Pascale Duchez

Flt3-ligand induces adhesion of haematopoietic progenitor cells via a very late antigen (VLA)-4- and VLA-5-dependent mechanism.

Summary. The adhesion of haematopoietic progenitor cells (HPC) to the bone marrow microenvironment is a process regulated by cytokines. In this study, we have shown that flt3‐ligand (FL), a growth factor that controls early haematopoiesis, regulated the function and expression of the beta‐1 integrins, very late antigen (VLA)‐4 and VLA‐5 on HPC. The modulation of the adhesiveness of HPC by FL was studied by adhesion assays on umbilical vein endothelial cells (HUVEC). Stimulation by FL induced two peaks of increased adhesiveness of HPC. The first peak was at around 30 min and was mechanistically related to an activation of the beta‐1 integrins, mainly VLA‐4 and VLA‐5. The second peak was at around 12 h and was related to increased expression of VLA‐4 and VLA‐5. The control of HPC adhesiveness by FL is a previously unreported property of FL that may be important for the homing and the retention of flt3‐expressing HPC within the bone marrow microenvironment.

A clinical-scale expansion of mobilized CD34+ hematopoietic stem and progenitor cells by use of a new serum-free medium

BACKGROUND:  The autologous transplantation of CD34+ cells expanded ex vivo in serum‐free conditions dramatically reduces postmyeloablative 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 HP01 (Macopharma), was evaluated.

Hypoxia-preconditioned mesenchymal stromal cells improve cardiac function in a swine model of chronic myocardial ischaemia

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 × 10(6)/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.

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

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.

Whole-blood leukodepletion filters as a source of CD34+ progenitors potentially usable in cell therapy

BACKGROUND:  Used leukodepletion 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 CD34+ cells recovered from LDFs were investigated.

Definitive Setup of Clinical Scale Procedure for Ex Vivo Expansion of Cord Blood Hematopoietic Cells for Transplantation

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.

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

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 (O2) 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 O2 concentrations (20%, 12.5% and 3% O2. 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 O2 concentration, reaching its maximum at 3% O2, 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 O2 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 O2 (mistakenly called hypoxia), should be considered as an important environmental factor that significantly influences cytokine activity.

An efficient large-scale thawing procedure for cord blood cells destined for selection and ex vivo expansion of CD34+ cells.

THE MAIN DISADVANTAGES of transplantation of cord blood (CB) hematopoietic stem or progenitor cells (HSPC) are the delayed neutrophil and platelet reconstitution coupled with the difficulty in obtaining a large enough graft for patients weighing more than 30 kg (1). Both problems could be overcome by ex vivo expansion of CB progenitors. In most CB banks, the whole CB samples are stored in individual or double bags. The ex vivo expansion of progenitors in whole blood samples is inefficient; thus, a CD341 selection before an expansion procedure is a prerequisite. However, this selection using frozen-thawed blood samples is impaired by aggregate formation (“clumping”). To overcome this problem, addition of recombinant human deoxyribonuclease (DNase) has been proposed during thawing of CB cells prior to transplantation (2) or expansion (3). We have extended this procedure to clinical-grade conditions incorporating subsequent CD341 cell selection and expansion in two-step culture conditions. Two bags [(Macopharma, Lille, France) GSR 7000 A), each containing 125 ml of CB and 125 ml of cryoprotecting solution 20% dimethylsulfoxide (DMSO), from a single CB and frozen by using a controlled-rate procedure and stored in liquid nitrogen, were thawed in a water bath at 37°C. DNase (Pulmozyme Roche, Neuilly, France) (3750 units) and the MgCl2 solution (Cooper, Mellin, France) (0.5 M, 250 ml) were added to each bag. Two samples were pooled and then rehydrated slowly (10 min at 20–22°C) by adding 250 ml of Dextran 40-sorbitol solution (Braun Medical, Boulogne, France) and 3.3 ml of Gamma IV (0.5 g LFB). The cell suspension was filtered, incubated with anti-CD34, and transferred into the separation chamber (system Isolex 300i, Baxter, Deerfield, IL). An additional 7500 Units of DNase and 500 ml of MgCl2 were added before the addition of immunomagnetic microbeads (Baxter kit). The suspension was processed on Isolex 300i. The CD341 cells were transferred into 50-ml conical tubes and centrifuged for 10 min at 1500 rpm. The supernatant was removed, and the cells were resuspended in 10 ml of serum-free culture medium (Irvine Scientific, Santa Ana, CA). The CD341 cells were expanded in the same serum-free medium (200 ml; 10,000 cells/ml) in gas-permeable bags (Opticyte 390 cm2, Baxter, Maurepas, France) in the presence of stem cell factor (SCF; Amgen, Thousand Oaks, CA), megakaryocyte growth and development factor (MGDF) (Amgen), Flt3 ligand (RD Median 3.333 106 n 5 5) CD341 cells per sample (Fig. 1A), i.e., a mean recovery of 48.5% with respect to the values found in fresh CB samples (Fig. 1B). The purity of these

Thrombopoietin to replace megakaryocyte-derived growth factor: impact on stem and progenitor cells during ex vivo expansion of CD34+ cells mobilized in peripheral blood

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).

Busulfan Administration Flexibility Increases the Applicability of Scid Repopulating Cell Assay in NSG Mouse Model

Background Xenotransplantation models allowing the identification and quantification of human Hematopoietic stem cells (HSC) in immunodeficient mice remain the only way to appropriately address human HSC function despite the recent progress in phenotypic characterization. However, these in vivo experiments are technically demanding, time consuming and expensive. Indeed, HSCs engraftment in mouse requires pre-conditioning of animals either by irradiation or cytotoxic drugs to allow homing of injected cells in specific stem cell niches and their subsequent expansion and differentiation in bone marrow. Recently, the development of busulfan pre-conditioning of animals improved the flexibility of experimentation in comparison with irradiation. Design and Methods In order to further facilitate the organization of these complex experiments we investigated the effect of extending the period between mice pre-conditioning and cell injection on the engraftment efficiency. In the meantime, we also explored the role of busulfan doses, mouse gender and intravenous injection route (caudal or retro orbital) on engraftment efficiency. Results and Conclusion We showed that a period of up to 7 days did not modify engraftment efficiency of human HSCs in NSG model. Moreover, retro orbital cell injection to female mice pre-conditioned with 2x25 mg/kg of busulfan seems to be the best adapted schema to detect the human HSC in xenotransplantation experiments.