Hypoxia/hypercapnia prevents iron-dependent cold injuries in cord blood stem and progenitor cells.

BACKGROUND: Cold-induced cell injuries are associated with an increase in the cellular labile iron pool (LIP) followed by lipid peroxidation and alteration of mitochondrial function, which lead to cell death. Recently, we showed that incubation in a hypoxic/hypercapnic (HH) gas mixture improved the survival of a population of cord blood hematopoietic progenitors and CD34+ hematopoietic progenitor and stem cells in severe hypothermia. To explain the underlying mechanism, here we test if this HH-induced cytoprotection in cold conditions is associated with the level of LIP and lysosome stability. METHODS: Cord blood CD34+ cells were incubated in air (20% O2/0.05% CO2) or in the hypoxic (5% O2)/hypercapnic (9% CO2) atmosphere for 7days at 4°C and analyzed. RESULTS: Incubation in HH condition maintained the day 0 (D-0) level of LIP detected using a bleomycin-dependent method. This was associated with preservation of lysosome integrity and a higher cell survival. Conversely, in the air condition LIP was significantly increased. Also, the presence of a moderate concentration of iron chelator deferoximine improves the conservation of total CD34+ cells and committed progenitors in air condition. Pre-treatment of CD34+ cells with the lysomotropic agent imidazole induces significant decrease in the lysosomal stability and in all conditions. This is associated with an important decrease of survival of conserved cells and an increase in the cellular LIP level. DISCUSSION: Our study showed that HH gas mixture cytoprotection during hypothermia maintains lysosome stability, which enables preservation of the cellular chelatable iron in the physiological ranges. These findings suggest a way to optimize cell conservation without freezing.

Steady state peripheral blood provides cells with functional and metabolic characteristics of real hematopoietic stem cells.

Hematopoietic stem cells (HSCs), which are located in the bone marrow, also circulate in cord and peripheral blood. Despite high availability, HSCs from steady state peripheral blood (SSPB) are little known and not used for research or cell therapy. We thus aimed to characterize and select HSCs from SSPB by a direct approach with a view to delineating their main functional and metabolic properties and the mechanisms responsible for their maintenance. We chose to work on Side Population (SP) cells which are highly enriched in HSCs in mouse, human bone marrow, and cord blood. However, no SP cells from SSBP have as yet been characterized. Here we showed that SP cells from SSPB exhibited a higher proliferative capacity and generated more clonogenic progenitors than non-SP cells in vitro. Furthermore, xenotransplantation studies on immunodeficient mice demonstrated that SP cells are up to 45 times more enriched in cells with engraftment capacity than non-SP cells. From a cell regulation point of view, we showed that SP activity depended on O2 concentrations close to those found in HSC niches, an effect which is dependent on both hypoxia-induced factors HIF-1α and HIF-2α. Moreover SP cells displayed a reduced mitochondrial mass and, in particular, a lower mitochondrial activity compared to non-SP cells, while they exhibited a similar level of glucose incorporation. These results provided evidence that SP cells from SSPB displayed properties of very primitive cells and HSC, thus rendering them an interesting model for research and cell therapy.

Repopulating hematopoietic stem cells from steady-state blood before and after ex vivo culture are enriched in the CD34+CD133+CXCR4low fraction.

The feasibility of ex vivo expansion allows us to consider the steady-state peripheral blood as an alternative source of hematopoietic stem progenitor cells for transplantation when growth factor-induced cell mobilization is contraindicated or inapplicable. Ex vivo expansion dramatically enhances the in vivo reconstituting cell population from steady-state blood. In order to investigate phenotype and the expression of homing molecules, the expression of CD34, CD133, CD90, CD45RA, CD26 and CD9 was determined on sorted CD34+ cells according to CXCR4 ("neg", "low" "bright") and CD133 expression before and after ex vivo expansion. Hematopoietic stem cell activity was determined in vivo on the basis of hematopoietic repopulation of primary and secondary recipients - NSG immuno-deficient mice. In vivo reconstituting cells in the steady-state blood CD34+ cell fraction before expansion belong to the CD133+ population and are CXCR4low or, to a lesser extent, CXCR4neg, while after ex vivo expansion they are contained only in the CD133+CXCR4low cells. The failure of the CXCR4bright population to engraft is probably due to the exclusive expression of CD26 by these cells. The limiting-dilution analysis showed that both repopulating cell number and individual proliferative capacity were enhanced by ex vivo expansion. Thus, steady-state peripheral blood cells exhibit a different phenotype compared to mobilized and cord blood cells, as well as to those issued from the bone marrow. These data represent the first phenotypic characterization of steady-state blood cells exhibiting short- and long-term hematopoietic reconstituting potential, which can be expanded ex vivo, a sine qua non for their subsequent use for transplantation.

Clinical-scale validation of a new efficient procedure for cryopreservation of ex vivo expanded cord blood hematopoietic stem and progenitor cells.

Survival of ex vivo expanded hematopoietic stem cells (HSC) and progenitor cells is low with the standard cryopreservation procedure. We recently showed that the efficiency of cryopreservation of these cells may be greatly enhanced by adding a serum-free xeno-free culture medium (HP01 Macopharma), which improves the antioxidant and biochemical properties of the cryopreservation solution. Here we present the clinical-scale validation of this cryopreservation procedure. The hematopoietic cells expanded in clinical-scale cultures were cryopreserved applying the new HP01-based procedure. The viability, apoptosis rate and number of functional committed progenitors (methyl-cellulose colony forming cell test), short-term repopulating HSCs (primary recipient NSG mice) and long-term HSCs (secondary recipient NSG mice) were tested before and after thawing. The efficiency of clinical-scale procedure reproduced the efficiency of cryopreservation obtained earlier in miniature sample experiments. Furthermore, the full preservation of short- and long-term HSCs was obtained in clinical scale conditions. Because the results obtained in clinical-scale volume are comparable to our earlier results in miniature-scale cultures, the clinical-scale procedure should be considered validated. It allows cryopreservation of the whole ex vivo expanded culture content, conserving full short- and long-term HSC activity.

Interleukin-6 enhances the activity of in vivo long-term reconstituting hematopoietic stem cells in "hypoxic-like" expansion cultures ex vivo.

BACKGROUND: Since interleukin (IL)-6 synergizes with the physiologically relevant O2 concentration in the maintenance of primitive hematopoietic stem cell (HSC) subpopulations, we hypothesized that its addition to our hypoxic response mimicking cultures (HRMCs), composed of an antioxidant-supplied serum-free xeno-free medium supplemented with the cytokines stabilizing hypoxia-inducible factor-1α and balancing HSC self-renewal and commitment, will result in a similar effect even if they are exposed to 20% O2 . STUDY DESIGN AND METHODS: HRMCs were exposed to 20 and 5% O2 with and without IL-6. Functional committed progenitors (colony-forming cells [CFCs]: CFU-GM, BFU-E, CFU-Mix, and CFU-Mk) were evaluated as well as the short- and long-term repopulating HSCs using in vivo NSG mice model (primary and secondary recipients, respectively). RESULTS: The addition of IL-6 to HRMCs exposed to 20% O2 did not significantly impact either the CFCs or in vivo short-term repopulating cells. However, it enhanced both the frequency and the individual proliferative capacity of the most primitive long-term repopulating cell population evidenced by the generation of human CFCs in the marrow of secondary recipient mice. The exposure of HRMCs to 5% O2 negatively affected the amplification of CFCs, which was not changed by the addition of IL-6 and exhibited a partial enhancing effect on the long-term repopulating cells. CONCLUSION: The addition of IL-6 to the cytokine cocktail further improves our expansion procedure based on atmospheric O2 concentration-exposed HRMCs by enhancing the maintenance of the most primitive HSCs without a negative impact on the less primitive HSC populations and CFCs.

Hypoxia/hypercapnia-induced adaptation maintains functional capacity of cord blood stem and progenitor cells at 4°C.

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.

Cryopreservation of hematopoietic stem and progenitor cells amplified ex vivo from cord blood CD34+ cells.

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.

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.

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.

Alpha Lipoic-Acid Potentiates Ex Vivo Expansion of Human Steady-State Peripheral Blood Hematopoietic Primitive Cells.

Steady state peripheral blood (SSPB) contains hematopoietic stem and progenitor cells (HSPCs) presenting characteristics of real hematopoietic stem cells, and thus represents an interesting alternative cell supply for hematopoietic cell transplantation. Development of ex vivo expansion strategies could overcome the low HSPC numbers usually rescued from SSPB. We investigated the effect of alpha lipoic acid (ALA) on ex vivo culture of SSPB CD34 positive (CD34pos) cells on primitive cell expansion, cell cycle, and oxidative metabolism as estimated by determining the ROS and GSH content. ALA increased the ex vivo expansion of total CD34pos cells and of phenotypically defined CD34pos HSPCs subpopulations that retained in vivo repopulating capacity, concomitantly to a decreased expansion of differentiating cells. ALA did not modify cell cycle progression nor the proliferation of ex vivo expanded CD34pos cells, and coherently did not affect the ROS level. On the contrary, ALA decreased the proliferation and disturbed cell cycle progression of cells reaching a differentiated status, a phenomenon that seems to be associated with a drop in ROS level. Nonetheless, ALA affected the redox status of hematopoietic primitive cells, as it reproducibly increased GSH content. In conclusion, ALA represents an interesting molecule for the improvement of ex vivo expansion strategies and further clinical application in hematopoietic cell transplantation (HCT).

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). 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.

α-Tocopherol Attenuates Oxidative Phosphorylation of CD34+ Cells, Enhances Their G0 Phase Fraction and Promotes Hematopoietic Stem and Primitive Progenitor Cell Maintenance.

Alpha tocopherol acetate (αTOA) is an analogue of alpha tocopherol (αTOC) that exists in the form of an injectable drug. In the context of the metabolic hypothesis of stem cells, we studied the impact of αTOA on the metabolic energetic profile and functional properties of hematopoietic stem and progenitor cells. In ex vivo experiments performed on cord blood CD34+ cells, we found that αTOA effectively attenuates oxidative phosphorylation without affecting the glycolysis rate. This effect concerns complex I and complex II of the mitochondrial respiratory chain and is related to the relatively late increase (3 days) in ROS (Reactive Oxygen Species). The most interesting effect was the inhibition of Hypoxia-Inducible Factor (HIF)-2α (Hexpression, which is a determinant of the most pronounced biological effect-the accumulation of CD34+ cells in the G0 phase of the cell cycle. In parallel, better maintenance of the primitive stem cell activity was revealed by the expansion seen in secondary cultures (higher production of colony forming cells (CFC) and Severe Combined Immunodeficiency-mice (scid)-repopulating cells (SRC)). While the presence of αTOA enhanced the maintenance of Hematopoietic Stem Cells (HSC) and contained their proliferation ex vivo, whether it could play the same role in vivo remained unknown. Creating αTOC deficiency via a vitamin E-free diet in mice, we found an accelerated proliferation of CFC and an expanded compartment of LSK (lineagenegative Sca-1+cKit+) and SLAM (cells expressing Signaling Lymphocytic Activation Molecule family receptors) bone marrow cell populations whose in vivo repopulating capacity was decreased. These in vivo data are in favor of our hypothesis that αTOC may have a physiological role in the maintenance of stem cells. Taking into account that αTOC also exhibits an effect on proliferative capacity, it may also be relevant for the ex vivo manipulation of hematopoietic stem cells. For this purpose, low non-toxic doses of αTOA should be used.

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. 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.

Normal Hematopoetic Stem and Progenitor Cells Can Exhibit Metabolic Flexibility Similar to Cancer Cells.

It is known that cancer stem cells (CSCs) with the largest proliferative capacity survive the anoxic and/or ischemic conditions present inside tumorous tissue. In this study we test whether normal stem cells can survive under the same conditions due to cancer cell-like metabolic adaptations. We cultivated a CD34+ population with a majority of hematopoietic progenitors, and a CD34+CD38lowCD133+CD90+CD45RA- population, highly enriched in hematopoietic stem cells (HSCs), under anoxic, anoxic/aglycemic ("ischemia-like"), or physiological conditions (3% O2). Results showed, despite a reduction in total cell fold expansion proportionate to the decrease in O2 concentration; CD34+ cells, aldehyde dehydrogenase-expressing primitive cells, and committed progenitors expanded, even in anoxia. Interestingly, under ischemia-like conditions, stem and CD34+ cell populations are maintained at day-0 level. Cell-cycle analysis further revealed an accumulation of cells in the G0/G1 phase in anoxia or anoxia/aglycemia, with a fraction of cells (~40%) actively cycling (SG2M phases). Also stem cell analysis showed that in these conditions a long-term Scid Repopulating activity was equal to that found with 3% O2. In addition stem cells with the highest proliferative capacity were maintained in anoxia/aglycemia and in anoxia. The estimated ATP profile, active mitochondrial content, and succinate accumulation are indicative of anaerobic mitochondrial respiration in both HSCs and CD34+ progenitors under ischemia-like conditions. We demonstrate here that primitive hematopoietic cells show similar metabolic flexibility to CSCs, allowing them to survive a lack of O2 and O2/glucose. Our study reveals that this feature is not the consequence of malignant transformation, but an attribute of stemness.

Characteristics of cells with engraftment capacity within CD34+ cell population upon G-CSF and Plerixafor mobilization.

In the context of hematopoietic cell transplantation, hematopoietic stem cells and progenitor cells (HSC and HPC) are usually collected by apheresis following their mobilization by G-CSF alone or in combination with Plerixafor® when patients fail to respond to G-CSF alone. In medical practice, the quality of the hematopoietic graft is based on CD34+ cell content that is used to define "Good Mobilizer (GM)" or "Poor Mobilizer (PM)" patients but does not report the real HSC content of grafts. In this study, we assessed the HSC content within the CD34+ fraction of graft samples from 3 groups of patients: 1-GM patients receiving G-CSF only (GMG-CSF), 2-PM patients receiving G-CSF only (PMG-CSF), 3-PM patients receiving G-CSF + Plerixafor (PMG-CSF+P). Although HSC from the 3 groups of patients displayed very similar phenotypic profiles, expression of "stemness" genes and metabolic characteristics, their capacity to engraft NSG mice differed revealing differences in terms of HSC between groups. Indeed according to mobilization regimen, we observed differences in migration capacity of HSC, as well as differences in engraftment intensity depending on the initial pathology (myeloma versus lymphoma) of patients. This suggests that mobilization regimen could strongly influence the long term engraftment efficiency of hematopoietic grafts.

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

BACKGROUND: During short-term storage of hematopoietic cells (HCs) at 4°C 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 4°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% O2 and approx. 0.05% CO2) or 3% O2/6% CO2 atmosphere, for 3, 5, and 7 days at 4°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 (SCI D-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% O2/6% CO2 than in air at all time points. Cells in G0 phase and SRC were equally preserved in both conditions. CONCLUSION: Atmosphere with low O2 and high CO2 concentration (3% O2/6% CO2) in hypothermia (+4°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.

A novel procedure to improve functional preservation of hematopoietic stem and progenitor cells in cord blood stored at +4°c before cryopreservation.

During storage and transportation of collected cord blood units (CBUs) to the bank prior to their processing and cryopreservation, it is imperative to preserve the functional capacities of a relatively small amount of cells of interest (stem and progenitor cells) which are critical for graft potency. To improve CBU storage efficiency, we conceived an approach based on the following two principles: (1) to provide a better nutritive and biochemical environment to stem and progenitor cells in CB and (2) to prevent the hyperoxygenation of these cells transferred from a low- (1.1%-4% O2 in the CB) to a high-oxygen (20%-21% O2 in atmosphere) concentration. Our hypothesis is confirmed by the functional assessment of stem cell (hematopoietic reconstitution capacity in immunodeficient mice-scid repopulating cell assay) and committed progenitor activities (capacity of in vitro colony formation and of ex vivo expansion) after the storage period with our medium (HP02) in gas-impermeable bags. This storage procedure maintains the full functional capacity of a CBU graft for 3 days with respect to day 0. Further, using this procedure, a graft stored 3 days at +4°C exhibits better functional capacities than one currently used in routine storage (CBUs stored at +4°C for 1 day in gas-permeable bags and without medium). We provided the proof of principle of our approach, developed a clinical-scale kit and performed a preclinical assay demonstrating the feasibility and efficiency of our CBU preservation protocol through all steps of preparation (volume reduction, freezing, and thawing).

Functional stability (at +4°C) of hematopoietic stem and progenitor cells amplified ex vivo from cord blood CD34+ cells.

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.

Long-term repopulating hematopoietic stem cells and "side population" in human steady state peripheral blood.

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.

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.