Differences amid bone marrow and cord blood hematopoietic stem/progenitor cell division kinetics

Human hematopoietic stem/progenitor cells (HSC) isolated based upon specific patterns of CD34 and CD38 expression, despite phenotypically identical, were found to be functionally heterogeneous, raising the possibility that reversible expression of these antigens may occur during cellular activation and/or proliferation. In these studies, we combined PKH67 tracking with CD34/CD38 immunostaining to compare cell division kinetics between human bone marrow (BM) and cord blood (CB)‐derived HSC expanded in a serum‐free/stromal‐based system for 14 days (d), and correlated CD34 and CD38 expression with the cell divisional history. CB cells began dividing 24 h earlier than BM cells, and significantly higher numbers underwent mitosis during the time in culture. By d10, over 55% of the CB‐cells reached the ninth generation, whereas BM‐cells were mostly distributed between the fifth and seventh generation. By d14, all CB cells had undergone multiple cell divisions, while 0.7–3.8% of BM CD34⁺ cells remained quiescent. Furthermore, the percentage of BM cells expressing CD34 decreased from 60.8 ± 6.3% to 30.6 ± 6.7% prior to initiating division, suggesting that downmodulation of this antigen occurred before commencement of proliferation. Moreover, with BM, all primitive CD34⁺CD38^? cells present at the end of culture arose from proliferating CD34⁺CD38⁺ cells that downregulated CD38 expression, while in CB, a CD34⁺CD38^? population was maintained throughout culture. These studies show that BM and CB cells differ significantly in cell division kinetics and expression of CD34 and CD38, and that the inherent modulation of these antigens during ex vivo expansion may lead to erroneous quantification of the stem cell content of the expanded graft. J. Cell. Physiol. 220: 102–111, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

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.     

Influence of a dual-injection regimen,plerixafor and CXCR4 on in utero hematopoietic stem cell transplantation and engraftment with use of the sheep model

Background aimsInadequate engraftment of hematopoietic stem cells (HSCs) after in utero HSC transplantation (IUHSCT) remains a major obstacle for the prenatal correction of numerous hereditary disorders. HSCs express CXCR4 receptors that allow homing and engraftment in response to stromal-derived factor 1 (SDF-1) ligand present in the bone marrow stromal niche. Plerixafor, a mobilization drug, works through the interruption of the CXCR4-SDF-1 axis.MethodsWe used the fetal sheep large-animal model to test our hypotheses that (i) by administering plerixafor in utero before performing IUHSCT to release fetal HSCs and thus vacating recipient HSC niches, (ii) by using human mesenchymal stromal/stem cells (MSCs) to immunomodulate and humanize the fetal BM niches and (iii) by increasing the CXCR4⁺ fraction of CD34⁺ HSCs, we could improve engraftment. Human cord blood-derived CD34⁺ cells and human bone marrow-derived MSCs were used for these studies.ResultsWhen MSCs were transplanted 1 week before CD34⁺ cells with plerixafor treatment, we observed 2.80% donor hematopoietic engraftment. Combination of this regimen with additional CD34⁺ cells at the time of MSC infusion increased engraftment levels to 8.77%. Next, increasing the fraction of CXCR4⁺ cells in the CD34⁺ population albeit transplanting at a late gestation age was not beneficial. Our results show engraftment of both lymphoid and myeloid lineages.ConclusionsPrior MSC and HSC cotransplantation followed by manipulation of the CXCR4–SDF-1 axis in IUHSCT provides an innovative conceptual approach for conferring competitive advantage to donor HSCs. Our novel approach could provide a clinically relevant approach for enhancing engraftment early in the fetus.     

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.     

Comparison of two single-platform ISHAGE-based CD34 enumeration protocols on BD FACSCalibur and FACSCanto flow cytometers

Background aimsEnumeration of viable CD34⁺ cells provides critical information for the bone marrow (BM) transplant physician. The single-platform ISHAGE protocol is the most reliable method currently available to quantitate accurately this important subset of cells. Previous studies have shown that 5 CD34⁺ cells/µL blood predicts the collection of at least 0.5 × 10⁶ CD34⁺ cells/kg patient weight. From the apheresis product, infusion of 2.5 × 10⁶ viable CD34⁺ cells (measured pre-cryopreservation)/kg patient weight will reliably permit engraftment of the hematopoietic system (as measured by the time to 20000 platelets/µL) by day 12–14 post-infusion.MethodsWe compared the CD34⁺ cell numbers derived from Flow Count-based Stem-Kit?; (Beckman Coulter) and Trucount? tube-based stem cell enumeration (SCE) kit (BD Biosciences) ISHAGE templates on BD FACSCalibur? and BD FACSCanto? cytometers on 12 granulocyte–colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) and 10 peripheral blood stem cell (PBSC) samples.ResultsComparison of results showed that there was no statistical difference between samples run with Stem-Kit on the FACSCalibur versus SCE kit-based assays on either the FACSCalibur or FACSCanto. Mean results for the Stem-Kit/Calibur combination were 137, for SCE kit/Calibur 140 and for SCE kit/Canto 137 cells/µL. Pair-wise comparison of data based on rank order showed no statistically significant difference and all correlation coefficients had an R²>0.98.ConclusionsThe two kits generated very similar data on a range of fresh samples regardless of instrument platform. These results confirm and extend the utility of the single-platform ISHAGE protocols with a variety of reagent kits and instrument platforms.     

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.     

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.     

Validation of a formula for predicting daily CD34(+) cell collection by leukapheresis

Background aimsThe ability to predict how many CD34⁺ cells a donor will collect on a given day is vital for efficient leukapheresis.MethodsWe validated a formula to predict daily CD34⁺ cell collections by leukapheresis, calculated as follows: (peripheral blood CD34⁺ cells/L) × (adjusted collection efficiency of 30%)/body weight (kg), multiplied by the number of liters processed. This validation was performed from 234 donors undergoing 30 L large volume leukapheresis (LVL) and 162 donors undergoing smaller collections (non-LVL). The LVL group consisted of 811 collection events (625 multiple myeloma, 186 non-myeloma). The non-LVL group consisted of 224 collection events (196 multiple myeloma, 28 non-myeloma). All predicted and observed CD34⁺ cell collection numbers were plotted (predicted versus observed) and assessed using linear regression analyses. Linear correlation coefficients (r-values), slopes and intercepts of the regression lines were evaluated.ResultsPredicted versus observed data points across all quantities of CD34⁺ cells/kg collected by both LVL and non-LVL had strong r-values of 0.947 and 0.913, respectively, demonstrating near perfect positive linear correlations. Data for LVL collections subgrouped by number of cells collected (poor, intermediate and good), mobilization regimen, collection day and diagnosis were analyzed the same way and showed consistent findings.ConclusionsWe have validated a formula with a strong ability to predict collection of CD34⁺ cells/kg that would allow for individualization of collection for any donor once the peripheral blood CD34⁺ cell count and optimal goal of collection were known; to date this has not been published by other groups.     

Ex vivo-expanded bone marrow CD34(+) for acute myocardial infarction treatment: in vitro and in vivo studies

Background aimsBone marrow (BM)-derived cells appear to be a promising therapeutic source for the treatment of acute myocardial infarction (AMI). However, the quantity and quality of the cells to be used, along with the appropriate time of administration, still need to be defined. We thus investigated the use of BM CD34⁺-derived cells as cells suitable for a cell therapy protocol (CTP) in the treatment of experimental AMI.MethodsThe need for a large number of cells was satisfied by the use of a previously established protocol allowing the expansion of human CD34⁺ cells isolated from neonatal and adult hematopoietic tissues. We evaluated gene expression, endothelial differentiation potential and cytokine release by BM-derived cells during in vitro culture. Basal and expanded CD34⁺ cells were used as a delivery product in a murine AMI model consisting of a coronary artery ligation (CAL). Cardiac function recovery was evaluated after injecting basal or expanded cells.ResultsGene expression analysis of in vitro-expanded cells revealed that endothelial markers were up-regulated during culture. Moreover, expanded cells generated a CD14⁺ subpopulation able to differentiate efficiently into VE-cadherin-expressing cells. In vivo, we observed a cardiac function recovery in mice sequentially treated with basal and expanded cells injected 4 h and 7 days after CAL, respectively.ConclusionsOur data suggest that combining basal and expanded BM-derived CD34⁺ cells in a specific temporal pattern of administration might represent a promising strategy for a successful cell-based therapy.     

Evaluation of methods for cultivating limbal mesenchymal stromal cells

Background aimsMesenchymal stromal cells (MSC) with similar properties to bone marrow-derived mesenchymal stromal cells (BM-MSC) have recently been grown from the limbus of the human cornea. We have evaluated methods for culturing human limbal MSC (L-MSC).MethodsFour basic strategies were compared: serum-supplemented medium (10% fetal bovine serum; FBS), standard serum-free medium supplemented with B-27, epidermal growth factor and fibroblast growth factor 2, or one of two commercial serum-free media, defined keratinocyte serum-free medium (Invitrogen) and MesenCult-XF® (Stem Cell Technologies). The resulting cultures were examined using photography, flow cytometry (for CD34, CD45, CD73, CD90, CD105, CD141 and CD271), immunocytochemistry (alpha-smooth muscle actin; α-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis) and co-culture experiments with human limbal epithelial (HLE) cells.ResultsWhile all techniques supported the establishment of cultures to varying degrees, sustained growth and serial propagation were only achieved in 10% FBS medium or MesenCult-XF medium. Cultures established in 10% FBS medium were 70–80% CD34^? CD45^? CD90⁺ CD73⁺ CD105⁺, approximately 25% α-sma⁺ and displayed multipotency. Cultures established in MesenCult-XF were > 95% CD34^? CD45^? CD90⁺ CD73⁺ CD105⁺, 40% CD141⁺, rarely expressed α-sma, and displayed multipotency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of MesenCult-XF-grown L-MSC. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker ?Np63, along with the corneal differentiation marker cytokeratin 3.ConclusionsMesenCult-XF is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells.     

Cryopreservation and thawing of hematopoietic stem cell CD34-induced apoptosis through caspase pathway activation: Key role of granulocytes

IntroductionCell damage inescapably occurs during both the freezing and the thawing graft processes for autologous hematopoietic stem cell (HSC) transplantation. To estimate HSC injury, a quality control is performed including: (i) CD34⁺ quantification; (ii) percentage of CD34⁺ viability and (iii) evaluation of HSC functional ability to form colony forming unit–granulocyte macrophage (CFU-GM). Apoptosis involves complex pathways such as caspase enzymes. Here, we assess the extent of apoptosis that is caspase-dependent before and after cryoconservation of CD34⁺, using a Fluorescent Labeled Inhibitor of CAspases (FLICA).MethodsCaspase pathway activation status was evaluated in 46 patients (multiple myeloma [n = 24], lymphoma [n = 22]), by flow cytometry, using a 7-aminoactinomycin-D (7AAD)/FLICA staining test, in CD34⁺, CD3⁺, CD14⁺ and CD56⁺ cells. Viable 7AAD^?/FLICA⁺ cells were then correlated with various parameters.ResultsWe showed a significant caspase pathway activation, with 23% CD34⁺/7AAD^?/FLICA⁺ cells after thawing, compared with the 2% described in fresh CD34⁺ cells (P < 0.0001). Moreover, caspase pathway was significantly activated in thawing CD3⁺, CD56⁺ and CD14⁺ cells. We also report a significant correlation between the rate of CD34⁺/7AAD^?/FLICA⁺ cells and post-thawing granulocytes count (P = 0.042) and their potential to be differentiated into CFU-GM (P = 0.004).DiscussionOur results show substantial cell death, induced by the increase of caspase pathway activation, secondary to the thawing process, and across all study cell types. This observation may affect the immune response quality during recipient aplasia, without detecting a clinical impact. Moreover, caspase pathway activation through CD3⁺ and CD56⁺ subpopulations could modify the therapeutic result of donor lymphocytes infusion (DLI).     

Aging impairs human bone marrow function and cardiac repair following myocardial infarction in a humanized chimeric mouse

Ventricular remodeling following myocardial infarction (MI) is a major cause of heart failure, a condition prevalent in older individuals. Following MI, immune cells are mobilized to the myocardium from peripheral lymphoid organs and play an active role in orchestrating repair. While the effect of aging on mouse bone marrow (BM) has been studied, less is known about how aging affects human BM cells and their ability to regulate repair processes. In this study, we investigate the effect aging has on human BM cell responses post‐MI using a humanized chimeric mouse model. BM samples were collected from middle aged (mean age 56.4 ± 0.97) and old (mean age 72.7 ± 0.59) patients undergoing cardiac surgery, CD34^+/− cells were isolated, and NOD‐scid‐IL2rγ^null (NSG) mice were reconstituted. Three months following reconstitution, the animals were examined at baseline or subjected to coronary artery ligation (MI). Younger patient cells exhibited greater repopulation capacity in the BM, blood, and spleen as well as greater lymphoid cell production. Following MI, CD34⁺ cell age impacted donor and host cellular responses. Mice reconstituted with younger CD34⁺ cells exhibited greater human CD45⁺ recruitment to the heart compared to mice reconstituted with old cells. Increased cellular responses were primarily driven by T‐cell recruitment, and these changes corresponded with greater human IFNy levels and reduced mouse IL‐1β in the heart. Age‐dependent changes in BM function led to significantly lower survival, increased infarct expansion, impaired host cell responses, and reduced function by 4w post‐MI. In contrast, younger CD34⁺ cells helped to limit remodeling and preserve function post‐MI.     

Granulocyte-macrophage colony-stimulating factor increases the proportion of circulating dendritic cells after autologous but not after allogeneic hematopoietic stem cell transplantation

Background aimsGranulocyte–macrophage (GM) colony-stimulating factor (CSF) has been used as an adjuvant in cancer immunotherapy. We tested the hypothesis that GM-CSF (Leukine^®; sargramostim) improves immune reconstitution after hematopoietic stem cell transplantation (HSCT) based on our prior in vitro work that demonstrated the pro-inflammatory effects of GM-CSF on dendritic cells (DC).MethodsGM-CSF was administered to donors, along with standard granulocyte (G) CSF, during stem cell mobilization, and to recipients from the day prior to transplant until engraftment. Eighteen patients consented to the GM-CSF⁺ protocol and were compared with 17 matched controls undergoing HSCT during the same time period (GM-CSF^?).ResultsNumbers of white blood cells (WBC) and CD34⁺ stem cells in the graft were comparable to controls. Surprisingly, contrary to our hypothesis, the allogeneic donor graft had significantly decreased numbers of CD3⁺ T cells and their subsets (CD4⁺, CD4⁺ CD45RA⁺, CD4⁺ CD45RO⁺, CD8⁺ and CD8⁺ CD45RO⁺), DC (both myeloid and plasmacytoid) and natural killer (NK) cells (CD16⁺ CD56⁺). In the GM-CSF arm, following allogeneic transplantation, the levels of DC, T cells and NK cells did not increase with treatment. Conversely, autologous transplant patients receiving GM-CSF had a higher proportion of DC at the time of engraftment.ConclusionsThese findings demonstrate that administration of GM-CSF improves DC reconstitution after autologous rather than allogeneic HSCT.     

Multi-site evaluation of the BD Stem Cell Enumeration Kit for CD34+ cell enumeration on the BD FACSCanto II and BD FACSCalibur flow cytometers

Background aimsEvaluation of the BD Stem Cell Enumeration Kit was conducted at four clinical sites with flow cytometry CD34⁺ enumeration to assess agreement between two investigational methods: (i) the BD FACSCanto II and BD FACSCalibur systems and (ii) the predicate method (Beckman Coulter StemKit and StemTrol, Immunotech SAS, Beckman Coulter, Marseille Cedex 9, France).MethodsLeftover and delinked specimens (n = 1032) from clinical flow cytometry testing were analyzed on the BD FACSCanto II (n = 918) and BD FACSCalibur (n = 905) in normal and mobilized blood, frozen and thawed bone marrow and leucopheresis and cord blood anticoagulated with citrate phosphate dextrose, anticoagulant citrate dextrose—solution A, heparin and ethylenediaminetetraacetate, alone or in combination. Fresh leucopheresis analysis addressed site equivalency for sample preparation, testing and analysis.ResultsThe mean relative bias showed agreement within predefined parameters for the BD FACSCanto II (−2.81 to 4.31 ±7.1) and BD FACSCalibur (−2.69 to 5.2 ±7.9). Results are reported as absolute and relative differences compared with the predicate for viable CD34⁺, percentage of CD34⁺ in CD45⁺ and viable CD45⁺ populations (or gates). Bias analyses of the distribution of the predicate low, mid and high bin values were done using BD FACSCanto II optimal gating and BD FACSCalibur manual gating for viable CD34⁺, percentage of CD34⁺ in CD45⁺ and viable CD45⁺. Bias results from both investigational methods show agreement. Deming regression analyses showed a linear relationship with R² > 0.92 for both investigational methods.DiscussionIn conclusion, the results from both investigational methods demonstrated agreement and equivalence with the predicate method for enumeration of absolute viable CD34⁺, percentage of viable CD34⁺ in CD45⁺ and absolute viable CD45⁺ populations.     

The Use of MAGE C1 and Flow Cytometry to Determine the Malignant Cell Type in Multiple Myeloma

The malignant cell phenotype of Multiple Myeloma (MM) remains unclear with studies proposing it to be either clonotypic B or proliferating plasma cells. Cancer/testis antigen MAGE C1 is being extensively studied in MM and it has been suggested that it is involved in the pathogenesis of the cancer. Therefore, we report on the use of MAGE C1 to determine the malignant cell phenotype in MM using flow cytometry. Bone marrow aspirate (BM) and peripheral blood (PB) was collected from twelve MM patients at diagnosis, as well as three MM disease-free controls. Mononuclear cells were isolated using density-gradient centrifugation, and stabilized in 80% ethanol, before analysis via flow cytometry using relevant antibodies against B cell development cell-surface markers and nuclear MAGE C1. MAGE C1 expression was observed consistently in the early stem cells (CD34⁺) and early pro-B to pre-B cells (CD34^+/−/CD19⁺), as well as the proliferating plasma cells in both the MM PB and BM, while no expression was observed in the corresponding control samples. Monoclonality indicated a common origin of these cell types suggesting that the CD34⁺/MAGE C1⁺ are the primary malignant cell phenotype that sustains the downstream B cell maturation processes. Furthermore, this malignant cell phenotype was not restricted to the BM but also found in the circulating PB cells.     

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 blood CD34+ cells from patients with severe coronary artery disease as a source of endothelial progenitor cells

Background aimsThe distinction between hematopoietic stem cells (HSC) and endothelial progenitor cells (EPC) is poorly defined. Co-expression of CD34 antigen with vascular endothelial growth factor (VEGF) receptor (VEGFR2) is currently used to define EPC (1).MethodsWe evaluated the phenotypic and genomic characteristics of peripheral blood-derived CD34⁺ cells in 22 granulocyte–colony-stimulating factor (G-CSF)-mobilized patients with severe coronary artery disease and assessed the influence of cell selection and storage on CD34⁺ cell characteristics.ResultsThe median CD34⁺ cell contents in the products before and after enrichment with the Isolex 300i Magnetic Cell Selection System were 0.2% and 82.5%, respectively. Cell-cycle analysis showed that 80% of CD34⁺ cells were in G0 stage; 70% of the isolated CD34⁺ cells co-expressed CD133, a marker for more immature progenitors. However, less than 5% of the isolated CD34⁺ cells co-expressed the notch receptor Jagged-1 (CD339) and only 2% of the isolated CD34⁺ population were positive for VEGFR2 (CD309). Molecular assessment of the isolated CD34⁺ cells demonstrated extremely low expression of VEGFR2 and endothelial nitric oxide synthase (eNOS) and high expression of VEGF-A. Overnight storage at 4°C did not significantly affect CD34⁺ cell counts and viability. Storage in liquid nitrogen for 7 weeks did not affect the percentage of CD34⁺ cells but was associated with a 26% drop in cell viability.ConclusionsWe have demonstrated that the majority of isolated CD34⁺ cells consist of immature and quiescent cells that lack prototypic markers of EPC. High VEGF-A gene expression might be one of the mechanisms for CD34⁺ cell-induced angiogenesis.     

Non-multipotent stroma inhibit the proliferation and differentiation of mesenchymal stromal cells in vitro

Background aimsThe ability to expand and maintain bone marrow (BM)-derived mesenchymal stem cells (MSC) in vitro is an important aspect of their therapeutic potential. Despite this, the exact composition of stromal cell types within these cultures and the potential effects of non-stem cells on the maintenance of MSC are poorly understood.MethodsC57BL/6J BM stroma was investigated as a model to determine the relationship between MSC and non-multipotent cells in vitro. Whole BM and single-cell derived cultures were characterized using flow cytometry and cell sorting combined with multipotent differentiation. Proliferation of individual stromal populations was evaluated using BrdU.ResultsAt a single-cell level, MSC were distinguished from committed progenitors, and cells lacking differentiation ability, by the expression of CD105 (CD105⁺). A 3-fold reduction in the percentage of CD105⁺ cells was detected after prolonged culture and correlated with loss of MSC. Depletion of CD105⁺ cells coincided with a 10–20% increase in the frequency of proliferating CD105^? cells. Removal of CD105^? stroma caused increased proliferation in CD105⁺ cells, which could be diminished by conditioned media from parent cultures. Comparison of the multipotent differentiation potential in purified and non-purified CD105⁺ cells determined that MSC were detectable for at least 3 weeks longer when cultured in the absence of CD105^? cells.ConclusionsThis work identifies a simple model for characterizing the different cellular components present in BM stromal cultures and demonstrates that stromal cells lacking multipotent differentiating capacity greatly reduce the longevity of MSC.     

The umbilical cord matrix is a better source of mesenchymal stem cells (MSC) than the umbilical cord blood

Many studies have drawn attention to the emerging role of MSC (mesenchymal stem cells) as a promising population supporting new clinical concepts in cellular therapy. However, the sources from which these cells can be isolated are still under discussion. Whereas BM (bone marrow) is presented as the main source of MSC, despite the invasive procedure related to this source, the possibility of isolating sufficient numbers of these cells from UCB (umbilical cord blood) remains controversial. Here, we present the results of experiments aimed at isolating MSC from UCB, BM and UCM (umbilical cord matrix) using different methods of isolation and various culture media that summarize the main procedures and criteria reported in the literature. Whereas isolation of MSC were successful from BM (10:10) and (UCM) (8:8), only one cord blood sample (1:15) gave rise to MSC using various culture media [DMEM (Dulbecco's modified Eagle's medium) +5% platelet lysate, DMEM+10% FBS (fetal bovine serum), DMEM+10% human UCB serum, MSCGM®] and different isolation methods [plastic adherence of total MNC (mononuclear cells), CD3⁺/CD19⁺/CD14⁺/CD38⁺‐depleted MNC and CD133⁺‐ or LNGFR⁺‐enriched MNC]. MSC from UCM and BM were able to differentiate into adipocytes, osteocytes and hepatocytes. The expansion potential was highest for MSC from UCM. The two cell populations had CD90⁺/CD73⁺/CD105⁺ phenotype with the additional expression of SSEA4 and LNGFR for BM MSC. These results clearly exclude UCB from the list of MSC sources for clinical use and propose instead UCM as a rich, non‐invasive and abundant source of MSC.