Cell recovery comparison between plasma depletion/reduction- and red cell reduction-processing of umbilical cord blood

Background aimsLimited cell dose has hampered the use of cord blood transplantation (CBT) in adults. One method of minimizing nucleated cell loss in cord blood (CB) processing is to deplete or reduce plasma but not red blood cells - plasma depletion/reduction (PDR).MethodsThe nucleated cell loss of PDR was studied, and determined to be less than 0.1% in the discarded supernatant plasma fraction in validation experiments. After testing and archival sampling, the median nucleated cell recovery for PDR processing was 90%, and median CD34⁺ cell recovery 88%. In a CB bank inventory of 12 339 products with both pre- and post-processing total nucleated cells (TNC), PDR processing resulted in median post-processing TNC recoveries of 90.0% after testing and archival samples removal. Using the same 10 CB units divided into two halves, we compared directly the recovery of PDR against hydroxyethyl starch red cell reduction (RCR) for TNC, CD34⁺ cells and colony-forming units (CFU-GM, CFU-E, CFU-GEMM and total CFU) after parallel processing. We also compared the loss of very small embryonic-like stem cells (VSEL).ResultsWe demonstrated significantly higher recoveries using PDR for TNC (124%), CD34⁺ cells (121%), CFU-GM (225%), CFU-GEMM (201%), total CFU (186%) and VSEL (187%). The proportion of high TNC products was compared between 10 912 PDR and 38 819 RCR CB products and found to be 200% higher for products that had TNC ≥150 × 10⁷ (P = 0.0001) for the PDR inventory.ConclusionsOur data indicate that PDR processing of CB provides a significantly more efficient usage of this valuable and scarce resource.     

Initial cord blood unit volume affects mononuclear cell and CD34+ cell-processing efficiency in a non-linear fashion

Background aimsUmbilical cord blood (UCB) is a source of hematopoietic stem cells that initially was used exclusively for the hematopoietic reconstitution of pediatric patients. It is now suggested for use for adults as well, a fact that increases the pressure to obtain units with high cellularity. Therefore, the optimization of UCB processing is a priority.MethodsThe present study focused on parameters influencing total nucleated cell (TNC), mononucleated cell (MNC) and CD34 ⁺ cell (CD34C) recovery after routine volume reduction of 1553 UCB units using hydroxyethyl starch-induced sedimentation with an automated device, under routine laboratory conditions.ResultsWe show that the unit volume rather than the TNC count significantly affects TNC, MNC and CD34C processing efficiency (PEf), and this in a non-linear fashion: when units were sampled according to the collection volume, including pre-loaded anticoagulant (gross volume), PEf increased up to a unit volume of 110–150 mL and decreased thereafter. Thus units with initial gross volumes < 90 mL and > 170 mL similarly exhibited a poor PEf.ConclusionsThese data identify unit gross volume as a major parameter influencing PEf and suggest that fractionation of large units should be contemplated only when the resulting volume of split units is > 90 mL.     

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.     

Intra-operative preparation of autologous bone marrow-derived CD34-enriched cellular products for cardiac therapy

Background and AimsWith the advent of regenerative therapy, there is renewed interest in the use of bone marrow as a source of adult stem and progenitor cells, including cell subsets prepared by immunomagnetic selection. Cell selection must be rapid, efficient and performed according to current good manufacturing practices. In this report we present a methodology for intra-operative preparation of CD34⁺ selected autologous bone marrow for autologous use in patients receiving coronary artery bypass grafts or left ventricular assist devices.Methods and ResultsWe developed a rapid erythrocyte depletion method using hydroxyethyl starch and low-speed centrifugation to prepare large-scale (mean 359 mL) bone marrow aspirates for separation on a Baxter Isolex 300i immunomagnetic cell separation device. CD34 recovery after erythrocyte depletion was 68.3 ± 20.2%, with an average depletion of 91.2 ± 2.8% and an average CD34 content of 0.58 ± 0.27%. After separation, CD34 purity was 64.1 ± 17.2%, with 44.3 ± 26.1% recovery and an average dose of 5.0 ± 2.7 × 10⁶ CD34⁺ cells/product. In uncomplicated cases CD34-enriched cellular products could be accessioned, prepared, tested for release and administered within 6 h. Further analysis of CD34⁺ bone marrow cells revealed a significant proportion of CD45^? CD34⁺ cells.ConclusionsIntra-operative immunomagnetic separation of CD34-enriched bone marrow is feasible using rapid low-speed Hetastarch sedimentation for erythrocyte depletion. The resulting CD34-enriched product contains CD45^? cells that may represent non-hematopoietic or very early hematopoietic stem cells that participate in tissue regeneration.     

Positive immunomagnetic CD34+ cell selection in haplo-identical transplants in β-thalassemia patients: removal of platelets using an automated system

Background aimsImmunomagnetic CD34⁺ cell selection (ICS) is utilized in autologous and allogeneic transplants. In the first case it is used to reduce the neoplastic contamination of concentrates, while in the second case it is needed to carry out a T-depletion of cell concentrates in order to reduce the incidence of graft-versus-host disease (GvHD) in patients who have undergone haplo-identical transplants.MethodsThe efficacy of CliniMACS technology, after reduction of platelet contamination, incubation of monoclonal antibodies (MAb) and successive washings of concentrates, performed in 16 ICS using the standard method without reducing platelet content, was compared with the use of the automated system CytoMate, which was carried out in 46 ICS.ResultsIn the group of ICS carried out after automatic manipulation, a significant statistical difference in purity was noted (91.39% versus 83.57, P = 0.017) compared with the group of ICS carried out with the standard procedure. The same significant difference was noted in relation to the remaining percentages of CD3⁺ and CD19⁺ cells (2.31% versus 5.68%, P = 0.012, and 1.58% versus 2.71%, P = 0.014, respectively). Recovery of CD34+ cells overlapped in the two groups (70.49% versus 68.39%, P = 0.774).ConclusionsImmunomagnetic selection carried out using the automated procedure was more efficient, producing a purer sample, more efficient T-depletion and optimal reduction of B cells, without influencing cell recovery. Furthermore, conforming to good manufacturing practice (GMP) guidelines, the entire procedure with CytoMate took place in a contamination-controlled environment.     

Lymphocyte subset analysis for the assessment of treatment-related complications after autologous stem cell transplantation in multiple myeloma

Background aimsThe aim of this study was to investigate the correlation between infused lymphocyte populations and lymphocyte subsets at engraftment, and the early clinical implications of lymphocyte subset recovery after autologous stem cell transplantation (ASCT) in multiple myeloma (MM).MethodsWe examined the lymphocyte populations of infused autografts and the lymphocyte subsets of peripheral blood at engraftment from 50 patients using flow cytometry. Each subset was grouped as low (below median) and high (above median) to examine the correlation with mucositis of grade 3 or more and the occurrence of infections and cytomegalovirus (CMV) reactivation.ResultsUsing Spearman correlation coefficients, we found that cell doses of infused CD8⁺ (P = 0.042) and CD19⁺ cells (P = 0.044) were significantly associated with the absolute lymphocyte count (ALC) at engraftment. The dose of infused CD34⁺ cells was not associated with the change of lymphocyte subsets except for an inverse correlation with CD4⁺ cells (P = 0.006). After adjusting for potential variables in univariate analysis, multivariate analyzes revealed that the lower ratio of infused CD4⁺ to CD8⁺ cells (P = 0.030) was an independent factor for severe mucositis. Of lymphocyte subsets at engraftment, a higher frequency of CD3⁺ (P = 0.024) and a lower frequency of CD56⁺ (P = 0.020) were independent predictors for infections after engraftment. A higher frequency of CD8⁺ cells (P = 0.041) and a lower ratio of CD4⁺ to CD8⁺ (P = 0.021) were independent predictors for CMV reactivation.ConclusionsOur data suggest that lymphocyte subset analysis of infused autograft and peripheral blood at engraftment may provide new predictors for early complications after ASCT in patient with MM.     

Influence of related donor age on outcomes after peripheral blood stem cell transplantation

Background aimsThe rising use of allogeneic transplantation in older recipients necessitates considering older related donors. The effect of related donor age for peripheral blood stem cell allografts (PBSC) on graft maintenance and outcomes, independent of CD34⁺cell dose, has not been well-characterized.MethodsHLA-related donors (98% siblings) underwent a uniform filgrastim-based mobilization regimen aiming to collect and infuse 5 × 10⁶ CD34⁺ cells/recipient kg. Donor and recipient age were modeled in multiple ways to account for the correlation, and outcomes reported by decade of donor age.ResultsThe median donor and recipient ages were 52 years and 54 years, respectively. The mean CD34⁺ cell dose infused was 5.6 × 10⁶ CD34⁺/kg and 75% of patients received a narrow range between 4.4 and 6.6 × 10⁶ CD34⁺ cells/kg. Neither better PBSC mobilization nor higher CD34⁺ content of allografts was significantly associated with engraftment or transplant outcomes. After adjusting for recipient age and other prognostic factors, older donor age by decade conferred a lower risk of non-relapse mortality (NRM) [hazard ratio (HR) = 0.64, 95% confidence interval (CI) 0.45–0.91, P = 0.013] and borderline improvement in overall survival (OS) (HR = 0.76, 95% CI 0.58–0.99, P = 0.045) without altering progression-free survival (PFS) (HR = 0.85, 95% CI 0.66–1.07, P = 0.18).ConclusionsOlder donor age does not worsen outcome after matched related donor PBSC transplantation in patients receiving a narrow range CD34⁺ cells. The relatively small sample size mandates that the finding of similar to improved outcomes for older related donor age must be confirmed in larger studies.     

Evaluation of mobilized peripheral stem cells according to CD34 and aldehyde dehydrogenase expression and effect of SSClo ALDHbr cells on hematopoietic recovery

Background aimsWe evaluated hematopoietic stem cells according to CD34 expression and aldehyde dehydrogenase (ALDH) activity in peripheral blood and apheresis product samples from patients after mobilization with granulocyte–colony-stimulating factor (G-CSF) alone or G-CSF after high-dose cyclophosphamide (4 g/m² once daily, intravenously on day 1). We also investigated the relationship between the number of SSC^lo CD45^dim CD34^hi cells, SSC^lo ALDH^br cells and engraftment.MethodsThirty patients (20 males and 10 females), who were candidates for autologous peripheral blood stem cell transplantation, were included in the study. Cyclophosphamide + G-CSF was used for 17 and G-CSF alone for 24 mobilizations. Primary diagnoses were multiple myeloma (n% = 14), Hodgkin's lymphoma (n% = 7), non-Hodgkin's lymphoma (n% = 2), acute myloid leukemia (n% = 2), chronic lymphocytic leukemia (n% = 1) and germ cell testis tumor (n% = 1).ResultsNumbers of SSC^lo CD45^dim CD34^hi cells and SSC^lo ALDH^br cells were highly correlated in both peripheral blood and apheresis products (P < 0.001). We could not find a relationship between the transplanted SSC^lo CD45^dim CD34^hi cell dose or SSC^lo ALDH^br cell dose and platelet or neutrophil recovery. The optimal thresholds for SSC^lo CD45^dim CD34^hi cells were 5.40 × 10⁶/kg for neutrophil recovery and 7.22 × 10⁶/kg for platelet recovery. The optimal thresholds for SSC^lo ALDH^br cells were 6.53 × 10⁶/kg for neutrophil recovery and 8.72 × 10⁶/kg platelet recovery.ConclusionsAccording to our data, numbers of SSC^lo ALDH^br cells are in very good agreement with numbers of SSC^lo CD45^dim CD34^hi cells and can be a predictor of stem cell mobilization.     

Single-platform quality control assay to quantify multipotential stromal cells in bone marrow aspirates prior to bulk manufacture or direct therapeutic use

Background aimsThe manufacture of multipotential stromal cell (MSC)-based products is costly; therefore, a rapid evaluation of bone marrow (BM) ‘quality’ with respect to MSC content is desirable. The aim of this study was to develop a rapid single-platform assay to quantify MSC in BM aspirates.MethodsAspirated MSC were enumerated using the CD45^?/low CD271^bright phenotype and AccuCheck counting beads and compared with a classic colony-forming unit–fibroblast (CFU-F) assay. The phenotype of CD45^?/low CD271^bright cells was defined using a range of MSC (CD73, CD105, CD90) and non-MSC (CD31, CD33, CD34, CD19) markers. The effect of aspirated BM volume on MSC yield was also determined.ResultsCD45^?/low CD271^bright cells had a classic MSC phenotype (CD73⁺ CD105⁺ CD90⁺ ). Their numbers correlated positively with CFU-F counted manually (R = 0.81, P < 0.001) or using automatic measurements of surface area occupied by colonies (R = 0.66, P < 0.001). Simultaneous enumeration of CD34 ⁺ cells revealed donor variability ranges compatible with standard International Society of Hematotherapy and Graft Engineering (ISHGE) protocols. Aspirating larger marrow volumes gave a significant several-fold reduction in the frequency of CFU-F and CD45^?/low CD271^bright cells per milliliter. Therefore aspirated MSC yields can be maximized through a standardized, low-volume harvesting technique.ConclusionsAbsolute quantification of CD45^?/low CD271^bright cells was found to be a reliable method of predicting CFU-F yields in BM aspirates. This rapid (< 40 min) procedure could be suitable for intra-operative quality control of BM aspirates prior to volume reduction/direct injection in orthopedics. In the production of culture-expanded MSC, this assay could be used to exclude samples containing low numbers of MSC, resulting in improved consistency and quality of manufactured MSC batches.     

CD34+ cell selection using small-volume marrow aspirates: a platform for novel cell therapies and regenerative medicine

Background aimsThis study was initiated to determine whether CD34⁺ cell selection of small-volume bone marrow (BM) samples could be performed effectively on the Isolex^® 300i Magnetic Cell Selection System^® device and whether the results obtained from these samples were comparable with results from large standard-volume samples. The impact on CD34⁺ recovery using a full versus half vial of Isolex^® CD34 reagent and the effects of shipping a post-selection product were evaluated.MethodsA protocol to evaluate CD34⁺ cell selection with two ranges of smaller volume BM samples (c. 50hmL and c. 100 mL) was developed and instituted at three Production Assistance for Cellular Therapies (PACT) facilities. The study was performed in two phases.ResultsIn phase I, the mean post-selection CD34⁺ recoveries from the two sizes of samples were 104.1% and 103.3% (smallest and largest volumes, respectively), and mean CD34⁺ recoveries were 115.6% and 88.7%, with full and half vials of reagent, respectively. Mean CD34⁺ recoveries for post-shipment smaller volume samples were 106.8% and for larger volume samples 116.4%; mean CD34⁺ recoveries were 99.9% and 127.4% for post-shipment samples processed with full and half vials of reagent, respectively. In phase II, mean CD34⁺ recovery was 76.8% for post-selection samples and 74.0% for post-shipment samples.ConclusionsThe results suggest that smaller volume BM sample processing on the Isolex^® system is as efficient or more efficient compared with standard-volume sample processing. Post-processing mean CD34⁺ recovery results obtained using a full or half vial of CD34 reagent were not significantly different.     

Effect of transcatheter renal arterial embolization combined with cryoablation on regulatory CD4+CD25+ T lymphocytes in the peripheral blood of patients with advanced renal carcinoma

ObjectiveTo analyze the effect of Argon-Helium cryosurgery (AHCS) combined with transcatheter renal arterial embolization (TRAE) on the differentiation of regulatory CD4⁺ CD25⁺ T cell (Treg) and its implication in patients with renal carcinoma.MethodsSeventy seven patients are included in the study, and divided into two groups: TRAE group (n = 45, receiving TRAE only) and TRAE + cryoablation group (n = 32, receiving cryoablation 2–3 weeks after TRAE). The percentage of Treg cells and T lymphocyte subsets (CD4⁺T, CD8⁺T, and CD4⁺T/CD8⁺T) in the peripheral blood is measured by flow cytometry previous to the therapy and 3 months after therapy. Meanwhile, the extent of tumor necrosis is measured by MRI or CT 1 month after therapy.ResultsThe percentages of Treg cells of patients in TRAE + cryoablation group decrease from (6.65 ± 1.22)% to (3.93 ± 1.16)%, (t = 42.768, P < 0.01), and the percentages of CD4⁺T and CD4⁺T/CD8⁺T increase significantly (P < 0.01). However, the results of patients in TRAE group show that the percentages of Treg, CD4⁺T, CD8⁺T and CD4⁺T/CD8⁺T increase slightly although the differences had no statistical significance (P > 0.05). The tumor necrosis rate of TRAE + cryoablation group is 57.5%, significantly higher than those of TRAE group, which shows 31.6% (t = 6.784, P < 0.01). The median survival duration of the TRAE + cryoablation group is 20 months, significantly longer than that of the TRAE group (χ² = 7.368, P < 0.01). The decreasing extent of Treg cells is correlated with tumor necrosis rates (r = 0.90, P < 0.01) and life time (r = 0.67, P < 0.01).ConclusionThe therapy of TRAE combined with cryoablation contributes to reduce the percentage of Treg cells and improve the immune situation of patients with renal cell carcinoma, which consequently increase tumor necrosis rate and prolong the patients‘ survival duration.     

Development and validation of a procedure to isolate viable bone marrow cells from the vertebrae of cadaveric organ donors for composite organ grafting

Background aimsDonor-derived vertebral bone marrow (BM) has been proposed to promote chimerism in solid organ transplantation with cadaveric organs. Reports of successful weaning from immunosuppression in patients receiving directed donor transplants in combination with donor BM or blood cells and novel peri-transplant immunosuppression has renewed interest in implementing similar protocols with cadaveric organs.MethodsWe performed six pre-clinical full-scale separations to adapt vertebral BM preparations to a good manufacturing practice (GMP) environment. Vertebral bodies L4–T8 were transported to a class 10 000 clean room, cleaned of soft tissue, divided and crushed in a prototype bone grinder. Bone fragments were irrigated with medium containing saline, albumin, DNAse and gentamicin, and strained through stainless steel sieves. Additional cells were eluted after two rounds of agitation using a prototype BM tumbler.ResultsThe majority of recovered cells (70.9 ± 14.1%, mean ± SD) were eluted directly from the crushed bone, whereas 22.3% and 5.9% were eluted after the first and second rounds of tumbling, respectively. Cells were pooled and filtered (500, 200 μm) using a BM collection kit. Larger lumbar vertebrae yielded about 1.6 times the cells of thoracic vertebrae. The average product yielded 5.2 ± 1.2 × 10¹⁰ total cells, 6.2 ± 2.2 × 10⁸ of which were CD45⁺ CD34⁺. Viability was 96.6 ± 1.9% and 99.1 ± 0.8%, respectively. Multicolor flow cytometry revealed distinct populations of CD34⁺ CD90⁺ CD117^dim hematopoietic stem cells (15.5 ± 7.5% of the CD34 ⁺ cells) and CD45^? CD73⁺ CD105⁺ mesenchymal stromal cells (0.04 ± 0.04% of the total cells).ConclusionsThis procedure can be used to prepare clinical-grade cells suitable for use in human allotransplantation in a GMP environment.     

A clinically adaptable method to enhance the cytotoxicity of natural killer cells against B-cell malignancies

Background aimsRetroviral transduction of anti-CD19 chimeric antigen receptors significantly enhances the cytotoxicity of natural killer (NK) cells against B-cell malignancies. We aimed to validate a more practical, affordable and safe method for this purpose.MethodsWe tested the expression of a receptor containing CD3ζ and 4-1BB signaling molecules (anti-CD19-BB-ζ) in human NK cells after electroporation with the corresponding mRNA using a clinical-grade electroporator. The cytotoxic capacity of the transfected NK cells was tested in vitro and in a mouse model of leukemia.ResultsMedian anti-CD19-BB-ζ expression 24 h after electroporation was 40.3% in freshly purified (n =18) and 61.3% in expanded (n = 31) NK cells; median cell viability was 90%. NK cells expressing anti-CD19-BB-ζ secreted interferon (IFN)-γ in response to CD19-positive target cells and had increased cytotoxicity. Receptor expression was detectable 6 h after electroporation, reaching maximum levels at 24–48 h; specific anti-CD19 cytotoxicity was observed at 96 h. Levels of expression and cytotoxicities were comparable with those achieved by retroviral transduction. A large-scale protocol was developed and applied to expanded NK cells (median NK cell number 2.5 × 10⁸, n = 12). Median receptor expression after 24 h was 82.0%; NK cells transfected under these conditions exerted considerable cytotoxicity in xenograft models of B-cell leukemia.ConclusionsThe method described here represents a practical way to augment the cytotoxicity of NK cells against B-cell malignancies. It has the potential to be extended to other targets beyond CD19 and should facilitate the clinical use of redirected NK cells for cancer therapy.     

CD133 immunomagnetic separation: effectiveness of the method for CD133(+) isolation from umbilical cord blood

Background aimsUmbilical cord blood (UCB) is a rich source of stem cells, the characterization and isolation of which requires specific stem cell markers and reliable and reproducible protocols.MethodsWe assessed CD133 isolation in 39 UCB samples, using a commercial immunomagnetic cell-sorting protocol, and, because of its non-reproducibility, we applied optimized protocols in an effort to improve it. These included extra-labeling of the selected CD133⁺ subpopulation and indirect labeling using anti-phycoerythrin (PE) microbeads, goat anti-mouse IgG microbeads or a combination of both. The CD34 isolation was used as a control.ResultsThe mononuclear cell fraction expressed 0.53 ± 0.06% CD133. The corresponding value for CD34 was 1.64 ± 0.15%. Following the manufacturer's instructions, the CD34 isolation resulted in a population expressing 93 ± 1.25% CD34 while, after the corresponding process, CD133⁺ expression ranged from 10% to 85% (median 60%). The optimized isolation protocols did not result in improved CD133⁺ yield. The variation in the purity of the CD133 population cannot be attributed to the different clones of CD133 used, because they do not cross-block, while other factors such as glycosylation, which could possibly interfere, do not apply in normal hematopoietic stem cells (HSC).ConclusionsCD34 isolation by the immunomagnetic method results in highly pure CD34⁺ population, while the efficient and reproducible yield of a pure CD133⁺ population is not feasible. Therefore quantification of the positive cells should follow each isolation procedure in order to confirm the number of CD133⁺ cells.     

Clinical-grade manufacturing of autologous mature mRNA-electroporated dendritic cells and safety testing in acute myeloid leukemia patients in a phase I dose-escalation clinical trial

Background aimsRNA-electroporated dendritic cell (DC)-based vaccines are rapidly gaining interest as therapeutic cancer vaccines. We report on a phase I dose-escalation trial using clinical-grade manufactured mature RNA-electroporated DC in acute myeloid leukemia (AML) patients.MethodsCD14⁺ cells were isolated from leukapheresis products by immunomagnetic CliniMACS separation and differentiated into mature DC (mDC). mDC were electroporated with clinical-grade mRNA encoding the Wilm's tumor (WT1) antigen, and tested for viability, phenotype, sterility and recovery. To test product safety, increasing doses of DC were administered intradermally four times at 2-week intervals in 10 AML patients.ResultsIn a pre-clinical phase, immunomagnetic monocyte isolation proved superior over plastic adherence in terms of DC purity and lymphocyte contamination. We also validated a simplified DC maturation protocol yielding a consistent phenotype, migration and allogeneic T-cell stimulatory capacity in AML patients in remission. In the clinical trial, highly purified CD14⁺ cells (94.5 ± 3.4%) were obtained from all patients. A monocyte-to-mDC conversion factor of 25 ± 10% was reached. All DC preparations exhibited high expression of mDC markers. Despite a decreased cell recovery of mDC after a combination of mRNA electroporation and cryopreservation, successful vaccine preparations were obtained in all AML patients. DC injections were well tolerated by all patients.ConclusionsOur method yields a standardized, simplified and reproducible preparation of multiple doses of clinical-grade mRNA-transfected DC vaccines from a single apheresis with consistent mature phenotype, recovery, sterility and viability. Intradermal injection of such DC vaccines in AML patients is safe.     

Cytokine-induced killer cells in the treatment of patients with solid carcinomas: a systematic review and pooled analysis

Background aimsThe aim of this study was to evaluate the efficacy and safety of cytokine-induced killer (CIK) cell therapy for solid carcinomas.MethodsWe performed a computerized search of phase II/III clinical trial databases of CIK cell-based therapy using a combination of the terms ‘cytokine-induced killer cells’, ‘tumor’ and ‘cancer’.ResultsTreatment with CIK cells was associated with a significantly improved half-year survival (P = 0.003), 1-year survival (P = 0.0005), 2-year survival (P  < 0.01) and mean survival time (MST) (P  < 0.001). Patients in the CIK group showed a prolonged half-year progression-free survival (PFS) (P  < 0.01), 1-year PFS (P < 0.01) and median time to progression (MTTP) (P < 0.001). A favored disease control rate (DCR) was observed in patients receiving CIK cell therapy, while the objective response rate (ORR) was not altered (P = 0.05) compared with the non-CIK group (P = 0.007). CIK cell therapy could also reduce the adverse effects of grade III and IV leukopenia caused by chemotherapy (P = 0.002) and diminish hepatitis B virus (HBV)-DNA content (P < 0.01). However, the incidence of fever in the CIK therapy group was significantly higher than in the non-CIK group (P = 0.02). The percentage of CD3⁺, CD4⁺, CD4⁺ CD8⁺, CD3^? CD56⁺ and CD3⁺ CD56⁺ T-lymphocyte subsets in the peripheral blood of cancer patients was significantly increased, whereas the percentage of CD8⁺ T-lymphocyte cells was significantly decreased in the CIK group compared with the non-CIK group (P < 0.01).ConclusionsCIK cell therapy has demonstrated a significant superiority in prolonging the MST, PFS, DCR and quality of life (QoL) of patients.     

Characterization of hematopoietic stem cell subsets from patients with multiple myeloma after mobilization with plerixafor

Background aimsPrevious studies have demonstrated that the combination of granulocyte–colony-stimulating factor (G-CSF) + plerixafor is more efficient in mobilizing CD34⁺ hematopoietic stem cells (HSC) into the peripheral blood than G-CSF alone. In this study we analyzed the impact of adding plerixafor to G-CSF upon the mobilization of different HSC subsets.MethodsWe characterized the immunophenotype of HSC subsets isolated from the peripheral blood of eight patients with multiple myeloma (MM) before and after treatment with plerixafor. All patients were supposed to collect stem cells prior to high-dose chemotherapy and consecutive autologous stem cell transplantation, and therefore received front-line mobilization with 4 days of G-CSF followed by a single dose of plerixafor. Samples of peripheral blood were analyzed comparatively by flow cytometry directly before and 12 h after administration of plerixafor.ResultsThe number of aldehyde dehydrogenase (ALDH)^bright and CD34⁺ cells was significantly higher after plerixafor treatment (1.2–5.0 and 1.5–6.0 times; both P < 0.01) and an enrichment of the very primitive CD34⁺ CD38^? and ALDH^bright CD34⁺ CD38^? HSC subsets was detectable. Additionally, two distinct ALDH⁺ subsets could be clearly distinguished. The small ALDH^high subset showed a higher number of CD34⁺ CD38^? cells in contrast to the total ALDH^bright subpopulation and probably represented a very primitive subpopulation of HSC.ConclusionsA combined staining of ALDH, CD34 and CD38 might represent a powerful tool for the identification of a very rare and primitive hematopoietic stem cell subset. The addition of plerixafor mobilized not only more CD34⁺ cells but was also able to increase the proportion of more primitive stem cell subsets.     

Lifespan of human amniotic fluid-derived multipotent mesenchymal stromal cells

Background aimsHuman multipotent mesenchymal stromal cells (hMSC) have become one of the main interests in regenerative medicine because of their ability to differentiate into different lineages. Human amniotic fluid is reported to contain MSC (hAMSC) and therefore may be a useful source of cells for clinical applications. However, our understanding of the behavior of these cells in indefinite in vitro culture conditions is very limited.MethodsWe systematically evaluated and characterized, throughout their whole lifespan, the expansion potential, chromosomal stability, surface and intracellular phenotype and differentiation potential of fibroblastoid hAMSC (F-type hAMSC).ResultsNine F-type hAMSC cultures could be expanded in in vitro culture conditions for 223.25 ± 24.44 days (mean ± SD), during which time 28.96 ± 1.5 passages were made giving rise to 54.95 ± 3.17 population doublings (PD) and an estimated number of accumulated cells of between 1.0 × 10²² and 9.7 × 10²³, with no visible alterations in the chromosome during their lifespan. All the cultures showed unchanged percentages of strongly positive expressions of the surface markers CD29, CD44, CD73, CD90, CD95, CD105 and HLA-ABC, as well as the embryonic intracellular markers Nanog and Sox2, during their lifespan, whereas the expression of the embryonic surface markers SSEA3, SSEA4, TRA-1-60 and TRA-1-81 fell until it disappeared with progression of the culture. These cells retained their differentiation capacities to adipogenic, chondrogenic and osteogenic lineages throughout their lifespan.ConclusionsF-type hAMSC exhibit reproducible biologic characteristics, confirming that these cells are ideal candidates for use in regenerative medicine.     

The use of plerixafor in hematopoietic progenitor cell collection in pediatric patients: a single center experience

Background aimsPlerixafor was recently approved for use in combination with granulocyte–colony-stimulating factor (G-CSF) for hematopoietic progenitor cell (HPC) collection by apheresis in adults with multiple myeloma (MM) or non-Hodgkin lymphoma (NHL). However, its efficacy in pediatric patients is not well-studied; thus, we report on our institutional experience with this population. Methods. A retrospective observational analysis was performed using both stem cell-processing laboratory information as well as apheresis charts and medical records on all pediatric patients who received plerixafor as part of the mobilization regimen between December 2006 and December 2010. The primary outcome was collection yield. Secondary outcomes included the ability to undergo autologous hematopoietic stem cell transplantation (auto-HSCT) and engraftment status. Results. Eighteen HPC collections by apheresis representing seven mobilization courses were performed on five pediatric patients with poor mobilization status (three males, two females; median age 14 years). Median pre-harvest peripheral blood CD34⁺ cell (PB CD34⁺) count was 6.88/μL. A strong correlation between pre-harvest PB CD34⁺ count and collection yield was observed. Median total collection yield was 2.26 × 10⁶ CD34⁺ cells/kg. Four patients achieved a minimum collection of 2 × 10⁶ CD34⁺ cells/kg. Three patients underwent auto-HSCT with a median neutrophil and platelet engraftment of 12 and 34 days, respectively. No major adverse events with plerixafor administration or apheresis collections were reported. Conclusions. Plerixafor in combination with G-CSF is a safe and potentially helpful mobilization agent in poor mobilizers. Further studies should be done to evaluate the true efficacy of plerixafor in the pediatric population.     

Interleukin (IL)-15 in combination with IL-2, fms-like tyrosine kinase-3 ligand and anti-CD3 significantly enhances umbilical cord blood natural killer (NK) cell and NK-cell subset expansion and NK function

Background aimsInterleukin (IL)-15 and fms-like tyrosine kinase-3 (FLT-3) are crucial factors for the development of human and murine natural killer (NK) cells. Previously, we have demonstrated significant ex vivo expansion and activation of unrelated cord blood (UCB) NK cells with an antibody/cytokine cocktail consisting of anti-CD3 + IL-2 + IL-12 + IL-7 and anti-CD3 + IL-2 + IL-12 + IL-18.MethodsIn the current experiments, we investigated the effects of short-term culture with anti-CD3 + IL-2 + FLT-3 + IL-15 on cord blood (CB) NK cell and NK-cell subset expansion and function. CB mononuclear cells were cultured for 48 h in AIM-V media or AIM-V + IL-2 (5 ng/mL) + anti-CD3 (50 ng/mL) + FLT-3 (50 ng/mL) ± escalating doses of IL-15 (1, 10 or 100 ng/mL). Flow cytometric analysis was performed using various fluorescent-conjugated monoclonal antibodies. In vitro cytotoxicity was determined with a standard europium assay against K562 and Daudi cells.ResultsThere was a 4.8-fold significant increase in NK-cell population (CD3^?/16⁺/56⁺; P < 0.03), 21-fold significant increase in CD3^?/56⁺/158a⁺ (KIR2DL1/S1; P < 0.002), 46-fold significant increase in CD3^?/56⁺/158b⁺ (KIR2DL1/S2; P < 0.002) and 11.5-fold significant increase in CD3^?/56⁺/NKB1⁺ (KIR3DL1; P < 0.01). We also noted a significant increase in both NK and lymphokine-activated killer (LAK) cytotoxicity with IL-2 + anti-CD3 + FLT-3 + IL-15 (100 ng/mL) compared with IL-2 + anti-CD3 + FLT-3 and media alone against K562 (P < 0.01) and Daudi (P < 0.001), respectively.ConclusionsWe have demonstrated a significant increase in UCB NK cells and NK cells expressing a variety of killer immunoglobulin-like receptor (KIR) receptors after short-term culture with anti-CD3, IL-2, FLT-3 and IL-15. Furthermore, there was a significant increase in in vitro NK/LAK cell cytotoxicity.