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Guitart A.V.,Bordeaux University | Guitart A.V.,French National Center for Scientific Research | Hammoud M.,Aquitaine Limousin Branch of French Blood Institute | Hammoud M.,University of Franche Comte | And 4 more authors.
Experimental Hematology | Year: 2010

Objective: Regulation of hematopoiesis depends on cytokines, cellular interactions, transcription, and metabolic factors. Among the latter, O2 has been neglected for a long time. Recently, an increasing number of publications evidenced the regulatory role of physiological low O2 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 O2 and hematopoiesis. Materials and Methods: 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. Results: A critical analysis of some key publications provides partial answers to three important questions: is the term hypoxia appropriate to describe physiological low O2 concentrations? Is a very low O2 level sufficient to control the quiescence/slow cycling balance of hematopoietic stem cells? Is the O2 concentration able to modify the effect of cytokines on hematopoietic stem cells? Conclusions: We propose to use in situ normoxia instead of the confusing term hypoxia when working with normal cells at physiological low O2 concentrations. We suggest that a very low O2 concentration is necessary but not sufficient to induce hematopoietic stem cell quiescence. We review some articles showing that O2 variations modify the effect of cytokines. © 2010 ISEH - Society for Hematology and Stem Cells.


Hammoud M.,Aquitaine Limousin Branch of French Blood Institute | Hammoud M.,French National Center for Scientific Research | Hammoud M.,University of Franche Comte | Vlaski M.,Aquitaine Limousin Branch of French Blood Institute | And 14 more authors.
Journal of Cellular Physiology | Year: 2012

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. © 2011 Wiley Periodicals, Inc.


PubMed | Aquitaine Limousin Branch of French Blood Institute
Type: Evaluation Studies | Journal: Transfusion | Year: 2011

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).CD34+ cells of myeloma patients were expanded for 10 days in serum-free cultures with SCF, G-CSF, or MGDF (100ng/mL) or with TPO (2.5, 10, 20, 50, and 100ng/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.TPO in doses of 2.5 and 10ng/mL exhibits an effect comparable to that of MGDF (100ng/mL) on total, CD34+, and CFCs amplification. Compared to MGDF, TPO (starting at 10ng/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.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 100ng/mL it could enhance both the stem cell activity and the percentage of megakaryocyte lineage cells in expansion product.


PubMed | Aquitaine Limousin Branch of French Blood Institute
Type: Journal Article | Journal: Transfusion | Year: 2011

During short-term storage of hematopoietic cells (HCs) at 4C a substantial decline in number and in functional capacity of progenitors occurs after 3 days. We hypothesized that physiologic O2 and CO2 concentrations of hematopoietic tissue microenvironment (approx. 3% O2 and approx. 6% CO2) could improve cell viability and functionality during storage at 4C.Mobilized peripheral blood (PB) CD34+ cells from multiple myeloma or non-Hodgkins lymphoma patients were stored in flasks containing air (approx. 20% O2 and approx. 0.05% CO2) or 3% O2/6% CO2 atmosphere, for 3, 5, and 7 days at 4C. 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 (SCI D-repopulating cell [SRC] assay).The total number of viable cells and cells in G1 phase as well as the number of total CFCs were significantly higher at 3% O2/6% CO2 than in air at all time points. Cells in G0 phase and SRC were equally preserved in both conditions.Atmosphere with low O2 and high CO2 concentration (3% O2/6% CO2) in hypothermia (+4C) 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.

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