), only one half of total nucleated and CD34+ cells and 30% of committed progenitors survived after 24 h. This condition has also an evident negative impact on stem cells in expansion product as demonstrated on the basis of reconstitution of NSG mice bone marrow by human CD45, CD33, CD19+ cells as well as by human committed progenitors (CFU). Surprisingly, if the cells were stored 48 h at +4°C in culture medium, very good survival of total and CD34+ cells (90 to 100%) and colony forming unit cells (CFCs; around 70%) was obtained, as well as the maintenance of stem cells (the same in vivo assay with NSG mice). These data point to the possibility of the maintenance of the full functional capacity of expanded grafts for 2 days, the time allowing for its transportation to any transplantation center worldwide.

To investigate the role of reduced-intensity allogeneic (RIC-allo) stem cell transplant (SCT) as postremission therapy in adult intermediate-risk patients with acute myelogenous leukemia (AML) with FLT3-ITD or wild-type NPM1 and CEBPA without FLT3-ITD, we conducted a single-center retrospective study between January 2001 and December 2010. Sixty-six patients were included: 37 treated with RIC-alloSCT and 29 with nonallogeneic SCT therapies. Both groups were comparable concerning age, WBC count at diagnosis, gender, karyotype, genotype, and number of courses of chemotherapy to reach complete remission (CR1). Median follow-up after CR1 was 37 months (range, 11-112 months) and 48 months (range, 9-83 months) in the allo and no-allo groups, respectively. In the allo versus no-allo groups, the 3-year cumulative incidence of relapse (CIR) rates were 25% ± 8% versus 61% ± 9%; P = .005. The 3-year nonrelapse mortality (NRM), overall survival (OS), and relapse-free survival (RFS) were 22% ± 7% versus 4% ± 4% (P = .005), 52% ± 9% versus 44% ± 10% (P = .75), and 53% ± 9% versus 35% ± 9% (P = .28), respectively. Multivariate analysis indicated that CIR was reduced by allo (hazard ratio [HR], 0.32; P = .01). A landmark analysis performed at day 185 after CR1 confirmed a lower CIR after allo. RIC-allo reduces the risk of relapse, suggesting a potent graft-versus-leukemia (GVL) effect in these patients at a high risk of relapse., (Copyright © 2012 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

The aim of this study was to identify a candidate formulation for further development of a home or near-home administrable paediatric rectal form of a broad-spectrum antibiotic - specially intended for (emergency) use in tropical rural settings, in particular for children who cannot take medications orally and far from health facilities where injectable treatments can be given. Azithromycin, a broad-spectrum macrolide used orally or intravenously for the treatment of respiratory tract, skin and soft tissue infections, was selected because of its pharmacokinetic and therapeutic properties. Azithromycin in vitro solubility and stability in physiologically relevant conditions were studied. Various pharmaceutical forms, i.e. rectal suspension, two different rectal gels, polyethylene glycol (PEG) suppository and hard gelatin capsule (HGC) were assessed for in vitro dissolution and in vivo bioavailability in the rabbit. Azithromycin PEG suppository appears to be a promising candidate., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Subclones homozygous for JAK2V617F are more common in polycythemia vera (PV) than essential thrombocythemia (ET), but their prevalence and significance remain unclear. The JAK2 mutation status of 6495 BFU-E, grown in low erythropoietin conditions, was determined in 77 patients with PV or ET. Homozygous-mutant colonies were common in patients with JAK2V617F-positive PV and were surprisingly prevalent in JAK2V617F-positive ET and JAK2 exon 12-mutated PV. Using microsatellite PCR to map loss-of-heterozygosity breakpoints within individual colonies, we demonstrate that recurrent acquisition of JAK2V617F homozygosity occurs frequently in both PV and ET. PV was distinguished from ET by expansion of a dominant homozygous subclone, the selective advantage of which is likely to reflect additional genetic or epigenetic lesions. Our results suggest a model in which development of a dominant JAK2V617F-homzygous subclone drives erythrocytosis in many PV patients, with alternative mechanisms operating in those with small or undetectable homozygous-mutant clones.

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 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., (Copyright © 2011 Wiley Periodicals, Inc.)

For the past 30years, pressure inactivation of microorganisms has been developed in biosciences, in particular for foods and more recently for biological products, including pharmaceutical ones. In many past studies, the effect of high hydrostatic pressure (HHP) processes on pathogens focused mainly on the effect of an increase of the pressure value. To assure the safety of pharmaceutical products containing fragile therapeutic components, development of new decontamination processes at the lowest pressure value is needed to maintain their therapeutic properties. The aim of this study was therefore to evaluate the impact of the process parameters characterizing high-pressure treatments [such as the pressurization rate (PR) and the application mode (AM)] on the inactivation of pathogens, in particular to determine how these parameters values could help decrease the pressure value necessary to reach the same inactivation level. The effect of these physical parameters was evaluated on the inactivation of Staphylococcus aureus ATCC 6538 which is an opportunistic pathogen of important relevance in the medical, pharmaceutical and food domains. Human blood plasma was chosen as the suspension medium because of its physiological importance in the transfusion field. It was shown that the optimization of all the selected parameters could lead to a high inactivation level (≈5log(10) decrease of the initial bacterial load) at a pressure level as low as 200MPa, underlining some synergistic effects among these parameters. Complete inactivation of the initial bacterial population was achieved for the following conditions: PR=50MPas(-1), AM=5×2min, T≈-5°C and P=300MPa., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Granulocyte-colony stimulating factor (G-CSF) is used to prevent febrile neutropenia and support intense chemotherapy. However, its impact on long-term outcome in oncological patients including adults with acute lymphoblastic leukaemia (ALL) has not been determined so far., Methods: In the current study follow-up data from individual patients recruited in five multicentre, prospective, randomised trials were pooled to perform a joint analysis. Among 347 adults and adolescents with ALL, 185 were assigned to receive prophylactically G-CSF along with induction chemotherapy while 162 patients were treated without G-CSF support., Results: With the median follow-up of 5.3years, there was a tendency towards increased 5year probability of the overall survival for the G-CSF arm compared to the controls (32%±4% versus 23%±4%, p=.07), which reached statistical significance in a subgroup of T-ALL (51%±8% versus 29%±9%, p=.01) and among patients aged 21-40years (44%±6% versus 27%±6%, p=.03). The probability of leukaemia-free survival was 38%±4% and 24%±4% (p=.01) while the median remission duration equalled 33 and 17months (p=.007), respectively. In a multivariate analysis the prophylactic use of G-CSF was independently associated with reduced risk of relapse (hazard ratio (HR)=.64, p=.007) and treatment failure (HR=.67, p=.02)., Conclusions: The prophylactic use of G-CSF during induction of ALL is associated with improved long-term outcome and should be recommended especially in a setting of T-ALL and in 'young adults'. Our analysis provides the first direct evidence coming from prospective trials for the impact of primary G-CSF prophylaxis on disease-free survival of oncological patients., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We recently developed a clinical grade ex vivo cord blood expansion procedure enabling a massive amplification of hematopoietic progenitors without any loss of stem cell potential. This procedure, based on day 14 liquid cultures of cord blood CD34(+) cells, in medium Macopharma HP01 and in the presence of stem cell factor (SCF; 100 ng/ml), fms-related tyrosine kinase 3-ligand (Flt-3L; 100 ng/ml), megakaryocyte growth and developmental factor (MGDF; 100 ng/ml), and granulocyte colony-stimulating factor (G-CSF; 10 ng/ml) had to be modified due to the commercially unavailability of clinical grade MGDF molecule. So MGDF was replaced by thrombopoietin (TPO) in fivefold lower dose (20 ng/ml), and culture time was reduced to 12 days. That way, a mean expansion fold of 400, 80, and 150 was obtained for total cells, CD34(+) cells, and colony-forming cells (CFCs), respectively. This amplification was associated with a slight enhancing effect on stem cells [Scid repopulating cells (SRCs)]. These are the ultimate preclinical modifications of a clinical grade expansion protocol, which is already employed in an ongoing clinical trial.

High-dose chemotherapy with stem cells support has largely improved in terms of hematopoietic stem and progenitor cells harvest procedures as well as in those, which target or manipulate the cellular composition of autologous graft. Optimal preparative regimens and supportive care had lead to better use of autologous transplantation procedure. For other patients assigned to hematopoietic transplantation, availability of allogeneic donors appears to be an interesting alternative source of hematopoietic stem cells. Since three decades, hematopoietic growth factors development has allowed mobilization optimization and collection of peripheral hematopoietic stem cells leading to reduced days of hospitalization and less blood products requirements, being more cost-effective for patients in autologous transplantation settings and for stem cell collection facilities in allogeneic ones. New perspectives include, besides ex vivo manipulation of graft, development of mobilizing drugs in order to perform transplantation even in poor mobilizers patients. An important goal is achieved with the description of genetic polymorphisms related to optimal mobilization of stem cells. New approach using more promising and selective agents called chemokines, such as plerixafor the main leader among these agents are now available and appear complementary for alternative approach using cytokines alone (G-CSF, GM-CSF, SCF). The aim of this review is to assess the evolution of theses biotechnologies and their role in different steps of autologous transplantation and allogeneic stem cells collection., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Background: The first protocol of ex vivo expansion that enabled almost total abrogation of postmyeloablative chemotherapy neutropenia was based on a three-cytokine cocktail (stem cell factor [SCF], granulocyte-colony-stimulating factor [G-CSF], pegylated-megakaryocyte growth and development factor [PEG-MGDF]) in a serum-free medium. Since the clinical-grade molecule MGDF is no longer available on the market, we evaluated its substitution by thrombopoietin (TPO)., Study Design and Methods: CD34+ cells of myeloma patients were expanded for 10 days in serum-free cultures with SCF, G-CSF, or MGDF (100 ng/mL) or with TPO (2.5, 10, 20, 50, and 100 ng/mL) instead of MGDF. Day 10 amplifications of total nucleated cells, CD34+ cells, committed progenitors (CFCs), the capacity of engraftment of NOD/SCID mice (SCID repopulating cells [SRCs]), and the immunophenotype of cells in expansion product (CD13, CD14, CD33, CD41, CD61) were analyzed., Results: TPO in doses of 2.5 and 10 ng/mL exhibits an effect comparable to that of MGDF (100 ng/mL) on total, CD34+, and CFCs amplification. Compared to MGDF, TPO (starting at 10 ng/mL) enhances two- to threefold the percentage of megakaryocyte lineage cells (CD41+ and CD61+). Finally, TPO maintains or even enhances (depending on dose) SRC activity., Conclusions: The use of TPO instead of MGDF in our protocol is feasible without any negative effect on progenitor cell expansion. Furthermore, applied in dose of 10 or 100 ng/mL it could enhance both the stem cell activity and the percentage of megakaryocyte lineage cells in expansion product., (© 2010 American Association of Blood Banks.)

We developed a clinical-scale cord blood (CB) cell ex vivo procedure to enable an extensive expansion of committed progenitors--colony-forming cells (CFCs) without impairing very primitive hematopoietic stem cells (HSCs). CD34(++) cells, selected from previously cryopreserved and thawed CB units, were cultured in two steps (diluted 1:4 after 6 days) in the presence of stem cell factor (SCF), fms-related tyrosine kinase 3 ligand (Flt-3L), megakaryocyte growth and development factor (MGDF) (100 ng/ml each), granulocyte-colony stimulating factor (G-CSF) (10 ng/ml) in HP01 serum-free medium. HSC activity was evaluated in a serial transplantation assay, by detection of human cells (CD45, CD33, CD19 and CFC of human origin) in bone marrow (BM) of primary and secondary recipient NOD/SCID mice 6-8 weeks after transplantation. A wide amplification of total cells (∼350-fold), CD34(+) cells (∼100-fold), and CFC (∼130-fold) without impairing the HSC activity was obtained. The activity of a particular HSC subpopulation (SRC(CFC)) was even enhanced.Thus, an extensive ex vivo expansion of CFCs is feasible without impairing the activity of HSCs. This result was enabled by associating antioxidant power of medium with an appropriate cytokine cocktail (i.e., mimicking physiologic effects of a weak oxygenation in hematopoietic environment).

Optimized prophylaxis against graft-versus-host disease (GVHD) after unrelated reduced-intensity allogeneic transplantation when preceded by a conditioning regimen utilizing antithymocyte globulin (ATG) is poorly defined. To investigate the effects of methotrexate (MTX) in this treatment setting, we conducted a retrospective analysis. Sixty-three patients were selected based on the administration of a total dose of 5 mg/kg of ATG in the conditioning regimen and then separated into either group M+ (n = 39), which received MTX or group M- (n = 24), which did not. All patients received cyclosporine. In the M- and M+ groups, cumulative incidences (CI) of grade III-IV acute GVHD (aGVHD) were 43% and 10%, respectively (P = .002). Multivariate analysis indicated that grade III-IV aGVHD was favored by both the absence of MTX and the provision of a female donor for a male recipient. At 2 years, the M+ and M- groups exhibited, respectively: overall survival of 69% and 40% (P = .06), disease-free survival of 57% and 43% (P = .2), nonrelapse mortality of 20% and 44% (P = .1), and incidence of relapse of 27% and 35% (P = .6). These data suggest that MTX reduces the incidence of severe aGVHD without increasing the risk of relapse but with an accompanying trend toward improved survival after unrelated reduced-intensity transplantation with ATG in the conditioning regimen., (2011 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Objective: The steady increase of the blood demand since 2001 requires to study the clinical characteristics of blood components recipients. The objective was to describe patients transfused in 2006 in Bordeaux University Hospital, and to identify the diseases which justified the transfusion practice, using French hospital claims database., Study Design: Data from haemovigilance system were linked to hospital claims databases in order to describe patients transfused in 2006. To target diseases related to transfusion, a list of diagnoses considered as markers for transfusion was drawn up, and validated by physicians prescribing blood components., Results: Among the 100,004 patients admitted to hospital in 2006, 6275 (6.3%) received blood components; 46,727 blood units were transfused to these patients, including 67% of red blood cell, 13% of platelet concentrates and 20% of fresh-frozen plasma; 69% of blood units were prescribed in medical wards, 30% in surgery wards and 1% in gynaecology and obstetrics. The main diagnoses associated with blood transfusion were circulatory complications after cardiac surgery (80% of patients with this diagnosis were transfused), bone marrow aplasia (76% of patients), anaemia (55%), and gastro-intestinal bleeding (48%). The highest numbers of blood units were transfused to patients with hypovolemic, traumatic or postoperative shock, anaemia, hemopathy, or coagulation disorders., Conclusion: This study provided a clinical profile of the transfused patients. Data collected could be used to plan blood collection and to define objectives and resources of healthcare establishments., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

Mesenchymal stem cells (MSC) are multipotent postnatal stem cells, involved in the treatment of ischemic vascular diseases. We investigate the ability of MSC, exposed to short-term hypoxic conditions, to participate in vascular and tissue regeneration in an in vivo model of hindlimb ischemia. Transplantation of hypoxic preconditioned murine MSC (HypMSC) enhanced skeletal muscle regeneration at day 7, improved blood flow and vascular formation compared to injected nonpreconditioned MSC (NormMSC). These observed effects were correlated with an increase in HypMSC engraftment and a putative role in necrotic skeletal muscle fiber clearance. Moreover, HypMSC transplantation resulted in a large increase in Wnt4 (wingless-related MMTV integration site 4) expression and we demonstrate its functional significance on MSC proliferation and migration, endothelial cell (EC) migration, as well as myoblast differentiation. Furthermore, suppression of Wnt4 expression in HypMSC, abrogated the hypoxia-induced vascular regenerative properties of these cells in the mouse hindlimb ischemia model. Our data suggest that hypoxic preconditioning plays a critical role in the functional capabilities of MSC, shifting MSC location in situ to enhance ischemic tissue recovery, facilitating vascular cell mobilization, and skeletal muscle fiber regeneration via a paracrine Wnt-dependent mechanism.