Hyperbaric oxygen treatment effects on in vitro cultured umbilical cord blood CD34+ cells

Background aims

Umbilical cord blood (UCB) provides an alternative source for hematopoietic stem/progenitor cells (HSPCs) in the treatment of hematological malignancies. However, clinical usage is limited due to the low quantity of HSPCs in each unit of cord blood and defects in bone marrow homing. Hyperbaric oxygen (HBO) is among the more recently explored methods used to improve UCB homing and engraftment. HBO works by lowering the host erythropoietin before UCB infusion to facilitate UCB HSPC homing, because such UCB cells are not directly exposed to HBO. In this study, we examined how direct treatment of UCB-CD34⁺ cells with HBO influences their differentiation, proliferation and in vitro transmigration.

Quality evaluation of umbilical cord blood progenitor cells cryopreserved with a small-scale automated liquid nitrogen system

The performance of a small-scale automated cryopreservation and storage system (Mini-BioArchive system) used in the banking of umbilical cord blood (UCB) units was evaluated. After thawing the units, the viability and recovery of cells, as well as the recovery rate of hematopoietic progenitor cells (HPCs) such as CD34⁺ cells, colony-forming unit-granulocyte-macrophage (CFU-GM), and total CFU were analyzed. Twenty UCB units cryopreserved using the automated system and stored for a median of 34 days were analyzed. Mean CD34⁺ cell viabilities before freezing were 99.8 ± 0.5% and after thawing were 99.8 ± 0.4% in the large bag compartments and 99.7 ± 0.5% in the small compartments. The mean recovery values for total nucleated cells, CD34⁺ cells, CFU-GM, and total CFU were 94.8 ± 16.0%, 99.3 ± 18.6%, 103.9 ± 20.6%, and 94.3 ± 12.5%, respectively in the large compartments, and 95.8 ± 25.9%, 106.8 ± 23.9%, 101.3 ± 23.3%, and 93.8 ± 19.2%, respectively in the small compartments. A small-scale automated cryopreservation and storage system did not impair the clonogenic capacity of UCB HPCs. This cryopreservation system could provide cellular products adequate for UCB banking and HPC transplantation.     

Carbohydrate-mediated inhibition of ice recrystallization in cryopreserved human umbilical cord blood

Cryopreservation of human umbilical cord blood (UCB) typically involves the cryoprotectant dimethylsulfoxide (DMSO), however, infusional toxicity and reductions in cell viability remain a concern. Ice recrystallization (IR) is an important source of cryopreservation-induced cellular injury and limits the stem cell dose in UCB units. Carbohydrates have wide-ranging intrinsic IR inhibition (IRI) activity related to structural properties. We investigated the impact of carbohydrate IRI on cell viability, induction of apoptosis and hematopoietic progenitor function in cryopreserved UCB. Mononuclear cells (MNCs) from UCB were cryopreserved in storage media containing specific carbohydrates (200 mM) and compared to 5% DMSO. Samples were analyzed under conditions of high IR (‘slow’ thaw) and low IR (‘fast’ thaw). Thawed samples were analyzed for viability and apoptosis by flow cytometry and hematopoietic function using colony-forming unit (CFU) assays. IRI of carbohydrate solutions was determined using the ‘splat cooling’ assay. Greater IRI capacity of carbohydrates correlated with increased yield of viable MNCs (r² = 0.92, p = 0.004) and CD34(+) cells (r² = 0.96, p = 0.019) after thawing under conditions of high IR. The correlations were less apparent under conditions of low IR. Carbohydrates with greater IRI modulate the induction of early apoptosis during thawing, especially in CD34+ cells (r² = 0.96, p = 0.0001) as compared to total mononuclear cells (p = 0.006), and preserve CFU capacity in vitro (r² = 0.92, p = <0.0001). Our results suggest that carbohydrates with potent IRI increase the yield of non-apoptotic and functional hematopoietic progenitors and provide a foundation for the development of novel synthetic carbohydrates with enhanced IRI properties to improve cryopreservation of UCB.     

Evaluations of bioantioxidants in cryopreservation of umbilical cord blood using natural cryoprotectants and low concentrations of dimethylsulfoxide

Transplantation using hematopoietic stem cells from umbilical cord blood (UCB) is a life-saving treatment option for patients with select oncologic diseases, immunologic diseases, bone marrow failure, and others. Often this transplant modality requires cryopreservation and storage of hematopoietic stem cells (HSC), which need to remain cryopreserved in UCB banks for possible future use. The most widely used cryoprotectant is dimethylsulfoxide (Me₂SO), but at 37 °C, it is toxic to cells and for patients, infusion of cryopreserved HSC with Me₂SO has been associated with side effects. Freezing of cells leads to chemical change of cellular components, which results in physical disruption. Reactive oxygen species (ROS) generation also has been implicated as cause of damage to cells during freezing. We assessed the ability of two bioantioxidants and two disaccharides, to enhance the cryopreservation of UCB. UCB was processed and subjected to cryopreservation in solutions containing different concentrations of Me₂SO, bioantioxidants and disaccharides. Samples were thawed, and then analysed by: flow cytometry analysis, CFU assay and MTT viability assay. In this study, our analyses showed that antioxidants, principally catalase, performed greater preservation of: CD34+ cells, CD123+ cells, colony-forming units and cell viability, all post-thawed, compared with the standard solution of cryopreservation. Our present studies show that the addition of catalase improved the cryopreservation outcome. Catalase may act on reducing levels of ROS, further indicating that accumulation of free radicals indeed leads to death in cryopreserved hematopoietic cells.     

CAPE promotes the expansion of human umbilical cord blood-derived hematopoietic stem and progenitor cells in vitro

Due to the low number of collectable stem cells from single umbilical cord blood(UCB)unit,their initial uses were limited to pediatric therapies.Clinical applications of UCB hematopoietic stem and progenitor cells(HSPCs)would become feasible if there were a culture method that can effectively expand HSPCs while maintaining their self-renewal capacity.In recent years,numerous attempts have been made to expand human UCB HSPCs in vitro.In this study,we report that caffeic acid phenethyl ester(CAPE),a small molecule from honeybee extract,can promote in vitro expansion of HSPCs.Treatment with CAPE increased the percentage of HSPCs in cultured mononuclear cells.Importantly,culture of CD34+HSPCs with CAPE resulted in a significant increase in total colony-forming units and high proliferative potential colony-forming units.Burst-forming unit-erythroid was the mostly affected colony type,which increased more than 3.7-fold in 1μg mL 1CAPE treatment group when compared to the controls.CAPE appears to induce HSPC expansion by upregulating the expression of SCF and HIF1-α.Our data suggest that CAPE may become a potent medium supplement for in vitro HSPC expansion.     

Effective expansion of umbilical cord blood hematopoietic stem/progenitor cells by regulation of microencapsulated osteoblasts under hypoxic condition

The expansion of hematopoietic stem/progenitor cells (HSPCs) from umbilical cord blood (UCB) with the support of microencapsulated osteoblasts under hypoxia environment was investigated. The expansion of HSPCs was evaluated through the total number of UCB mononuclear cells (MNCs) produced, their repopulating potential with the colony-forming unit assay (CFU-Cs) and CD34⁺ phenotypic analysis with flow cytometry. At the end of 7 days of culture, the UCB-MNCs, CFU-Cs and CD34⁺ cells had achieved 18.7 ± 1.6, 11.6 ± 0.9 and 23.4 ± 2-fold expansions, respectively, in the test groups. These were significantly different from those in control groups. Microencapsulated osteoblasts under hypoxia conditions had therefore a significant effect on the expansion potential of HSPCs in vitro.     

Gene expression profiles of cryopreserved CD34 human umbilical cord blood cells are related to their bone marrow reconstitution abilities in mouse xenografts

Human umbilical cord blood (UCB) cells are an alternative source of hematopoietic stem cells for treatment of leukemia and other diseases. It is very difficult to assess the quality of UCB cells in the clinical situation. Here, we sought to assess the quality of UCB cells by transplantation to immunodeficient mice. Cryopreserved CD34⁺ UCB cells from twelve different human donors were transplanted into sublethally irradiated NOD/shi-scid Jic mice. In parallel, the gene expression profiles of the UCB cells were determined from oligonucleotide microarrays. UCB cells from three donors failed to establish an engraftment in the host mice, while the other nine succeeded to various extents. Gene expression profiling indicated that 71 genes, including HOXB4, C/EBP-β, and ETS2, were specifically overexpressed and 23 genes were suppressed more than 2-fold in the successful UCB cells compared to those that failed. Functional annotation revealed that cell growth and cell cycle regulators were more abundant in the successful UCB cells. Our results suggest that hematopoietic ability may vary among cryopreserved UCB cells and that this ability can be distinguished by profiling expression of certain sets of genes.     

Role of Reactive Oxygen Species in the Radiation Response of Human Hematopoietic Stem/Progenitor Cells

Hematopoietic stem/progenitor cells (HSPCs), which are present in small numbers in hematopoietic tissues, can differentiate into all hematopoietic lineages and self-renew to maintain their undifferentiated phenotype. HSPCs are extremely sensitive to oxidative stressors such as anti-cancer agents, radiation, and the extensive accumulation of reactive oxygen species (ROS). The quiescence and stemness of HSPCs are maintained by the regulation of mitochondrial biogenesis, ROS, and energy homeostasis in a special microenvironment called the stem cell niche. The present study evaluated the relationship between the production of intracellular ROS and mitochondrial function during the proliferation and differentiation of X-irradiated CD34⁺ cells prepared from human placental/umbilical cord blood HSPCs. Highly purified CD34⁺ HSPCs exposed to X-rays were cultured in liquid and semi-solid medium supplemented with hematopoietic cytokines. X-irradiated CD34⁺ HSPCs treated with hematopoietic cytokines, which promote their proliferation and differentiation, exhibited dramatically suppressed cell growth and clonogenic potential. The amount of intracellular ROS in X-irradiated CD34⁺ HSPCs was significantly higher than that in non-irradiated cells during the culture period. However, neither the intracellular mitochondrial content nor the mitochondrial superoxide production was elevated in X-irradiated CD34⁺ HSPCs compared with non-irradiated cells. Radiation-induced gamma-H2AX expression was observed immediately following exposure to 4 Gy of X-rays and gradually decreased during the culture period. This study reveals that X-irradiation can increase persistent intracellular ROS in human CD34⁺ HSPCs, which may not result from mitochondrial ROS due to mitochondrial dysfunction, and indicates that substantial DNA double-strand breakage can critically reduce the stem cell function.     

The circadecadal rhythm of oscillation of umbilical cord blood parameters correlates with geomagnetic activity – An analysis of long-term measurements (1999–2011)

Recently, we have shown that the contents of total nucleated cells (TNCs) and CD34⁺ hematopoietic stem and progenitor cells (CD34⁺ HSPCs) as well as the cord blood volume (CBV) in umbilical cord blood (UCB) show a circadecadal (~10 years) rhythm of oscillation. This observation was based on an analysis of 17,936 cord blood donations collected during 1999–2011. The aim of the present study was to investigate whether this circadecadal rhythm of oscillation in TNCs, CD34⁺ HSPCs and CBV is related to geomagnetic activity. For the analysis, the yearly averages of TNCs, CD34⁺ HSPCs and CBV in UCB were correlated with geomagnetic activity (Dcx index). Our analysis revealed that (i) all three UCB parameters were statistically significantly correlated with the level of geomagnetic activity, (ii) CBV showed a linear correlation with the Dcx index (r = 0.5290), (iii) the number of TNCs and CD34⁺ HSPCs were quadratic inversely correlated with the Dcx index (r = ?0.5343 and r = ?0.7749, respectively). Furthermore, (iv) CBV and the number of TNCs were not statistically significantly correlated with the number of either modest or intense geomagnetic storms per year, but (v) the number of CD34⁺ HSPCs was statistically significantly correlated with the number of modest (r = 0.9253) as well as intense (r = 0.8683) geomagnetic storms per year. In conclusion, our study suggests that UCB parameters correlate with the state of the geomagnetic field (GMF) modulated by solar activity. Possible biophysical mechanisms underlying this observation, as well as the outcome of these findings, are discussed.     

Optimization of cryopreservation condition for hematopoietic stem cells from umbilical cord blood

The conditions for cryopreservation of CD34⁺ hematopoietic stem cells (HSC) from umbilical cord blood (UCB) were optimized with a new cryo-medium containing 10% ethylene glycol (EG) and 2% dimethyl sulfoxide (Me₂SO) using a controlled-rate freezing (CRF) method. After the cryopreservation of mononuclear cells (MNC) from UCB, recoveries of MNC, CD34⁺ cells, and total colony-forming units (CFU) were significantly improved compared to those in the control cryo-medium containing 10% Me₂SO and 2% Dextran-40 (P < 0.05). This study shows that the new cryo-medium and CRF method provide better recoveries of MNC, HSC and total CFU than the control cryo-medium and isopropylalcohol freezing (IPA) method. Therefore, this cryo-medium, combined with the CRF method, is valuable for optimizing cryopreservation conditions for HSC from UCB to obtain satisfactory HSC recovery.     

Evaluation of trehalose and sucrose as cryoprotectants for hematopoietic stem cells of umbilical cord blood

Bone marrow transplantation (BMT) is a therapeutic procedure that involves transplantation of hematopoietic stem cells (HSC). To date, there are three sources of HSC for clinical use: bone marrow; mobilized peripheral blood; and umbilical cord blood (UCB). Depending on the stem cell source or type of transplantation, these cells are cryopreserved. The most widely used cryoprotectant is dimethylsulfoxide (Me₂SO) 10% (v/v), but infusion of Me₂SO-cryopreserved cells is frequently associated with serious side effects in patients. In this study, we assessed the use of trehalose and sucrose for cryopreservation of UCB cells in combination with reduced amounts of Me₂SO. The post-thawed cells were counted and tested for viability with Trypan blue, the proportion of HSC was determined by flow cytometry, and the proportion of hematopoeitic progenitor cells was measured by a colony-forming unit (CFU) assay. A solution of 30 mmol/L trehalose with 2.5% Me₂SO (v/v) or 60 mmol/L sucrose with 5% Me₂SO (v/v) produced results similar to those for 10% (v/v) Me₂SO in terms of the clonogenic potential of progenitor cells, cell viability, and numbers of CD45⁺/34⁺ cells in post-thawed cord blood cryopreserved for a minimum of 2 weeks. Thus, cord blood, as other HSC, can be cryopreserved with 1/4 the standard Me₂SO concentration with the addition of disaccharides. The use of Me₂SO at low concentrations in the cryopreservation solution may improve the safety of hematopoietic cell transplantation by reducing the side effects on the patient.     

Reduced dimethyl sulfoxide concentrations successfully cryopreserve human hematopoietic stem cells with multi-lineage long-term engraftment ability in mice

Background aimsThe cryopreservation of hematopoietic stem cells (HSCs) in dimethyl sulfoxide (DMSO) is used widely, but DMSO toxicity in transplant patients and the effects of DMSO on the normal function of cryopreserved cells are concerns. To address these issues, in vitro and clinical studies have explored using reduced concentrations of DMSO for cryopreservation. However, the effect of reducing DMSO concentration on the efficient cryopreservation of HSCs has not been directly measured.MethodsCryopreservation of human bone marrow using 10%, 7.5% and 5% DMSO concentrations was examined. Cell counting, flow cytometry and colony assays were used to analyze different cell populations. The recovery of stem cells was enumerated using extreme limiting dilution analysis of long-term multi-lineage engraftment in immunodeficient mice. Four different methods of analyzing human engraftment were compared to ascertain stem cell engraftment: (i) engraftment of CD33⁺ myeloid, CD19⁺ B-lymphoid, CD235a⁺ erythroid and CD34⁺ progenitors; (ii) engraftment of the same four populations plus CD41⁺CD42b⁺ platelets; (iii) engraftment of CD34⁺⁺CD133⁺ cells; and (iv) engraftment of CD34⁺⁺CD38⁻ cells.ResultsHematopoietic colony-forming, CD34^++/+, CD34⁺⁺CD133⁺ and CD34⁺⁺CD38⁻ cells were as well preserved with 5% DMSO as they were with the higher concentrations tested. The estimates of stem cell frequencies made in the xenogeneic transplant model did not show any significant detrimental effect of using lower concentrations of DMSO. Comparison of the different methods of gauging stem cell engraftment in mice led to different estimates of stem cell numbers, but overall, all measures found that reduced concentrations of DMSO supported the cryopreservation of HSCs.ConclusionCryopreservation of HSCs in DMSO concentrations as low as 5% is effective.     

Cryopreservation of human fetal liver hematopoietic stem/progenitor cells using sucrose as an additive to the cryoprotective medium

IntroductionHuman fetal liver (HFL) is a valuable source of hematopoietic stem/progenitor cells (HSCs) for the treatment of various hematological disorders. This study describes the effect of sucrose addition to a cryoprotective medium in order to reduce the Me₂SO concentration during cryopreservation of HFL hematopoietic cell preparations.MethodsHuman fetal liver (HFL) cells of 8–12 weeks of gestation were cryopreserved with a cooling rate of 1 °C/min down to −80 °C and stored in liquid nitrogen. The cryoprotectant solutions contained 2% or 5% Me₂SO (v/v) with or without sucrose at a final concentration of 0.05, 0.1, 0.2 or 0.3 M. The metabolic activity of HFL cells was determined using the alamar blue assay. For the determination of the number and survival of hematopoietic progenitors present, cells were stained with CD34 (FITC) and 7-AAD, and analyzed by flow cytometry. The colony-forming activity of HFL hematopoietic stem/progenitor cells after cryopreservation was assessed in semisolid methylcellulose.ResultsThe addition of sucrose to the cryoprotective medium produced a significant reduction in HFL cell loss during cryopreservation. The metabolic activity of HFL cells, cryopreserved with 5% Me₂SO/0.3 M sucrose mixture was comparable to cryopreservation in 5% Me₂SO/10% FCS. Although the inclusion of sucrose did not affect the survival of CD34⁺ cells in HFL after cryopreservation it did improve the functional capacity of hematopoietic stem/progenitor cells.ConclusionThe inclusion of sucrose as an additive to cryoprotective media for HFL cells enables a reduction in the concentration of Me₂SO, replacing serum and increasing the efficiency of cryopreservation.     

Increased apoptosis in cryopreserved autologous hematopoietic progenitor cells collected by apheresis and delayed neutrophil recovery after transplantation: a nested case-control study

Background aimsDelayed neutrophil recovery following autologous hematopoietic stem cell transplantation (aHSCT) increases transplant-related morbidity. Apoptosis induced by cryopreservation and thawing of hematopoietic progenitor cells collected by apheresis (HPC-A) was investigated in this nested case-control study as a factor associated with delayed neutrophil recovery following aHSCT.MethodsAmong patients with lymphoma who underwent aHSCT between 2000 and 2007 (n = 326), 13 cases of primary delayed neutrophil recovery and 22 age- and sex-matched controls were identified. Apoptosis and viability were measured using multiparameter flow cytometry, and colony-forming capacity was determined using semi-solid methylcellulose assays.ResultsHPC-A grafts from cases and controls had similar percentages of viable mononuclear cells (MNC) and CD34⁺progenitor cells, as determined by standard 7AAD dye exclusion methods measured before and after cryopreservation. Patients with delayed neutrophil recovery received increased numbers of apoptotic MNC (P = 0.02) but similar numbers of apoptotic CD34⁺ cells per kilogram measured after thawing. Apoptosis was more pronounced in MNC compared with CD34⁺ cells after thawing, and apoptosis was negligible in freshly collected HPC-A products. Patients with delayed neutrophil recovery had fewer total colony-forming unites (CFU) and CFU-granulocyte–macrophages (GM) per 10⁵ viable post-thaw MNC compared with controls (P < 0.05).ConclusionsIncreased numbers of apoptotic MNC in thawed HPC-A products are associated with delayed neutrophil recovery after aHSCT. Studies that address factors contributing to increased apoptosis are needed, and measuring apoptosis in thawed HPC-A may have a role in the assessment of graft adequacy.     

Effects of long-term cryopreservation on peripheral blood progenitor cells

Background aimsThe long-term stability of cryopreserved peripheral blood progenitor cells is an important issue for patients experiencing disease relapse. However, there is no consensus on how to evaluate the long-term effects of cryopreservation. We describe the effect of cryopreservation on viability and progenitor colony activity from 87 individual samples processed at the Scripps Green Hospital Stem Cell Processing Center (La Jolla, CA, USA).MethodsWe randomly selected 87 peripheral blood hematopoietic stem cell (PBHSC) samples from 60 patients and evaluated the effect of cryopreservation on sample viability and red and white cell colony activity after < 24 h and 7, 10 and 15 years of cryopreservation. Viability was assayed via trypan blue dye exclusion and activity was measured following 14 days of culture.ResultsAn age at collection older than 50 years may result in suboptimal activity and viability following long-term cryopreservation, while gender and disease status had no effect. Cryopreservation did not significantly affect white or red cell activity following 10 years of cryopreservation. However, for samples stored longer than 10 years, viability and activity significantly decreased. We noted a positive association between higher pre-cryopreservation %CD34 count and colony activity.ConclusionsCryopreservation of peripheral blood progenitor cells for up to 10 years results in no loss of clonogenic capacity, as determined by culture activity, although longer durations of storage may affect activity. Until validated methods are developed, cryopreserved grafts should be evaluated based on pre-freeze CD34⁺ cell counts as assayed by flow cytometry, and post-thaw sample evaluation should be reserved for patients identified as poor mobilizers.     

Fucosylation with fucosyltransferase VI or fucosyltransferase VII improves cord blood engraftment

Background aimsAdvantages associated with the use of cord blood (CB) transplantation include the availability of cryopreserved units, ethnic diversity and lower incidence of graft-versus-host disease compared with bone marrow or mobilized peripheral blood. However, poor engraftment remains a major obstacle. We and others have found that ex vivo fucosylation can enhance engraftment in murine models, and now ex vivo treatment of CB with fucosyltransferase (FT) VI before transplantation is under clinical evaluation (NCT01471067). However, FTVII appears to be more relevant to hematopoietic cells and may alter acceptor substrate diversity. The present study compared the ability of FTVI and FTVII to improve the rapidity, magnitude, multi-lineage and multi-tissue engraftment of human CB hematopoietic stem and progenitor cells (HSPCs) in vivo.MethodsCD34-selected CB HSPCs were treated with recombinant FTVI, FTVII or mock control and then injected into immunodeficient mice and monitored for multi-lineage and multi-tissue engraftment.ResultsBoth FTVI and FTVII fucosylated CB CD34⁺ cells in vitro, and both led to enhanced rates and magnitudes of engraftment compared with untreated CB CD34⁺ cells in vivo. Engraftment after treatment with either FT was robust at multiple time points and in multiple tissues with similar multi-lineage potential. In contrast, only FTVII was able to fucosylate T and B lymphocytes.ConclusionsAlthough FTVI and FTVII were found to be similarly able to fucosylate and enhance the engraftment of CB CD34⁺ cells, differences in their ability to fucosylate lymphocytes may modulate graft-versus-tumor or graft-versus-host effects and may allow further optimization of CB transplantation.     

Generation of Dendritic Cells from Fresh and Frozen Cord Blood CD34Cells

Dendritic cells (DCs) are professional antigen-presenting cells that are required for the initiation of the immune response. DCs have been shown to be generated from CD34⁺pluripotent hematopoietic progenitor cells in the bone marrow and cord blood (CB), but relatively little is known about the effect of cryopreservation on functional maturation of DCs from hematopoietic stem cells. In this work we report the generation of DCs from cryopreserved CB CD34⁺cells. CB CD34⁺cells were cryopreserved at −80°C for 2 days. Cryopreserved CB CD34⁺cells as well as freshly isolated CB CD34⁺cells cultured with granulocyte—macrophage colony-stimulating factor (GM-CSF)/stem cell factor (SCF)/tumor necrosis factor-α (TNF-α) for 14 days gave rise to CD1a⁺/CD4⁺/CD11c⁺/CD14⁻/CD40⁺/CD80⁺/CD83⁺/CD86⁺/HLA-DR⁺cells with dendritic morphology. DCs derived from cryopreserved CB CD34⁺cells showed a similar endocytic capacity for fluorescein isothiocyanate-labeled dextran and lucifer yellow when compared with DCs derived from freshly isolated CB CD34⁺cells. Flow cytometric analysis revealed that two CC chemokine receptors (CCRs), CCR-1 and CCR-3, were expressed on the cell surface of DCs derived from both cryopreserved and freshly isolated CB CD34⁺cells, and these DCs exhibited similar chemotactic migratory capacities in response to regulated on activation normal T-cell expressed and secreted. DCs derived from cryopreserved as well as freshly isolated CB CD34⁺cells were more efficient than peripheral blood mononuclear cells in the primary allogeneic T-cell response. These results indicate that frozen CB CD34⁺cells cultured with GM-CSF/TNF-α/SCF gave rise to dendritic cells which were morphologically, phenotypically and functionally similar to DCs derived from fresh CB CD34⁺cells.