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.     

CD271 enrichment does not help isolating mesenchymal stromal cells from G-CSF-mobilized peripheral blood

Reports on the isolation of mesenchymal stromal cells (MSCs) from granulocyte colony stimulating factor mobilized peripheral blood (G-CSF-mobilized PB) using regular culturing techniques are controversial. Enrichment techniques such as CD133 isolation have increased the success rates. CD271 is a wellknown marker for enrichment of MSCs from bone marrow (BM). In the present study, we aimed to find out whether CD271 enrichment can help isolation of MSCs from G-CSF-mobiiized PB. Five G-CSF-mobilized PB samples were collected from the remnant parts of the bags used for BM transplantation. Five BM samples were used as the control. Mononuclear cells (MNCs) from both resources were collected and underwent magnetic sorting for CD271-positive cells. The isolated cells were cultured, undergoing flowcytometry and differentiation assays to determine if they fulfill MSCs characteristics. CD271-positive portion of G-CSF-mobilized PB did not yield any cell outgrowth but the BM counterpart could successfully form MSC colonies. Although the percentage of CD271⁺ cells showed no difference between BM-MNCs and G-CSF-mobilized PB-MNCs, hematopoietic markers such as CD45, CD34 and CD133 composed a higher percentage of CD271-positive cells in the G-CSF-tnobiiized PB group. Results obtained indicated that CD271 enrichment does not help isolation of MSCs from G-CSF-mobilized PB. In this source, almost all of the CD271⁺ cells are from hematopoietic origin and the frequency of MSCs is so low that possibly during the process of cell isolation most of them are lost and the isolation fails.     

Hematopoietic Stem/Progenitor Cell Sources to Generate Reticulocytes for Plasmodium vivax Culture

The predilection of Plasmodium vivax (P. vivax) for reticulocytes is a major obstacle for its establishment in a long-term culture system, as this requires a continuous supply of large quantities of reticulocytes, representing only 1–2% of circulating red blood cells. We here compared the production of reticulocytes using an established in vitro culture system from three different sources of hematopoietic stem/progenitor cells (HSPC), i.e. umbilical cord blood (UCB), bone marrow (BM) and adult peripheral blood (PB). Compared to CD34⁺-enriched populations of PB and BM, CD34⁺-enriched populations of UCB produced the highest amount of reticulocytes that could be invaded by P. vivax. In addition, when CD34⁺-enriched cells were first expanded, a further extensive increase in reticulocytes was seen for UCB, to a lesser degree BM but not PB. As invasion by P. vivax was significantly better in reticulocytes generated in vitro, we also suggest that P. vivax may have a preference for invading immature reticulocytes, which should be confirmed in future studies.     

Immunogenicity of dendritic cells pulsed with MAGE3, Survivin and B-cell maturation antigen mRNA for vaccination of multiple myeloma patients

The introduction of autologous stem cell transplantation (SCT) and novel drugs has improved overall survival in multiple myeloma (MM) patients. However, minimal residual disease (MRD) remains and most patients eventually relapse. Myeloma plasma cells express tumor-associated antigens (TAA), which are interesting targets for immunotherapy. In this phase 1 study, we investigated the safety and immunological effects of TAA-mRNA-loaded dendritic cell (DC) vaccination for treatment for MRD in MM after SCT. Mature monocyte-derived DCs were pulsed with keyhole limpet hemocyanin (KLH) and electroporated with MAGE3, Survivin or B-cell maturation antigen (BCMA) mRNA. Twelve patients were vaccinated three times with intravenous (5–22 × 10⁶ DCs) and intradermal vaccines (4–11 × 10⁶ DCs), at biweekly intervals. Immunological responses were monitored in blood and delayed-type hypersensitivity (DTH) biopsies. All patients developed strong anti-KLH T-cell responses, but not KLH antibodies. In 2 patients, vaccine-specific T cells were detected in DTH biopsies. In one patient, we found MAGE3-specific CD4⁺ and CD8⁺ T cells, and CD3⁺ T cells reactive against BCMA and Survivin. In the other patient, we detected low numbers of MAGE3 and BCMA-reactive CD8⁺ T cells. Vaccination was well tolerated with limited toxicity. These findings illustrate that TAA-mRNA-electroporated mature DCs are capable of inducing TAA-T-cell responses in MM patients after SCT.     

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.     

Concomitant overexpression of triple antioxidant enzymes selectively increases circulating endothelial progenitor cells in mice with limb ischaemia

Endothelial progenitor cells (EPCs) are a group of heterogeneous cells in bone marrow (BM) and blood. Ischaemia increases reactive oxygen species (ROS) production that regulates EPC number and function. The present study was conducted to determine if ischaemia‐induced ROS differentially regulated individual EPC subpopulations using a mouse model concomitantly overexpressing superoxide dismutase (SOD)1, SOD3 and glutathione peroxidase. Limb ischaemia was induced by femoral artery ligation in male transgenic mice with their wild‐type littermate as control. BM and blood cells were collected for EPCs analysis and mononuclear cell intracellular ROS production, apoptosis and proliferation at baseline, day 3 and day 21 after ischaemia. Cells positive for c‐Kit⁺/CD31⁺ or Sca‐1⁺/Flk‐1⁺ or CD34⁺/CD133⁺ or CD34⁺/Flk‐1⁺ were identified as EPCs. ischaemia significantly increased ROS production and cell apoptosis and decreased proliferation of circulating and BM mononuclear cells and increased BM and circulating EPCs levels. Overexpression of triple antioxidant enzymes effectively prevented ischaemia‐induced ROS production with significantly decreased cell apoptosis and preserved proliferation and significantly increased circulating EPCs level without significant changes in BM EPC populations, associated with enhanced recovery of blood flow and function of the ischemic limb. These data suggested that ischaemia‐induced ROS was differentially involved in the regulation of circulating EPC population.     

Bone Marrow Alterations and Lower Endothelial Progenitor Cell Numbers in Critical Limb Ischemia Patients

Background

Critical limb ischemia (CLI) is characterized by lower extremity artery obstruction and a largely unexplained impaired ischemic neovascularization response. Bone marrow (BM) derived endothelial progenitor cells (EPC) contribute to neovascularization. We hypothesize that reduced levels and function of circulating progenitor cells and alterations in the BM contribute to impaired neovascularization in CLI.

Methods

Levels of primitive (CD34⁺ and CD133⁺) progenitors and CD34⁺KDR⁺ EPC were analyzed using flow cytometry in blood and BM from 101 CLI patients in the JUVENTAS-trial ({"type":"clinical-trial","attrs":{"text":"NCT00371371","term_id":"NCT00371371"}}NCT00371371) and healthy controls. Blood levels of markers for endothelial injury (sE-selectin, sICAM-1, sVCAM-1, and thrombomodulin), and progenitor cell mobilizing and inflammatory factors were assessed by conventional and multiplex ELISA. BM levels and activity of the EPC mobilizing protease MMP-9 were assessed by ELISA and zymography. Circulating angiogenic cells (CAC) were cultured and their paracrine function was assessed.

Results

Endothelial injury markers were higher in CLI (P<0.01). CLI patients had higher levels of VEGF, SDF-1α, SCF, G-CSF (P<0.05) and of IL-6, IL-8 and IP-10 (P<0.05). Circulating EPC and BM CD34⁺ cells (P<0.05), lymphocytic expression of CXCR4 and CD26 in BM (P<0.05), and BM levels and activity of MMP-9 (P<0.01) were lower in CLI. Multivariate regression analysis showed an inverse association between IL-6 and BM CD34⁺ cell levels (P = 0.007). CAC from CLI patients had reduced paracrine function (P<0.0001).

Conclusion

CLI patients have reduced levels of circulating EPC, despite profound endothelial injury and an EPC mobilizing response. Moreover, CLI patients have lower BM CD34⁺-cell levels, which were inversely associated with the inflammatory marker IL-6, and lower BM MMP-9 levels and activity. The results of this study suggest that inflammation-induced BM exhaustion and a disturbed progenitor cell mobilization response due to reduced levels and activity of MMP-9 in the BM and alterations in the SDF-1α/CXCR4 interaction contribute to the attenuated neovascularization in CLI patients.     

In vitro generation of murine myeloid dendritic cells from CD34-positive precursors

Dendritic cells (DCs) link the innate and adaptive immune system. Currently, murine DCs for cell biology investigations are developed from MHC class II-negative bone marrow (BM) precursor cells, non-depleted BM cells or BM monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Here we demonstrate an isolation procedure of functionally intact myeloid CD11c⁺ CD11b⁺ DCs derived from murine CD34-positive precursors. DCs derived from CD34⁺ cells show functional internalization, maturation, cytokine secretion, MHC-restricted antigen presentation, and MHCII retrograde transport of antigens from the lysosomes to the cell surface. In comparison to the established method, the advantages of this isolation procedure are a shorter cultivation period, a superior transfection efficiency, the yield of a purer and more homogeneous population of immature DCs, and less consumption of cell culture medium and GM-CSF. The new isolation procedure and the functional quality of CD34⁺ cell-derived murine myeloid DCs make them ideally suited for immunology and cell biology studies.     

CD34+ cells homing: quantitative expression of adhesion molecules and adhesion of CD34+ cells to endothelial cells exposed to shear stress

To investigate the function of the main adhesion receptors (CD62L, CD49d, CD49e, CD11b and CD18) on CD34+ cells during homing, their expression was quantified by flow cytometry using calibration beads. CD34+ cells were isolated from bone-marrow (BM), cord blood (CB) or peripheral blood (PB) from patients with myeloma. As this process might mimic the mature leukocyte migration, we also observed the effect of exposing endothelial cells to shear stress (7 dyn/cm(2)) on the adhesion of CB CD34+ cells.The proportion of CD34+/CD62L+ cells was greater in PB than in BM (p<0.05). Likewise, we found a significantly greater expression of CD62L receptor on PB cells compared to BM cells (p<0.05) and on BM cells compared to CB cells (p<0.05). The proportions of CD34+/CD49d+ cells and CD34+/CD49e+ cells were significantly higher in the BM and CB than in PB. However, no significant difference in CD49d or CD49e antigen densities was observed. The beta_2 integrins (CD11b and CD18) receptors are also implicated in CD34+ cells homing to BM. No significant variation in CD34+/CD11b+ and CD34+/CD18+ cells frequency was noted. However quantitative analysis revealed that CD18 was more strongly expressed on BM cells than on PB and CB cells.The adhesion assay showed that fluid flow may favour a firm adhesion of CB CD34+ cells to endothelial cells whereas static conditions just allowed CD34+ cells sedimentation.In conclusion, quantitative expression of the main receptors on CD34+ cells indicates that the three main sources of CD34+ cells currently used for transplantation have neither the same phenotype nor the same number of antigenic sites for a receptor. So, we hypothesize that migrational capacity of these cells might be different. Moreover, it seems that shear stress could favor adhesion of CD34+ cells to endothelial cells.     

Peripheral CD8+ T cell proliferation is prognostic for patients with advanced thoracic malignancies

There is a complex interplay between the immune system and a developing tumor that is manifest in the way that the balance of T cell subsets in the local tumor environment reflects clinical outcome. Tumor infiltration by CD8⁺ T cells and regulatory T cells (Treg) is associated with improved and reduced survival, respectively, in many cancer types. However, little is known of the prognostic value of immunological parameters measured in peripheral blood. In this study, peripheral CD8⁺ T cells and Treg from 43 patients with malignant mesothelioma or advanced non-small-cell lung cancer scheduled to commence palliative chemotherapy were assessed by flow cytometry and evaluated for association with patient survival. Patients had a higher proportion of peripheral Treg, proliferating CD8⁺ T cells and CD8⁺ T cells with an activated effector phenotype compared with age-matched healthy controls. Higher proportions of Treg and proliferating CD8⁺ T cells were both associated with poor survival in univariate analyses (hazard ratio [HR] 3.81, 95 % CI 1.69–8.57; p < 0.01 and HR 2.86, 95 % CI 1.26–6.50; p < 0.05, respectively). CD8⁺ T cell proliferation was independently predictive of reduced survival in multivariate analysis (HR 2.58, 95 % CI 1.01–6.61; p < 0.05). These findings suggest that peripheral CD8⁺ T cell proliferation can be a useful prognostic marker in patients with thoracic malignancies planned for palliative chemotherapy.     

Early activated Th-1 type and dominantly diverse natural killer T (CD3⁺CD161⁺Vα24⁻) cells in bone marrow among visceral leishmaniasis patients

Lipid antigens of Leishmania donovani-like lipophosphoglycans (LPG) are demonstrated to be a potent ligand for natural killer T (NKT) cell activation. Little is known about the phenotype or function of these cells and their trafficking pattern to the bone marrow (BM) of visceral leishmaniasis (VL) patients. Their precise role in humans still requires pathological validation. The study included 42 parasitologically confirmed patients (mean age 24.80 ± 16.26 years; range 3-70 years; 25 males and 17 females), 33 healthy contact subjects (family/non-family members) and normal BM specimens (NBM; n = 9). Enumeration of NKT cells and quantification of parasites (before and after therapy) were performed for the recruited patients. Results established that non-CD1d restricted, diverse cells are the dominant population among resident but not enriched NKT (CD3⁺CD161⁺) cells at the disease site (BM). Expression profiles for various markers are indicative of their early activated (CD69⁺, CD62L^low, CD11a^high) CCR5⁺ phenotype at the BM. Functionally, BM-derived NKT cells were dominantly producing IFN-γ in response to L. donovani antigen in vitro. Given these observations, these data indicate that CD3⁺CD161⁺ diverse NKT cells are heterogeneous in function and of the dominant Th-1 phenotype at the disease site.     

Testosterone influences renal electrolyte excretion in SHR/y and WKY males

Background

Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH⁺ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH⁺ cellular subsets in bone marrow from pure red cell aplasia patients.

Results

In normal bone marrow, we identified three populations of cells, namely ALDH⁺CD34⁺, ALDH^-CD34⁺ and ALDH⁺CD34^- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH^-CD34⁺ cells, ALDH⁺CD34⁺ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH⁺CD34^- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH⁺CD34^- cells showed a CD71^bright, CD105⁺, CD45^- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH⁺CD34^- precursors were not detectable in patients with pure red cell aplasia (PRCA).

Conclusion

Our study, comparing surface antigen expression of ALDH⁺/CD34⁺, ALDH^-/CD34⁺ and ALDH⁺/CD34^- progenitor cell subsets in human bone marrow, clearly indicated that ALDH⁺CD34^- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia.     

Expression of early hematopoietic markers in cord and mobilized blood

G-CSF mobilized peripheral blood and cord blood are major sources of hematopoietic progenitor cells. These cells are characterized by expression of “early” antigens. We have evaluated the coexpression of hematopoietic markers CD34, CD133, CD90, CDCP1, and CD117 and activation antigen CD38 using multicolor flow cytometry. We showed that cells positive for each particular antigen generate a separate population. The percentage of cells expressing each particular “early” antigen is twice as high in the cord blood as in the mobilized blood. The cell number with complex progenitor phenotype (CD34⁺/CD38^?/CD117^?, CD133⁺/CD34⁺/CD38^?, CDCP1⁺/CD34⁺/CD38^?, etc.) is equal in mobilized and cord blood. There is a strong positive correlation between CD34, CD133, CD117, and CDCP1 expression in both groups. Positive correlation was observed between CD90 and CD34, CD133, CDCP1, and CD117 expression only in cord blood; it was not significant in mobilized blood. The analyses of early antigens’ coexpression with activation CD38 marker did not confirm the hypothesis of sequential activation and loss of expression of the aforementioned antigens. We assume that there is global regulation of CD34, CD133, CDCP1, and CD117 expression. Expression of CD38 may be reversibly suppressed during maturation of the hematopoietic cells, and CD117 may be expressed on not only myeloid cells.     

Cytokine effect on ex vivo expansion of haemopoietic stem cells from different human sources

Human pluripotential stem cells (PSC) are currently the target for transplantation attempts and genetic manipulation. We have therefore investigated the frequency and the expansion potential of PSC’s in different types of blood samples. CD 34⁺ cells were thus obtained from human bone marrow (BM), as well as from peripheral blood (PB) and cord blood (CB) samples. After immuno-magnetic separation the highest yields of CD 34⁺ cells were from BM (1.08–2.25%) and CB (0.42–1.32%) while PB samples gave much lower values. Suspension cultures of PSC’s from the three sources were then set up, in the presence of combinations of haemopoietic growth factors. A remarkable amplification of the nucleated cell pool was observed reaching a maximum between 10 and 15 days of culture; earliest and maximum expansion (up to 220-fold) was achieved when Erythropoietin (Epo) was added to the culture medium, but this resulted in reduction of colony-forming cells and differentiation into erythroid progenitors. Clonogenic tests for BFU-E’s derived colonies showed a peak value at 5 days of liquid culture. Further studies are advisable to establish the best cytokine combination for a valuableex vivo expansion, coupled with preservation of stem cell properties.     

Non-Invasive Imaging Provides Spatiotemporal Information on Disease Progression and Response to Therapy in a Murine Model of Multiple Myeloma

Background

Multiple myeloma (MM) is a B-cell malignancy, where malignant plasma cells clonally expand in the bone marrow of older people, causing significant morbidity and mortality. Typical clinical symptoms include increased serum calcium levels, renal insufficiency, anemia, and bone lesions. With standard therapies, MM remains incurable; therefore, the development of new drugs or immune cell-based therapies is desirable. To advance the goal of finding a more effective treatment for MM, we aimed to develop a reliable preclinical MM mouse model applying sensitive and reproducible methods for monitoring of tumor growth and metastasis in response to therapy.

Material and Methods

A mouse model was created by intravenously injecting bone marrow-homing mouse myeloma cells (MOPC-315.BM) that expressed luciferase into BALB/c wild type mice. The luciferase in the myeloma cells allowed in vivo tracking before and after melphalan treatment with bioluminescence imaging (BLI). Homing of MOPC-315.BM luciferase+ myeloma cells to specific tissues was examined by flow cytometry. Idiotype-specific myeloma protein serum levels were measured by ELISA. In vivo measurements were validated with histopathology.

Results

Strong bone marrow tropism and subsequent dissemination of MOPC-315.BM luciferase⁺ cells in vivo closely mimicked the human disease. In vivo BLI and later histopathological analysis revealed that 12 days of melphalan treatment slowed tumor progression and reduced MM dissemination compared to untreated controls. MOPC-315.BM luciferase⁺ cells expressed CXCR4 and high levels of CD44 and α4β1 in vitro which could explain the strong bone marrow tropism. The results showed that MOPC-315.BM cells dynamically regulated homing receptor expression and depended on interactions with surrounding cells.

Conclusions

This study described a novel MM mouse model that facilitated convenient, reliable, and sensitive tracking of myeloma cells with whole body BLI in living animals. This model is highly suitable for monitoring the effects of different treatment regimens.     

Mobilization of CD34~+CD38~- hematopoietic stem cells after priming in acute myeloid leukemia

AIM: To evaluate quantitatively and qualitatively the different CD34+cell subsets after priming by chemotherapy granulocyte colony-stimulating factor(± G-CSF)in patients with acute myeloid leukemia.METHODS: Peripheral blood and bone marrow sampleswere harvested in 8 acute myeloid leukemia patients during and after induction chemotherapy. The CD34/CD38 cell profile was analyzed by multi-parameter flow cytometry. Adhesion profile was made using CXC chemokine receptor 4(CXCR4)(CD184), VLA-4(CD49d/CD29) and CD47.RESULTS: Chemotherapy ± G-CSF mobilized immature cells(CD34+CD38 population), while the more mature cells(CD34+CD38lowand CD34+CD38+populations) decreased progressively after treatment. Circulating CD34+cells tended to be more sensitive to chemotherapy after priming with G-CSF. CD34+cell mobilization was correlated with a gradual increase in CXCR4 and CD47expression, suggesting a role in cell protection and the capacity of homing back to the marrow.CONCLUSION: Chemotherapy ± G-CSF mobilizes into the circulation CD34+bone marrow cells, of which, the immature CD34+CD38-cell population. Further manipulations of these interactions may be a means with which to control the trafficking of leukemia stem cells to improve patients’ outcomes.     

The proliferation index of specific bone marrow cell compartments from myelodysplastic syndromes is associated with the diagnostic and patient outcome

Myelodysplastic syndromes (MDS) are clonal stem cell disorders which frequently show a hypercellular dysplastic bone marrow (BM) associated with inefficient hematopoiesis and peripheral cytopenias due to increased apoptosis and maturation blockades. Currently, little is known about the role of cell proliferation in compensating for the BM failure syndrome and in determining patient outcome. Here, we analyzed the proliferation index (PI) of different compartments of BM hematopoietic cells in 106 MDS patients compared to both normal/reactive BM (n = 94) and acute myeloid leukemia (AML; n = 30 cases) using multiparameter flow cytometry. Our results show abnormally increased overall BM proliferation profiles in MDS which significantly differ between early/low-risk and advanced/high-risk cases. Early/low-risk patients showed increased proliferation of non-lymphoid CD34⁺ precursors, maturing neutrophils and nucleated red blood cells (NRBC), while the PI of these compartments of BM precursors progressively fell below normal values towards AML levels in advanced/high-risk MDS. Decreased proliferation of non-lymphoid CD34⁺ and NRBC precursors was significantly associated with adverse disease features, shorter overall survival (OS) and transformation to AML, both in the whole series and when low- and high-risk MDS patients were separately considered, the PI of NRBC emerging as the most powerful independent predictor for OS and progression to AML. In conclusion, assessment of the PI of NRBC, and potentially also of other compartments of BM precursors (e.g.: myeloid CD34⁺ HPC), could significantly contribute to a better management of MDS.     

Characteristics of circulating CD31+ cells from patients with coronary artery disease

Recently, we reported the properties of CD31‐expressing cells in healthy individuals. However, the characteristics of CD31‐expressing cells derived from coronary artery disease (CAD) patients remain unknown. This study aimed to investigate the relationship between circulating CD31⁺ cells and CAD as well as their biological characteristics. Analysis with flow cytometry revealed that CD31⁺ cells (C‐CD31) from the peripheral blood (PB) of CAD patients exhibited low levels of T‐cell marker and high levels of macrophage marker compared with the PB‐CD31⁺ cells from healthy individuals (H‐CD31). In addition, the expression levels of multiple pro‐angiogenic and chemokine genes were significantly down‐regulated in C‐CD31. However, inflammatory gene IL‐1α was highly up‐regulated in C‐CD31. Patients with unstable angina (UA) had significantly more CD31⁺ cells in the PB than healthy control group (P < 0.001). Moreover, there were significant correlations between the number of CD31⁺ cells and cardiovascular (CV) disease activity (R = 0.318, P = 0.006) and the number of diseased coronaries (R = 0.312, P = 0.005). For the diagnostic category of UA, the area under curve was 0.803 (P < 0.001). In conclusion, C‐CD31 have impaired angiogenic potential and the number of circulating CD31⁺ cells were correlated with CV risk. These findings may contribute to the understanding of the pathogenesis of CAD.     

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.