Functional single-cell analyses of mesenchymal stromal cell proliferation and differentiation using ALDH-activity and mitochondrial ROS content.

BASKGROUND: Previous research has unveiled a stem cell-like transcriptome enrichment in the aldehyde dehydrogenase-expressing (ALDHhigh) mesenchymal stromal cell (MStroC) fraction. However, considering the heterogeneity of MStroCs, with only a fraction of them presenting bona fide stem cells (MSCs), the actual potency of ALDH as an MSC-specific selection marker remains an issue. METHODS: To address this, the proliferative and differentiation potential of individual ALDHhigh and ALDHlow MStroCs incubated at low oxygen concentrations, estimated to mimic stem cell niches (0.1% O2), were assayed using single-cell clonal analysis, compared to standard conditions (20% O2). RESULTS: We confirm that a high proliferative capacity and multi-potent MSCs are enriched in the ALDHhigh MStroC population, especially when cells are cultured at 0.1% O2. Measurements of reduced/oxidized glutathione and mitochondrial superoxide anions with MitoSoX (MSX) indicate that this advantage induced by low oxygen is related to a decrease in the oxidative and reactive oxygen species (ROS) levels in the stem cell metabolic setup. However, ALDH expression is neither specific nor exclusive to MSCs, as high proliferative capacity and multi-potent cells were also found in the ALDHlow fraction. Furthermore, single-cell assays performed after combined cell sorting based on ALDH and MSX showed that the MSXlow MStroC population is enriched in stem/progenitor cells in all conditions, irrespective of ALDH expression or culture oxygen concentration. Importantly, the ALDHhighMSXlow MStroC fraction exposed to 0.1% O2 was almost exclusively composed of genuine MSCs. In contrast, neither progenitors nor stem cells (with a complete absence of colony-forming ability) were detected in the MSXhigh fraction, which exclusively resides in the ALDHlow MStroC population. CONCLUSION: Our study reveals that ALDH expression is not exclusively associated with MSCs. However, cell sorting using combined ALDH expression and ROS content can be utilized to exclude MStroCs lacking stem/progenitor cell properties.

Bioenergetic Changes Underline Plasticity of Murine Embryonic Stem Cells.

Murine embryonic stem cells (mESCs) are endowed by a time-dependent window of plasticity during their early commitment steps. Indeed, while mESCs deprived of leukemia inhibitory factor (LIF) for 24 hours revert to their naive pluripotent state after subsequent LIF readdition, cells deprived of LIF for 48 hours are no longer efficient in reverting, upon LIF addition, and undergo irreversible differentiation. We investigated undisclosed bioenergetic profiles of early mESC-derived committed cells versus their undifferentiated states in order to reveal specific bioenergetic changes associated with mESC plasticity. Multiparametric bioenergetic analysis revealed that pluripotent (+LIF) and reversibly committed cells (-LIF24h) are energetically flexible, depending on both oxidative phosphorylation (OXPHOS) and glycolysis. They exhibit high mitochondrial respiration in the presence of the main energetic substrates and can also rely on glycolysis in the presence of OXPHOS inhibitor. Inhibition of the glycolysis or mitochondrial respiration does not change drastically the expression of pluripotency genes, which remain well expressed. In addition, cells treated with these inhibitors keep their capacity to differentiate efficiently upon embryoid bodies formation. Transition from metabolically active mESCs to irreversibly committed cells is associated with a clear change in mitochondrial network morphology, to an increase of adenosine triphosphate (ATP) produced from glycolysis and a decline of ATP turnover and of the mitochondrial activity without change in the mitochondrial mass. Our study pointed that plasticity window of mESCs is associated with the bivalent energetic metabolism and potency to shift to glycolysis or OXPHOS on demand. LIF removal provokes glycolytic metabolic orientation and consecutive loss of the LIF-dependent reversion of cells to the pluripotent state. Stem Cells 2019;37:463-475.

Discarded plasma obtained after cord blood volume reduction as an alternative for fetal calf serum in mesenchymal stromal cells cultures.

BACKGROUND: Utilization of the fetal calf serum (FCS) carries a potential health risk and raises growing economic and ethical problems. Umbilical cord blood volume reduction, required for banking, provides clinical-grade umbilical cord blood plasma (UCBP) discarded as a waste. The aim of this study was to test whether serum derived from UCBP could replace FCS for the amplification of mesenchymal stromal cells (MSCs). STUDY DESIGN AND METHODS: To this end, the amplification of the MSCs and mesenchymal progenitors was estimated in the presence of serum derived from UCBP and its cytokine content was determined by cytometric bead array and enzyme-linked immunosorbent assay techniques. As a comparison, other sources of clinical-grade human serum were tested in parallel: serum derived from solvent/detergent-treated fresh-frozen plasma (S/D-FFP) and from platelet (PLT)-rich and PLT-poor umbilical plasma. RESULTS: Serum derived from UCBP-supplemented culture sustains identical amplification of MSCs and their progenitors as in the case of FCS addition. Furthermore, the assays reveal the presence in the serum derived from UCBP of cytokines influencing the properties of MSCs (basic fibroblast growth factor, transforming growth factor-ß, vascular endothelial growth factor, and interleukin-8) or involved in the development of the myeloid lineage (thrombopoietin, erythropoietin, granulocyte-colony-stimulating factor, and granulocyte-macrophage-colony-stimulating factor). Also, our study indicates important differences between neonatal and adult-derived serum. Poor cytokine content in the S/D-FFP makes a less efficient replacement of FCS comparing to other human blood-derived supplements. CONCLUSION: Our work shows that the discarded human cord blood plasma from volume reduction is an easily obtainable and greatly available, xeno-free source of serum that is a highly efficient replacement of FCS in sustaining MSC growth.

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.

Human amniotic membrane for guided bone regeneration of calvarial defects in mice.

Due to its biological properties, human amniotic membrane (hAM) is widely studied in the field of tissue engineering and regenerative medicine. hAM is already very attractive for wound healing and it may be helpful as a support for bone regeneration. However, few studies assessed its potential for guided bone regeneration (GBR). The purpose of the present study was to assess the potential of the hAM as a membrane for GBR. In vitro, cell viability in fresh and cryopreserved hAM was assessed. In vivo, we evaluated the impact of fresh versus cryopreserved hAM, using both the epithelial or the mesenchymal layer facing the defect, on bone regeneration in a critical calvarial bone defect in mice. Then, the efficacy of cryopreserved hAM associated with a bone substitute was compared to a collagen membrane currently used for GBR. In vitro, no statistical difference was observed between the conditions concerning cell viability. Without graft material, cryopreserved hAM induced more bone formation when the mesenchymal layer covered the defect compared to the defect left empty. When associated with a bone substitute, such improved bone repair was not observed. These preliminary results suggest that cryopreserved hAM has a limited potential for GBR.

Reliability of ROS and RNS detection in hematopoietic stem cells--potential issues with probes and target cell population.

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.

A new clinical-scale serum-free xeno-free medium efficient in ex vivo amplification of mesenchymal stromal cells does not support mesenchymal stem cells.

BACKGROUND: 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. STUDY DESIGN AND METHODS: 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. RESULTS: 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 α-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. CONCLUSION: 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.

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.

Concise review: the role of oxygen in hematopoietic stem cell physiology.

Molecular dioxygen, O(2), is an important element in cellular microenvironment in vivo, and often overlooked in standard in vitro and ex vivo cell culture systems. Molecular oxygen is the ultimate electron acceptor in oxidative cellular respiration, and also a signal that regulates cell fate through concentration gradients. Recent advances in physiology of oxygen and adult stem cell research have shown that apart from being important for oxidative phosphorylation, thus energy metabolism, oxygen is also important as a signaling molecule and an integral part of the stem cell niche. This review article covers the influence of physiologically relevant oxygen levels on adult stem cells through highlighting the research on the effect of oxygen concentration on hematopoietic stem cell maintenance, proliferation and differentiation. This is important particularly to understand the embryonic and adult stem cell biology and physiology. The new discoveries in this field will help to further improve current tissue engineering and clinical applications. In addition, understanding the relationship between oxygen and stemness is invaluable for the advanced treatments of neoplastic diseases. Authors believe that in the future, active and programmed dynamic of oxygen levels will be routinely used for the programmed in vitro and ex vivo expansion of different adult stem cell types and tissue regeneration purposes.

Neonatal sex and weight influence CD34(+) cell concentration in umbilical cord blood but not stromal cell-derived factor 1-3'A polymorphism.

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.

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).

Regulatory Crosstalk between Physiological Low O2 Concentration and Notch Pathway in Early Erythropoiesis.

Physiological low oxygen (O2) concentration (<5%) favors erythroid development ex vivo. It is known that low O2 concentration, via the stabilization of hypoxia-induced transcription factors (HIFs), intervenes with Notch signaling in the control of cell fate. In addition, Notch activation is implicated in the regulation of erythroid differentiation. We test here if the favorable effects of a physiological O2 concentration (3%) on the amplification of erythroid progenitors implies a cooperation between HIFs and the Notch pathway. To this end, we utilized a model of early erythropoiesis ex vivo generated from cord blood CD34+ cells transduced with shHIF1α and shHIF2α at 3% O2 and 20% O2 in the presence or absence of the Notch pathway inhibitor. We observed that Notch signalization was activated by Notch2R−Jagged1 ligand interaction among progenitors. The inhibition of the Notch pathway provoked a modest reduction in erythroid cell expansion and promoted erythroid differentiation. ShHIF1α and particularly shHIF2α strongly impaired erythroid progenitors' amplification and differentiation. Additionally, HIF/NOTCH signaling intersects at the level of multipotent progenitor erythroid commitment and amplification of BFU-E. In that, both HIFs contribute to the expression of Notch2R and Notch target gene HES1. Our study shows that HIF, particularly HIF2, has a determining role in the early erythroid development program, which includes Notch signaling.

Autophagy Targeting and Hematological Mobilization in FLT3-ITD Acute Myeloid Leukemia Decrease Repopulating Capacity and Relapse by Inducing Apoptosis of Committed Leukemic Cells.

Targeting FLT3-ITD in AML using TKI against FLT3 cannot prevent relapse even in the presence of complete remission, suggesting the resistance and/or the persistence of leukemic-initiating cells in the hematopoietic niche. By mimicking the hematopoietic niche condition with cultures at low oxygen concentrations, we demonstrate in vitro that FLT3-ITD AML cells decrease their repopulating capacity when Vps34 is inhibited. Ex vivo, AML FLT3-ITD blasts treated with Vps34 inhibitors recovered proliferation more slowly due to an increase an apoptosis. In vivo, mice engrafted with FLT3-ITD AML MV4-11 cells have the invasion of the bone marrow and blood in 2 weeks. After 4 weeks of FLT3 TKI treatment with gilteritinib, the leukemic burden had strongly decreased and deep remission was observed. When treatment was discontinued, mice relapsed rapidly. In contrast, Vps34 inhibition strongly decreased the relapse rate, and even more so in association with mobilization by G-CSF and AMD3100. These results demonstrate that remission offers the therapeutic window for a regimen using Vps34 inhibition combined with mobilization to target persistent leukemic stem cells and thus decrease the relapse rate.

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.