Improved immunomagnetic enrichment of CD34(+) cells from umbilical cord blood using the CliniMACS cell separation system

Background aimsCD34+ enrichment from cord blood units (CBU) is used increasingly in clinical applications involving ex vivo expansion. The CliniMACS instrument from Miltenyi Biotec is a current good manufacturing practice (cGMP) immunomagnetic selection system primarily designed for processing larger numbers of cells: a standard tubing set (TS) can process a maximum of 60 billion cells, while the larger capacity tubing set (LS) will handle 120 billion cells. In comparison, most CBU contain only 1–2 billion cells, raising a question regarding the optimal tubing set for CBU CD34+ enrichment. We compared CD34+ cell recovery and overall viability after CliniMACS processing of fresh CBU with either TS or LS.MethodsForty-six freshly collected CBU (≤ 36 h) were processed for CD34+ enrichment; 22 consecutive units were selected using TS and a subsequent 24 processed with LS. Cell counts and immunophenotyping were performed pre- and post-selection to assess total nucleated cells (TNC), viability and CD34+ cell content.ResultsTwo-sample t-tests of mean CD34+ recovery and viability revealed significant differences in favor of LS (CD34+ recovery, LS = 56%, TS = 45%, P = 0.003; viability, LS = 74%, TS = 59%, P = 0.011). Stepwise linear regression, considering pre-processing unit age, viability, TNC and CD34+ purity, demonstrated statistically significant correlations only with the tubing set used and age of unit.ConclusionsFor CD34+ enrichment from fresh CBU, LS provided higher post-selection viability and more efficient recovery. In this case, a lower maximum TNC specification of TS was not predictive of better performance. The same may hold for smaller scale enrichment of other cell types with the CliniMACS instrument.     

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.     

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.     

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.     

A cross-sectional study of umbilical cord blood donor profiles and their influence on umbilical cord blood collection in a Brazilian hospital

Background aimsUmbilical cord blood (UCB) cells are a new alternative to bone marrow source for hematopoietic stem cell transplantation and their use has increased in the last decade. Thus efforts are being made to improve the umbilical cord blood unit's quality. Besides compatibility, other factors, such as the total nucleated cell (TNC) count and the percentage of CD34⁺ cells in the product, are very important for a successful transplant outcome. Our aim was to describe our donor population and assess the best cord blood collection technique at Hospital Israelita Albert Einstein's cord blood bank (São Paulo, Brazil).MethodsThis was a retrospective study in which all analyses were performed simultaneously. A Student's t-test was used for qualitative variables for non-matched samples. For qualitative analyses, we used either the chi-square test or the exact Fisher's test.ResultsThe stratification of the population characteristics allowed us to determine which ones had an impact on unit volume, TNC count and percentage of CD34⁺ cells. A significant correlation was observed between donor characteristics and the quality of UCB units as related to maternal and gestational age, type of pregnancy, route of delivery, cord blood collection technique and birth weight.ConclusionsWe found that cord blood collection technique and newborn weight were significantly correlated with the TNC content. The collection technique used at our institution significantly improved the UCB unit volume and consequently the TNC count. Some findings, such as the impact of maternal age and newborn weight, have led us to re-evaluate our protocol in order to achieve better results.     

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.     

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.     

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.     

Cryopreserved mobilized autologous blood progenitors stored for more than 2 years successfully support blood count recovery after high-dose chemotherapy

Background aimsThe ability of hematopoietic progenitor cells–apheresis (HPC-A) that have been stored for many years after cryopreservation to reconstitute hematopoiesis following high-dose chemo/radiotherapy has not been well-documented.MethodsIn this retrospective study, eight Canadian centers contributed data from 53 autologous stem cell transplants (ASCT) performed using HPC-A that had undergone long-term storage (>2 years, range 2–7 years) and 120 ASCT using HPC-A stored for <6 months (short-term storage).ResultsThe doses of nucleated and CD34⁺ cells per kilogram recipient weight were similar between the short- (mean ± SD, 4.7 ± 4.9 × 10⁸ and 6.8 ± 4.3 × 10⁶, respectively) and long- (4.0 ± 4.9 × 10⁸ and 6.1 ± 3.4 × 10⁶, respectively) term storage groups. The median days to neutrophils (absolute neutrophil count; ANC) >0.5 × 10⁹/L (median 11 days for both short- and long-term storage) and platelets >20 × 10⁹/L (median 12 and 11 for short- and long-term storage, respectively) post-ASCT were not significantly different between the two groups. When ASCT performed with <5 × 10⁶/kg CD34⁺ cells was compared there was also no difference in ANC or platelet recovery (median 12 days for both after short-term storage, and 12 and 11 days, respectively, after long-term storage). Fourteen HPC-A products stored for >5 years also showed similar count recoveries as the entire long-term storage group (median 11 days for both ANC and platelets).ConclusionsCryopreserved HPC-A can be stored for at least 5 years with no apparent loss in their ability to support hematopoietic reconstitution after high-dose chemotherapy.     

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.     

Epidermal growth factor, basic fibroblast growth factor and platelet-derived growth factor-bb can substitute for fetal bovine serum and compete with human platelet-rich plasma in the ex vivo expansion of mesenchymal stromal cells derived from adipose tissue

Background aimsHuman mesenchymal stromal cells (MSC) are multipotent cells possessing self-renewal capacity, long-term viability and multilineage potential. We analyzed the effect of four different medium supplements on the expansion and differentiation of adipose tissue-derived MSC (ADSC) in order to avoid the use of xenogeneic serum.MethodsWe compared fetal bovine serum (FBS) with 10% human platelet-rich plasma (hPRP), 3% human platelet-poor plasma (hPPP) and with a cytokine cocktail composed of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and platelet-derived growth factor-bb (PDGFbb) added to 3% hPPP. This mixture was developed testing EGF, bFGF, granulocyte–colony-stimulating factor (G-CSF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF-I), PDGFbb and transforming growth factor (TGF)-β1 added alone or in combination with hPPP.ResultsOur data demonstrate that the addition of EGF, bFGF and PDGFbb, in a medium supplemented with hPPP, obtainable from 150–200 mL whole autologous blood, supports ADSC expansion better than FBS, as confirmed by cumulative population doublings (cPD; 15.0 ± 0.5 versus 9.4 ± 2.8). The addition of human platelet-rich plasma (hPRP) further improved ADSC proliferation (cPD 20.0 ± 1.2), but the achievement of hPRP presented a major drawback, requiring 1000–1200 mL autologous or donor whole blood. The medium supplements did not influence ADSC phenotype: they expressed CD105, CD90 and CD44 lacking hematopoietic antigens. The exposure to the proposed cocktail or to hPRP increased adipogenic and osteogenic differentiation.ConclusionsThe addition of EGF, bFGF and PDGFbb to hPPP could ensure a sufficient number of ADSC for clinical applications, avoiding the use of animal serum and representing a novel approach in regenerative medicine.     

Analysis and cryopreservation of hematopoietic stem and progenitor cells from umbilical cord blood

BACKGROUND: Umbilical cord blood (UCB) is an important source of hematopoietic stem and progenitor cells (HSC/HPC) for the reconstitution of the hematopoietic system after clinical transplantation. Cryopreservation of these cells is critical for UCB banking and transplantation as well as for research applications by providing readily available specimens. The objective of this study was to optimize cryopreservation conditions for CD34+ HSC/HPC from UCB. METHODS: Cryopreservation of CD34+ HSC/HPC from UCB after mononuclear cell (MNC) preparation was tested in a research-scale setup. Experimental variations were concentration of the cryoprotectant, the protein additive and cell concentration. In addition, protocols involving slow, serial addition and removal of DMSO were compared with standard protocols (fast addition and removal of DMSO) in order to avoid osmotic stress for the cryopreserved cells. Viability and recoveries of MNC, CD34+ cells and total colony-forming units (CFU) were calculated as read-outs. In addition, sterility testing of the collected UCB units before further processing was performed. RESULTS: The optimal conditions for cryopreservation of CD34+ HPC in MNC preparations were 10% DMSO and 2% human albumin at high cell concentrations (5 x 10(7) MNC/mL) with fast addition and removal of DMSO. After cryopreservation using a computer-controlled freezer, high viabilities (89%) and recoveries for CD34+ cells (89%) as well as for CFU (88%) were observed. Microbial contamination of the collected UCB samples was reduced to a rate of 6.4%. DISCUSSION: Optimized cryopreservation conditions were developed for UCB MNC in respect of the composition of the cryosolution. In addition, our results showed that fast addition of DMSO is essential for improved cryopreservation and post-thaw quality assessment results, whereas the speed of DMSO removal after thawing has little influence on the recoveries of CD34+ cells and CFU.     

Predictive factors for poor peripheral blood stem cell mobilization and peak CD34(+) cell count to guide pre-emptive or immediate rescue mobilization

Background aimsFailure in mobilization of peripheral blood (PB) stem cells is a frequent reason for not performing hematopoietic stem cell transplantation (HSCT). Early identification of poor mobilizers could avoid repeated attempts at mobilization, with the administration of pre-emptive rescue mobilizationMethodsData from the first mobilization schedule of 397 patients referred consecutively for autologous HSCT between 2000 and 2010 were collected. Poor mobilization was defined as the collection of < 2 × 10⁶ CD34⁺cells/kg body weight (BW).ResultsThe median age was 53 years (range 4–70) and 228 (57%) were males. Diagnoses were multiple myeloma in 133 cases, non-Hodgkin's lymphoma in 114, acute myeloid leukemia or myelodysplastic syndrome in 81, Hodgkin's lymphoma in 42, solid tumors in 17 and acute lymphoblastic leukemia in 10. The mobilization regimen consisted of recombinant human granulocyte–colony-stimulating factor (G-CSF) in 346 patients (87%) and chemotherapy followed by G-CSF (C + G-CSF) in 51 (13%). Poor mobilization occurred in 105 patients (29%), without differences according to mobilization schedule. Diagnosis, previous therapy with purine analogs and three or more previous chemotherapy lines were predictive factors for poor mobilization. A CD34⁺cell count in PB > 13.8/μL was enough to ensure ≥ 2 × 10⁶ CD34⁺cells/kg, with high sensitivity (90%) and specificity (91%).ConclusionsThe prevalence of poor mobilization was high, being associated with disease type, therapy with purine analogs and multiple chemotherapy regimens. The threshold of CD34⁺ cell count in PB identified poor mobilizers, in whom the administration of immediate or pre-emptive plerixafor could be useful to avoid a second mobilization.     

GMP-manufactured density gradient media for optimized mesenchymal stromal/stem cell isolation and expansion

Background aimsBone marrow (BM) mesenchymal stromal/stem cells (MSC) are therapeutic tools in regenerative medicine and oncology. MSC isolation is often performed starting from a separation step based on research-grade 1.077 g/mL density gradient media (DGM). However, MSC clinical application should require the introduction of good manufacturing practice (GMP) reagents. We took advantage of two novel GMP DGM with densities of 1.077 and 1.073 g/mL (Ficoll-Paque? PREMIUM and Ficoll-Paque PREMIUM 1.073, respectively) to test whether these reagents could isolate MSC efficiently while simultaneously comparing their performance.MethodsBM samples were processed using either 1.077 or 1.073 g/mL GMP DGM. BM mononucleated cell (MNC) fractions were analyzed for viability, immunophenotype, clonogenic potential, ex vivo expansion and differentiation potential.ResultsNo differences were noticed in cell recovery and viability between the groups. Fluorescence-activated cell-sorting (FACS) analyzes on freshly isolated cells indicated that the 1.073 g/mL GMP DGM more efficiently depleted the CD45⁺ fraction in comparison with 1.077 GMP DGM. Moreover, in the 1.073 group, fibroblastic colony-forming units (CFU-F) were 1.5 times higher and the final MSC yield 1.8 times increased after four passages. Both reagents isolated MSC with the expected phenotype; however, 1.073-isolated MSC showed a higher expression of CD90, CD146 and GD2. Additionally, MSC from both groups were capable of fully differentiating into bone, adipose cells and cartilage.ConclusionsBoth GMP DGM enriched MSC from BM samples, suggesting that these reagents would be suitable for clinical-grade expansions. In addition, the density of 1.073 g/mL provides a significant advantage over 1.077 g/mL GMP DGM, impacting the quantity of MSC obtained and reducing the ex vivo expansion time for optimized cell-based clinical applications.     

Sphingomyelin synthase 2 over-expression induces expression of aortic inflammatory biomarkers and decreases circulating EPCs in ApoE KO mice

AimsThis study sought to assess the effect of sphingomyelin synthase 2 (SMS2) over-expression on plaque component and endothelial dysfunction in atherosclerosis.Main methodsWe generated recombinant adenovirus vectors containing human SMS2 cDNA (AdV-SMS2) or control gene GFP cDNA (AdV-GFP). Both AdVs were injected (i.v.) into ApoE KO mice to establish SMS2 over-expressing and control mice models, respectively. The mice were fed a high fat diet for 30 days. We then examined their plasma lipid levels, expression levels of aortic inflammatory biomarkers critical for the plaque's stability, and numbers of peripheral endothelial progenitor cells (EPC).Key findingsCompared with the control mice, SMS2 over-expression had significantly (1) increased aortic matrix metalloproteinase-2 (MMP-2), monocyte chemoattractant protein-1 (MCP-1), tissue factor (TF) and cyclooxygenase-2 (COX-2) mRNA levels (1.9-fold, 2.2-fold, 2.6-fold and 3.2-fold, respectively, P < 0.01) and protein levels (2.2-fold, 1.9-fold, 1.9-fold and 2.1-fold, respectively, P < 0.01); (2) increased MMP-2, COX-2 in situ expression in aortic root (2.6-fold and 2.3-fold, respectively, P < 0.01); (3) decreased aortic COX-1 mRNA levels (65%, P < 0.01) and protein levels (64%, P < 0.01); and (4) decreased CD34/KDR-positive cells (33%, P < 0.01), circulating angiogenic cells (CACs) (50%, P < 0.05), and colony forming units (CFUs) (40%, P < 0.05) in circulation.SignificanceSMS2 over-expression was probably associated with increased expression of aortic inflammatory biomarkers, as well as decreased numbers of CD34/KDR-positive cells, CACs and CFUs in circulation. Therefore, SMS2 over-expression might correlate with endothelial dysfunction and aggravate atherosclerotic plaque instability in ApoE KO mice.     

Prediction of CD34(+) cell yield in hematopoietic cell products from children by peripheral blood CD34(+) cell counts

Background aimsPeripheral blood stem cells (PBSC) are increasingly used as an alternative to bone marrow in autologous transplantations. In adult patients, the peripheral blood CD34 ⁺ cell count is a good predictor of CD34 ⁺ cell yield in apheresis. However, the determinants of stem cell yield in the pediatric population have not been well established.MethodsWe retrospectively studied 396 apheresis procedures in 301 pediatric patients. Receiver operating characteristic (ROC) curves based on pre-apheresis peripheral blood CD34 ⁺ cell counts were generated to facilitate prediction of the optimal timing of PBSC collection. The associations between CD34 ⁺ cell yield and age and mobilization regimen were analyzed.ResultsSignificant differences in CD34 ⁺ cell yield among different age groups were observed. Furthermore, higher CD34 ⁺ cell yields were obtained in patients receiving chemotherapy as part of the mobilization regimen than those without chemotherapy. A correlation was noted between the CD34 ⁺ cell yield and blood surrogate markers, including white blood cell count, absolute neutrophil count and pre-apheresis peripheral blood CD34 ⁺ cell count. Cut-off values of > 35 CD34 ⁺ cells/μL in patients < 15 years old and > 45 CD34 ⁺ cells/μL in patients ≥ 15 years old were strong predictors of an adequate PBSC collection in one apheresis session. For clinical use, ROC curves and tables were generated to assist advance planning for PBSC collection.ConclusionsThe pre-apheresis peripheral blood CD34 ⁺ cell count is most useful in predicting PBSC yield. Our new cut-off values have better operating characteristics for children than the conventional value of 20 CD34 ⁺ cells/μL used for adults.     

Megakaryocyte development is normal in mice with targeted disruption of Tescalcin

BackgroundTescalcin is an EF-hand calcium-binding protein that interacts with the Na+/H + exchanger 1 (NHE1). Levay and Slepak recently proposed a role for tescalcin in megakaryopoiesis that was independent of NHE1 activity. Their studies using K562 and HEL cell lines, and human CD34 + hematopoietic stem cells suggested an essential role for tescalcin in megakaryocyte differentiation.ObjectiveTo study the role of tescalcin in megakaryocyte development using a murine model of megakaryopoiesis.MethodsWe generated a mouse with targeted disruption of tescalcin and investigated megakaryocyte development.ResultsTescalcin-deficient mice had a normal number of megakaryocytes and platelets. The morphology, polyploidization profile, and expression of Fli-1 in bone marrow-derived megakaryocytes were also normal.ConclusionTescalcin does not appear to be necessary for normal megakaryocyte development.     

Plasma renin and aldosterone levels in obese and non-obese women with polycystic ovary syndrome

ObjetiveTo assess plasma renin and aldosterone levels in obese and non-obese women with polycystic ovary syndrome (PCOS).MethodsObese women (body mass index [BMI] > 30 kg/m2; group A, n = 34) and non-obese women (BMI < 25 kg/m2; group B, n = 13) with PCOS were selected. The control group (group C, n =47) consisted of age-matched women with regular menses and normal ultrasonographic ovaries. Luteinizing hormone, follicle-stimulating hormone, androstenedione, testosterone, sex hormone-binding globulin, serum glucose, insulin, renin, plasma renin activity, and aldosterone levels were measured.ResultsObese and non-obese women with PCOS had higher luteinizing hormone, follicle-stimulating hormone, androstenedione, testosterone, and insulin levels as compared to women in the control group (p < 0.05). Women with PCOS had significantly higher renin levels (group A: 50.2 ± 4.9 picoU/mL, group B: 39.9 ± 2.7 picoU/mL, and group C: 24.6 ± 2.6 picoU/mL), plasma renin activity (group A: 3.7 ± 0.3 ng/mL/h, group B: 3.6 ± 0.3 ng/mL/h, and group C: 2.2 ± 0.4 ng/mL/h), and aldosterone levels (group A: 31.2 ± 3.3 ng/dL, group B: 29.3 ± 2.9 ng/dL, and group C: 22.2 ± 3.9 ng/dL) as compared with controls.ConclusionSignificant differences exist in plasma renin and aldosterone levels between obese and non-obese women as compared with polycystic ovary syndrome and normal controls.