Enhancement of proliferation of human umbilical cord blood–derived CD34+ hematopoietic stem cells by a combination of hyper-interleukin-6 and small molecules

Umbilical cord blood (UCB) is an alternative source of allogeneic hematopoietic stem cells (HSCs) for transplantation to treat various hematological disorders. The major limitation to the use of UCB-derived HSCs (UCB–HSCs) in transplantation, however, is the low numbers of HSCs in a unit of cord blood. To overcome this limitation, various cytokines or small molecules have been used to expand UCB-HSCs ex vivo. In this study, we investigated a synergistic effect of the combination of HIL-6, SR1, and UM171 on UCB-HSC culture and found that this combination resulted in the highest number of CD34⁺ cells. These results suggest that the combination of SR1, UM171 and HIL-6 exerts a synergistic effect in the proliferation of HSCs from UCB and thus, SR1, UM171 and HIL-6 is the most suitable combination for obtaining HSCs from UCB for clinical transplantation.     

Short-term exposure of umbilical cord blood CD34+ cells to granulocyte–macrophage colony-stimulating factor early in culture improves ex vivo expansion of neutrophils

Background aimsDespite the availability of modern antibiotics/antimycotics and cytokine support, neutropenic infection accounts for the majority of chemotherapy-associated deaths. While transfusion support with donor neutrophils is possible, cost and complicated logistics make such an option unrealistic on a routine basis. A manufactured neutrophil product could enable routine prophylactic administration of neutrophils, preventing the onset of neutropenia and substantially reducing the risk of infection. We examined the use of pre-culture strategies and various cytokine/modulator combinations to improve neutrophil expansion from umbilical cord blood (UCB) hematopoietic stem and progenitor cells (HPC).MethodsEnriched UCB HPC were cultured using either two-phase pre-culture strategies or a single phase using various cytokine/modulator combinations. Outcome was assessed with respect to numerical expansion, cell morphology, granulation and respiratory burst activity.ResultsPre-culture in the absence of strong differentiation signals (e.g. granulocyte colony-stimulating factor; G-CSF) failed to provide any improvement to final neutrophil yields. Similarly, removal of differentiating cells during pre-culture failed to improve neutrophil yields to an appreciable extent. Of the cytokine/modulator combinations, the addition of granulocyte–macrophage (GM)-colony-stimulating factor (CSF) alone gave the greatest increase. In order to avoid production of monocytes, it was necessary to remove GM-CSF on day 5. Using this strategy, neutrophil expansion improved 2.7-fold.ConclusionsAlthough all cytokines and culture strategies employed have been reported previously to enhance HPC expansion, we found that the addition of GM-CSF alone was sufficient to improve total cell yields maximally. The need to remove GM-CSF on day 5 to avoid monocyte differentiation highlights the context and time-dependent complexity of exogenous signaling in hematopoietic cell differentiation and growth.     

TNF-α and IL-1β inhibit RUNX2 and collagen expression but increase alkaline phosphatase activity and mineralization in human mesenchymal stem cells

AimsJoint inflammation leads to bone erosion in rheumatoid arthritis (RA), whereas it induces new bone formation in spondyloarthropathies (SpAs). Our aims were to clarify the effects of tumour necrosis factor α (TNF-α) and interleukin 1β (IL-1β) on osteoblast differentiation and mineralization in human mesenchymal stem cells (MSCs).Main methodsIn MSCs, expression of osteoblast markers was assessed by real-time PCR and ELISA. Activity of tissue-nonspecific alkaline phosphatase (TNAP) and mineralization were determined by the method of Lowry and alizarin red staining respectively. Involvement of RUNX2 in cytokine effects was investigated in osteoblast-like cells transfected with a dominant negative construct.Key findingsTNF-α (from 0.1 to 10 ng/ml) and IL-1β (from 0.1 to 1 ng/ml) stimulated TNAP activity and mineralization in MSCs. Addition of 50 ng/ml of IL-1 receptor antagonist in TNF-α-treated cultures did not reverse TNF-α effects, indicating that IL-1 was not involved in TNF-α-stimulated TNAP activity. Both TNF-α and IL-1β decreased RUNX2 expression and osteocalcin secretion, suggesting that RUNX2 was not involved in mineralization. This hypothesis was confirmed in osteoblast-like cells expressing a dominant negative RUNX2, in which TNAP expression and activity were not reduced. Finally, since mineralization may merely rely on increased TNAP activity in a collagen-rich tissue, we investigated cytokine effects on collagen expression, and observed that cytokines decreased collagen expression in osteoblasts from MSC cultures.SignificanceThe different effects of cytokines on TNAP activity and collagen expression may therefore help explain why inflammation decreases bone formation in RA whereas it induces ectopic ossification from collagen-rich entheses during SpAs.     

The in vitro growth of a cord blood–derived cell population enriched for CD34+ cells is influenced by its cell cycle status and treatment with hydroxyurea

Objective

Cell cycle plays a fundamental role in the physiology of hematopoietic stem and progenitor cells. In the present study we used a negative selection system to obtain an immature cell population—enriched for cord blood–derived CD34⁺ cells—and we determined its proliferation, expansion and differentiation patterns as a function of the cell cycle status. The effects of hydroxyurea (HU) were also assessed.

CD4+CD25+FOXP3+ T cells,Foxp3 gene and protein expression contribute to antiasthmatic effects of San’ao decoction in mice model of asthma

ObjectiveSan’ao decoction (SAD) is a commonly used traditional combinatorial formula composed of Herba Ephedrae, Radix Glycyrrhizae and Amygdalus Communis Vas. Early studies showed that in the OVA sensitization asthmatic mice model its compatibility could lower airway reactivity and airway inflammatory cell infiltration. Based on the above results, this study mainly discussed San’ao decoction's immunomodulatory effects on Tregs.MethodsUPLC–PDA–TOF-MS was applied to analyze chemicals of SAD, and under the optimized chromatographic and MS condition, the major components in SAD were well separated and detected within 22 min. An asthma model was established in BALB/c mice that were sensitized and challenged with ovalbumin. After 2 weeks’ treatment, peripheral blood and bronchoalveolar lavage fluid (BALF) were assessed for inflammatory cell counts; histological change of lung tissue were detected; flow cytometry detection of splenic CD4⁺CD25⁺Foxp3⁺ Treg cells of the mice were counted; Foxp3 expression in lung tissues were examined as well.Results22 Peaks signal chemical components in SAD were identified by UPLC–QTO-MS method. In terms of the percentage of eosinophile in peripheral blood and bronchoalveolar lavage fluid (BALF), SAD groups were significantly lower (p < 0.01) than model group. Compared with model group, lung histological changes of SAD groups were reduced; the proportion of CD4⁺CD25⁺Foxp3⁺ Treg cells in CD4⁺ cells of asthmatic mice also decreased; SAD significantly increased the proportion of CD4⁺CD25⁺Foxp3⁺ Treg cells and promoted Foxp3 expression in a mouse model of asthma.ConclusionThese results suggest that the antiasthmatic effects of SAD are at least partially associated with CD4⁺CD25⁺Foxp3⁺ Treg cells.     

Enumeration of viable CD34(+) cells by flow cytometry in blood, bone marrow and cord blood: results of a study of the novel BD™ stem cell enumeration kit

Background aimsEnumeration of CD34⁺ cells in leukocyte-rich cell suspensions is important for clinical decision-making in stem cell transplantation. Single-platform flow cytometry assays offer the significant advantages of speed and reproducibility, and have therefore become the gold standard in stem cell enumeration. The clinical community has recently defined the need for stem cell enumeration kits that incorporate viability dyes. The purpose of this study was to evaluate a novel assay, BD Biosciences’ (BD) stem cell enumeration kit (SCE kit³), in relation to Beckman Coulter's (BC) commercially available BC Stem-Kit?.MethodsFresh/freeze-thawed samples from leukapheresis, bone marrow and cord blood, and fresh normal/mobilized blood, were analyzed with both assays (simultaneous detection of side/forward scatter and three fluorescence signals) on two flow cytometry platforms, BD FACSCanto II and BD FACSCalibur.ResultsResults from both assays were highly congruent, with an overall r² ≥ 0.99 (all specimen types included), a linear correlation across all CD34⁺ cell frequencies and concentrations, and an almost ideal steepness of the trend line.ConclusionsBoth assays functioned reliably. Being based on single-platform International Society of Hematotherapy and Graft Engineering (ISHAGE) guidelines and similar staining methods, both assays essentially come to identical results. For most specimen types, the viability of CD34⁺ cells was equal to overall leukocyte viability. In summary, in the hands of an experienced technician, the BD? SCE kit and the BC Stem-Kit are equivalent. The infrequent user might derive benefit from the fact that counting spheres are pre-pipetted into the Trucount tube for the SCE kit, making this assay less susceptible to pipetting inaccuracy.     

Identification of a NF-кB site in the negative regulatory element (ε-NRAII) of human ε-globin gene and its binding protein NF-кB p50 in the nuclei of K562 cells

INTRODUCTIONThe human P-like globin genes are arranged as acluster of five genes(e, Gry, Ary, 8 and P) in the orderof their temporal expression. The human embryonicE-globin gene is expressed in the blood island of theembryonic yolk sac and is silenced completely at 6~8w of gestation in the fetal liverI11. Studies on trans-genic mice suggested that the regulation of 5-globingene expression is autonomous. The activating andsilencing of 5-globin gene expression rely on distallocus control …     

Effects of tobacco smoke on IL-16 in CD8+ cells from human airways and blood: a key role for oxygen free radicals?

Chronic exposure to tobacco smoke leads to an increase in the frequency of infections and in the number of CD8(+) and CD4(+) cells as well as the CD4(+) chemoattractant cytokine IL-16 in the airways. Here, we investigated whether tobacco smoke depletes intracellular IL-16 protein and inhibits de novo production of IL-16 in CD8(+) cells from human airways and blood while increasing extracellular IL-16 and whether oxygen free radicals (OFR) are involved. Intracellular IL-16 protein in CD8(+) cells and mRNA in all cells was decreased in bronchoalveolar lavage (BAL) samples from chronic smokers. This was also the case in human blood CD8(+) cells exposed to water-soluble tobacco smoke components in vitro, in which oxidized proteins were markedly increased. Extracellular IL-16 protein was increased in cell-free BAL fluid from chronic smokers and in human blood CD8(+) cells exposed to water-soluble tobacco smoke components in vitro. This was not observed in occasional smokers after short-term exposure to tobacco smoke. A marker of activation (CD69) was slightly increased, whereas other markers of key cellular functions (membrane integrity, apoptosis, and proliferation) in human blood CD8(+) cells in vitro were negatively affected by water-soluble tobacco smoke components. An OFR scavenger prevented these effects, whereas a protein synthesis inhibitor, a β-adrenoceptor, a glucocorticoid receptor agonist, a phosphodiesterase, a calcineurin phosphatase, and a caspase-3 inhibitor did not. In conclusion, tobacco smoke depletes preformed intracellular IL-16 protein, inhibits its de novo synthesis, and distorts key cellular functions in human CD8(+) cells. OFR may play a key role in this context.