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Godfrey A.L.,University of Cambridge | Godfrey A.L.,Addenbrookes Hospital | Chen E.,University of Cambridge | Pagano F.,University of Cambridge | And 18 more authors.
Blood | Year: 2012

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. © 2012 by The American Society of Hematology.


Vlaski M.,Etablissement Francais du Sang Aquitaine Limousin | Vlaski M.,University of Bordeaux Segalen | Negroni L.,University of Bordeaux Segalen | Kovacevic-Filipovic M.,University of Belgrade | And 13 more authors.
Journal of Cellular Physiology | Year: 2014

We analyzed the effect of exposure to hypoxic/hypercapnic (HH) gas mixture (5% O2/9% CO2) on the maintenance of functional cord blood CD34+ hematopoietic stem and progenitor cells in severe hypothermia (4°C) employing the physiological and proteomic approaches. Ten-day exposure to HH maintained the Day 0 (D-0) level of hematopoietic stem cells as detected in vivo on the basis of hematopoietic repopulation of immunodeficient mice-short-term scid repopulating cells (SRC). Conversely, in the atmospheric air (20% O2/0.05% CO2), usual condition used for cell storage at 4°C, stem cell activity was significantly decreased. Also, HH doubled the survival of CD34+ cells and committed progenitors (CFCs) with respect to the atmospheric air (60% vs. 30%, respectively). Improved cell maintenance in HH was associated with higher proportion of aldehyde dehydrogenase (ALDH) positive cells. Cell-protective effects are associated with an improved maintenance of the plasma and mitochondrial membrane potential and with a conversion to the glycolytic energetic state. We also showed that HH decreased apoptosis, despite a sustained ROS production and a drop of ATP amount per viable cell. The proteomic study revealed that the global protein content was better preserved in HH. This analysis identified: (i) proteins sensitive or insensitive to hypothermia irrespective of the gas phase, and (ii) proteins related to the HH cell-protective effect. Among them are some protein families known to be implicated in the prolonged survival of hibernating animals in hypothermia. These findings suggest a way to optimize short-term cell conservation without freezing. © 2014 Wiley Periodicals, Inc.


Guitart A.V.,University of Bordeaux 1 | Guitart A.V.,French National Center for Scientific Research | Debeissat C.,University of Bordeaux 1 | Debeissat C.,French National Center for Scientific Research | And 10 more authors.
Cell Death and Differentiation | Year: 2011

Oxygen (O2) concentrations in bone marrow vary from 4% in capillaries to 0.1% in subendosteum, in which hematopoietic stem cells reside in specific niches. Culture at low O2 concentrations (3, 1 and 0.1%) influences hematopoietic stem and progenitor cells survival, proliferation and differentiation, depending on their level of differentiation. Culture of human CD34 cells at low O2 concentrations (O2 3%) maintains stem cell engraftment potential better than at 20% O2 (NOD/Scid xenograft model). In contrast, progenitors disappear from cultures at/or 1% O2 concentrations. A very low O2 concentration (0.1%) induces CD34 quiescence in G 0. The exploration of molecules and mechanisms involved in hematopoietic stem and progenitor cells quiescence and differentiation related to low O2 concentrations is unfeasible with primary CD34 cells. Therefore, we performed it using murine hematopoietic nonleukemic factor-dependent cell Paterson (FDCP)-Mix progenitor cell line. The culture of the FDCP-Mix line at 0.1% O2 induced in parallel G 0 quiescence and granulo-monocytic differentiation of most cells, whereas a minority of undifferentiated self-renewing cells remained in active cell cycle. Hypoxia also induced hypophosphorylation of pRb and increased the expression of p27 KIP1, the two proteins that have a major role in the control of G 0 and G 1 to S-phase transition. © 2011 Macmillan Publishers Limited All rights reserved.


Jaussaud J.,French Institute of Health and Medical Research | Biais M.,French Institute of Health and Medical Research | Biais M.,Bordeaux University Hospital Center | Biais M.,University of Bordeaux 1 | And 13 more authors.
European Journal of Cardio-thoracic Surgery | Year: 2013

Objectives: Cell loss during cardiac injection and hostility of the host-tissue microenvironment have the potential to diminish the overall effect of stem cell therapy. The purposes of this study were to evaluate the effect of a hypoxic preconditioning of mesenchymal stromal cells (MSC), to determine its safety and effectiveness, and to improve the efficacy of cell therapy using MSC in the setting of chronic myocardial ischaemia in swine. Methods: Myocardial ischaemia was induced by an ameroid constrictor. Human MSC were cultured under normoxic (20% O2) or hypoxic conditions (1.5% O2) before transplantation. One month after ischaemia, pigs were randomly assigned to saline injection (sham), and 1 × 106/kg normoxic or hypoxic MSC transplantation into the ischaemic inferior-lateral zone. RESULTS: Twenty-seven pigs were operated on and the mortality rate was 33.3%. The remaining 18 animals were randomly assigned to sham (n = 4), normoxic (n = 8) or hypoxic MSC (n = 6) treatment. Global systolic (left ventricle ejection fraction, P = 0.04) and diastolic (E/Ea, P = 0.008) functions were increased in the hypoxic group compared with other groups. The peak of 2-dimensional longitudinal strain was less altered in the hypoxic group compared with other groups (P < 0.001). Haemodynamic data showed that dP/dT max was improved in the hypoxic group compared with the other group (P < 0.01). Capillary density was increased in the hypoxic group (P = 0.001). MSC density was significantly higher in the ischaemic zone in the hypoxic group (P < 0.01). CONCLUSION: MSC engraftment with hypoxic preconditioning significantly improves capillary density and cell survival, resulting in improvement in global, regional and diastolic left ventricular functions. This highlights the therapeutic potential of transplanting hypoxic-preconditioned MSC in the setting of chronic ischaemic heart failure. © The Author 2012. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.


Duchez P.,Etablissement Francais du Sang Aquitaine Limousin | Duchez P.,University of Bordeaux Segalen | Chevaleyre J.,Etablissement Francais du Sang Aquitaine Limousin | Chevaleyre J.,University of Bordeaux Segalen | And 8 more authors.
Cell Transplantation | Year: 2012

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. © 2012 Cognizant Comm. Corp.


Vlaski-Lafarge M.,Etablissement Francais du Sang Aquitaine Limousin | Vlaski-Lafarge M.,University of Bordeaux Segalen | Ivanovic Z.,Etablissement Francais du Sang Aquitaine Limousin | Ivanovic Z.,University of Bordeaux Segalen
Journal of Cell Science | Year: 2015

Many studies have provided evidence for the crucial role of the reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the regulation of differentiation and/or self-renewal, and the balance between quiescence and proliferation of hematopoietic stem cells (HSCs). Several metabolic regulators have been implicated in the maintenance of HSC redox homeostasis; however, the mechanisms that are regulated by ROS and RNS, as well as their downstream signaling are still elusive. This is partially owing to a lack of suitable methods that allow unequivocal and specific detection of ROS and RNS. In this Opinion, we first discuss the limitations of the commonly used techniques for detection of ROS and RNS, and the problem of heterogeneity of the cell population used in redox studies, which, together, can result in inaccurate conclusions regarding the redox biology of HSCs. We then propose approaches that are based on single-cell analysis followed by a functional test to examine ROS and RNS levels specifically in HSCs, as well as methods that might be used in vivo to overcome these drawbacks, and provide a better understanding of ROS and RNS function in stem cells. © 2015.


PubMed | Etablissement francais du sang Aquitaine Limousin, Etablissement francais du sang, Etablissement francais du sang Alpes Mediterranee, Aix - Marseille University and 2 more.
Type: Journal Article | Journal: Transfusion clinique et biologique : journal de la Societe francaise de transfusion sanguine | Year: 2014

Hepatitis E virus (HEV) is a non-enveloped RNA virus transmitted by the fecal-oral route. Autochthonous hepatitis E occurring in developed countries is caused by genotypes 3 and 4 and is a zoonotic infection. Humans are infected mostly after ingestion of undercooked meat from infected animals. Most HEV 3 and 4 infections are clinically inapparent. However, genotype 3 (HEV 3) can lead to chronic hepatitis in immuno-compromised patients such as organ-transplant recipients and patients with haematological malignancies. In Europe, HEV 3 is implicated in transfusion-transmitted HEV infection. In France, as observed in several European countries, prevalence of HEV RNA and specific IgG antibodies are high indicating that viral circulation is important. The systematic HEV NAT screening of blood donations used for preparation of solvent detergent plasma indicate that 1 to 2218 donation is infected by HEV RNA. The need or implementations impacts of safety measures to prevent HEV transmission by blood transfusion are under reflexion by Frenchs health authorities. The HEV NAT screening is the only available tool of prevention. Alternative strategies are under investigation including individual or mini pool NAT testing all or part of blood donations.


PubMed | Etablissement francais du sang Alpes Mediterranee, Etablissement francais du sang Aquitaine Limousin, Institute of Veille Sanitaire, Sanguine and Etablissement francais du sang
Type: Journal Article | Journal: Transfusion clinique et biologique : journal de la Societe francaise de transfusion sanguine | Year: 2014

The risk assessment for blood transfusion is an essential step that must precede any screening strategy of a pathogen transmitted by transfusion. After several cases of HEV transmission by transfusion in France, a risk assessment for this virus was performed.We used a method based on the prevalence of HEV-RNA in plasmas collected for the preparation of SD-plasma. To estimate the rate of HEV-RNA positive among all blood donations, data on SD-plasma were adjusted on the following HEV risk factors: gender, age group and region of residence. We assumed that HEV risk factors were the same in plasma donors and whole blood donors.Among 57,101 plasma donations tested for HEV-RNA in 2013, 24 were positive (crude rate of 4.2 per 10,000 donations). After adjustment, the total number of HEV-RNA positive blood donations was estimated at 788, accounting for a rate of 2.65 per 10,000 donations (95% CI: 1.6-3.7) or 1 in 3800 donations (1 in 6,200-1 in 2,700). This rate was 12 times higher in men than in women, increased with age, and varied according to region of residence.The risk of blood donation contamination by HEV has been estimated to be 1 in 3800 donations in 2013. An essential input is still missing to assess now the risk in recipients: the minimum infectious dose. Furthermore, the risk in recipients has to be analyzed according to characteristics of transfused patients: presence of anti-HEV immunity, existence of chronic liver disease or immunodeficiency.


PubMed | Etablissement Francais du Sang Aquitaine Limousin
Type: | Journal: Transfusion | Year: 2016

We evaluated a new serum-free, xeno-free medium (Xuri, GE HealthCare) in ex vivo cultures for amplification of mesenchymal stromal cells (MStroC) in comparison with classical culture supplemented with fetal calf serum and basic fibroblast growth factor.MStroC and mesenchymal stem cell (MSC) proliferative capacities were studied in bulk cultures and single-cell cultures with assay of secondary replating capacity of individual clones. Flow-cytometric phenotype analysis and proliferative history analysis were also performed.In cultures initiated with previously amplified and cryopreserved MStroC from human marrow, Xuri medium enabled a total cell expansion fold comparable to one obtained in control fetal calf serum (FCS)-supplemented culture. However, both the number and the proliferative capacity of colony-forming unit-fibroblast were greatly reduced in Xuri medium cultures. This is even more evident in single-cell cultures, where, in rare positive wells, only several cells were found in Xuri cultures, compared to abundant cell content in FCS and -minimal essential medium cultures. Replating these single-cell clones in secondary cultures (FCS in both cases) revealed a total exhaustion of MSC proliferative capacity after Xuri primary culture.Since in both conditions after a 7-day bulk culture, similar immunophenotype and proliferative history were found when the standard MSC immunophenotype panel was employed, the loss of proliferative capacity in Xuri medium shows that it cannot maintain functional MSC population. This is a drastic example showing that the real MSC activity can be completely unrelated to the immunophenotype considered as MSC phenotype.

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