RESUMO
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.
Assuntos
Técnicas de Cultura de Células , Meios de Cultura/química , Sangue Fetal/química , Células-Tronco Mesenquimais , Plasma/química , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Soroalbumina BovinaRESUMO
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.
Assuntos
Temperatura Baixa , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/patologia , Hipercapnia/patologia , Ferro/farmacologia , Antígenos CD34/metabolismo , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ferritinas/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismoRESUMO
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.
Assuntos
Antígenos CD/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Receptores CXCR4/metabolismo , Aloenxertos , Animais , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
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.
Assuntos
Células Sanguíneas/metabolismo , Metabolismo Energético , Células-Tronco Hematopoéticas/metabolismo , Células da Side Population/metabolismo , Animais , Antígenos CD34/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores/metabolismo , Células Sanguíneas/transplante , Hipóxia Celular , Proliferação de Células , Células Cultivadas , Feminino , Sangue Fetal/citologia , Glucose/metabolismo , Transplante de Células-Tronco Hematopoéticas , Xenoenxertos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Mitocôndrias/metabolismo , Fenótipo , Interferência de RNA , Células da Side Population/transplante , TransfecçãoRESUMO
Albeit tyrosine kinase inhibitors anti-Abl used in Chronic Myeloid Leukemia (CML) block the deregulated activity of the Bcr-Abl tyrosine kinase and induce remission in 90% of patients, they do not eradicate immature hematopoietic compartments of leukemic stem cells. To elucidate if autophagy is important for stem cell survival and/or proliferation, we used culture in low oxygen concentration (0.1% O2 for 7 days) followed back by non-restricted O2 supply (normoxic culture) to mimic stem cell proliferation and commitment. Knockdown of Atg7 expression, a key player in autophagy, in K562 cell line inhibited autophagy compared to control cells. Upon 7 days at 0.1% O2 both K562 and K562 shATG7 cells stopped to proliferate and a similar amount of viable cells remained. Back to non-restricted O2 supply K562 cells proliferate whereas K562 shATG7 cells exhibited strong apoptosis. Using immunomagnetic sorted normal and CML CD34+ cells, we inhibited the autophagic process by lentiviral infection expressing shATG7 or using a Vps34 inhibitor. Both, normal and CML CD34+ cells either competent or deficient for autophagy stopped to proliferate in hypoxia. Surprisingly, while normal CD34+ cells proliferate back to non restricted O2 supply, the CML CD34+ cells deficient for autophagy failed to proliferate. All together, these results suggest that autophagy is required for CML CD34+ commitment while it is dispensable for normal CD34 cells.
RESUMO
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.
Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Meios de Cultura/farmacologia , Sangue Fetal/citologia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Camundongos , Camundongos SCID , Transplante HeterólogoRESUMO
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.
Assuntos
Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Interleucina-6/farmacologia , Animais , Hipóxia Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Cinética , Células Progenitoras de Megacariócitos/citologia , Células Progenitoras de Megacariócitos/efeitos dos fármacos , CamundongosRESUMO
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.
Assuntos
Sangue Fetal/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Células-Tronco/fisiologia , Adaptação Fisiológica , Animais , Antígenos CD34/metabolismo , Dióxido de Carbono/farmacologia , Proliferação de Células/efeitos dos fármacos , Sangue Fetal/citologia , Sangue Fetal/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Hipercapnia/fisiopatologia , Hipóxia/fisiopatologia , Camundongos , Oxigênio/farmacologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacosRESUMO
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).
Assuntos
Temperatura Baixa , Criopreservação/métodos , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Animais , Antígenos CD34/metabolismo , Dióxido de Carbono/análise , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Crioprotetores/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Camundongos , Camundongos SCID , Oxigênio/análise , Padrões de ReferênciaRESUMO
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.
Assuntos
Bussulfano/farmacologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Imunodeficiência Combinada Severa , Condicionamento Pré-Transplante , Animais , Peso Corporal/efeitos dos fármacos , Bussulfano/administração & dosagem , Diferenciação Celular/efeitos dos fármacos , Feminino , Sobrevivência de Enxerto , Humanos , Masculino , Camundongos , Imunodeficiência Combinada Severa/mortalidade , Imunodeficiência Combinada Severa/terapia , Fatores Sexuais , Fatores de Tempo , Transplante HeterólogoRESUMO
New adult stem cell sources, devoid of the technical/ethical/economical barriers of those presently available, would favor the ongoing development of in vitro cell engineering and transplantation. Hematopoietic transplantation opened the way to and remains the most successful cell transplantation procedure. CD34+ cells that include hematopoietic stem cells (HSCs) and hematopoietic progenitors (HPs) are presently harvested from bone marrow (BM), cord blood or peripheral blood (after being mobilized from BM). The panel of potential donors, the quantities of collected cells and some other technical/medical problems still represent limiting factors to their transplantation in some patients. Steady state peripheral blood (SSPB) contains very low frequencies of CD34+ cells. They are trapped in leukoreduction filters (LRFs), which are discarded after the preparation of therapeutic red blood cell concentrates from individual blood donations. We recently developed a procedure allowing the easy and rapid elution of CD34+ cells from LRFs and we showed that they are functionally similar to those harvested from other sources. After providing an overview of the sources, interests and limitations of therapeutic HSCs presently available, we will provide arguments based on our and others' results suggesting that SSPB could become an attractive source of HSCs for hematopoietic transplantation and of other cell types for various research/development procedures.
Assuntos
Engenharia Celular/métodos , Células-Tronco Hematopoéticas/citologia , Procedimentos de Redução de Leucócitos/instrumentação , Animais , Preservação de Sangue , Citometria de Fluxo , Células-Tronco Hematopoéticas/imunologia , Humanos , Pesquisa com Células-TroncoRESUMO
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.
Assuntos
Células-Tronco Hematopoéticas/citologia , Células da Side Population/citologia , Animais , Separação Celular , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células da Side Population/imunologia , Células da Side Population/metabolismoRESUMO
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.
Assuntos
Antígenos CD34/metabolismo , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Temperatura , Animais , Proliferação de Células , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Camundongos SCIDRESUMO
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.
Assuntos
Antígenos CD34/metabolismo , Criopreservação/métodos , Sangue Fetal/citologia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Animais , Ensaio de Unidades Formadoras de Colônias , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Camundongos SCIDRESUMO
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.
Assuntos
Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Isquemia Miocárdica/cirurgia , Análise de Variância , Animais , Hipóxia Celular/fisiologia , Modelos Animais de Doenças , Coração/fisiopatologia , Hemodinâmica , Histocitoquímica , Humanos , Células-Tronco Mesenquimais/metabolismo , Isquemia Miocárdica/fisiopatologia , Distribuição Aleatória , SuínosRESUMO
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.
Assuntos
Sangue Fetal/citologia , Transplante de Células-Tronco Hematopoéticas/métodos , Células Cultivadas , Células-Tronco Hematopoéticas/citologia , HumanosRESUMO
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.
Assuntos
Comunicação Celular , Diferenciação Celular , Proliferação de Células , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Oxigênio/metabolismo , Aldeído Desidrogenase/metabolismo , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Técnicas de Cocultura , Células-Tronco Hematopoéticas/imunologia , Humanos , Interleucina-6/metabolismo , Células-Tronco Mesenquimais/imunologia , Fatores de TempoRESUMO
We developed a clinical-scale cord blood (CB) cell ex vivo procedure to enable an extensive expansion of committed progenitors--colony-forming cells (CFCs) without impairing very primitive hematopoietic stem cells (HSCs). CD34(++) cells, selected from previously cryopreserved and thawed CB units, were cultured in two steps (diluted 1:4 after 6 days) in the presence of stem cell factor (SCF), fms-related tyrosine kinase 3 ligand (Flt-3L), megakaryocyte growth and development factor (MGDF) (100 ng/ml each), granulocyte-colony stimulating factor (G-CSF) (10 ng/ml) in HP01 serum-free medium. HSC activity was evaluated in a serial transplantation assay, by detection of human cells (CD45, CD33, CD19 and CFC of human origin) in bone marrow (BM) of primary and secondary recipient NOD/SCID mice 6-8 weeks after transplantation. A wide amplification of total cells (â¼350-fold), CD34(+) cells (â¼100-fold), and CFC (â¼130-fold) without impairing the HSC activity was obtained. The activity of a particular HSC subpopulation (SRC(CFC)) was even enhanced.Thus, an extensive ex vivo expansion of CFCs is feasible without impairing the activity of HSCs. This result was enabled by associating antioxidant power of medium with an appropriate cytokine cocktail (i.e., mimicking physiologic effects of a weak oxygenation in hematopoietic environment).
Assuntos
Antígenos CD34/metabolismo , Técnicas de Cultura de Células/métodos , Linhagem da Célula , Sangue Fetal/citologia , Células-Tronco/citologia , Animais , Contagem de Células , Movimento Celular , Proliferação de Células , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Humanos , Imunofenotipagem , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Células-Tronco , Células-Tronco/metabolismoRESUMO
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.
Assuntos
Células-Tronco Hematopoéticas/efeitos dos fármacos , Mieloma Múltiplo/sangue , Polietilenoglicóis/farmacologia , Trombopoetina/farmacologia , Animais , Antígenos CD34/análise , Divisão Celular , Células Cultivadas/efeitos dos fármacos , Meios de Cultura Livres de Soro , Sobrevivência de Enxerto , Fator Estimulador de Colônias de Granulócitos/farmacologia , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/química , Células-Tronco Hematopoéticas/citologia , Humanos , Imunofenotipagem , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fator de Células-Tronco/farmacologia , Transplante HeterólogoRESUMO
BACKGROUND: Human CD34+ cells are mandatory to study many aspects of human hematopoiesis. Their low frequency in blood or marrow and ethical reasons limit their obtainment in large quantities. Leukoreduction filters (LRFs) are discarded after preparation of red blood cells. The CD34+ cell concentration in healthy donor blood is low (1×10(3) -4×10(3) /mL), but their number trapped in one LRF after filtration of 400 to 450mL of blood is high (0.4×10(6) -1.6×10(6) ). STUDY DESIGN AND METHODS: To develop a procedure allowing obtainment of purified CD34+ cells from LRFs with a good yield, white blood cell (WBC) recoveries after a 500-mL continuous or after sequential elution (50- or 20-mL fractions) were compared. Different WBC and mononuclear cell (MNC) centrifugation methods were tested to minimize their PLT contamination before the CD34+ cell immunomagnetic selection. Cell functionality was finally analyzed under various culture conditions. RESULTS: The 20-mL back-flushing of LRFs allowed the most efficient WBC recovery. The next steps (110×g centrifugation, MNC separation on Ficoll, and washes) resulted in a cell suspension in which the lymphocyte recovery was approximately 76±10% and the PLT contamination below 1.6%. After immunomagnetic selection, 4×10(5) to 6×10(5) cells containing approximately 85% of functional CD34+ cells were obtained. CONCLUSION: This procedure allows the easy, rapid (<5hr), and efficient preparation of large quantities of CD34+ cells having functional activities similar to those of CD34+ cells from other sources. Therefore, easily available and virally safe, LRFs represent an important and regular WBC source to work with human CD34+ cells, but also with other WBC types.