Adherent Cell Fraction Research Articles

Unraveling the molecular determinants of the anti-phagocytic protein cloak of plague bacteria.

The pathogenic bacterium Yersina pestis is protected from macrophage engulfment by a capsule like antigen, F1, formed of long polymers of the monomer protein, Caf1. However, despite the importance of this pathogen, the mechanism of protection was not understood. Here we demonstrate how F1 protects the bacteria from phagocytosis. First, we show that Escherichia coli expressing F1 showed greatly reduced adherence to macrophages. Furthermore, the few cells that did adhere remained on the macrophage surface and were not engulfed. We then inserted, by mutation, an “RGDS” integrin binding motif into Caf1. This did not change the number of cells adhering to macrophages but increased the fraction of adherent cells that were engulfed. Therefore, F1 protects in two separate ways, reducing cell adhesion, possibly by acting as a polymer brush, and hiding innate receptor binding sites needed for engulfment. F1 is very robust and we show that E. coli expressing weakened mutant polymers are engulfed like the RGDS mutant. This suggests that innate attachment sites on the native cell surface are exposed if F1 is weakened. Single-molecule force spectroscopy (SMFS) experiments revealed that wild-type F1 displays a very high mechanical stability of 400 pN. However, the mechanical resistance of the destabilised mutants, that were fully engulfed, was only 20% weaker. By only marginally exceeding the mechanical force applied to the Caf1 polymer during phagocytosis it may be that the exceptional tensile strength evolved to resist the forces applied at this stage of engulfment.

Functional flow cytometry of monocytes for routine diagnosis of innate primary immunodeficiencies

Functional flow cytometry of monocytes for routine diagnosis of innate primary immunodeficiencies

Microfluidic Shear Force Assay to Determine Cell Adhesion Forces.

Cell adhesion is implicated in many physiological settings such as the retention of hematopoietic stem cells (HSCs) in their bone marrow niches or their migration into the bloodstream. During HSC mobilization these adhesion sites are cleaved and have to be newly formed during HSC homing and engraftment. To determine the adhesive properties of HSCs on different extracellular matrix (ECM) molecules, we present a microfluidic shear force assay, where a laminar flow is used to detach a semi-adherent cell population, the HSC model cell line KG-1a, from an ECM protein-coated substrate. This technique combines the high throughput of population-based assays with the ability to observe cell detachment in real time. Additionally, it is suitable for weakly adherent cells, as the setup allows cell incubation on various substrates and application of shear stress ranging several orders of magnitude in one setup without additional washing or transfer steps. As a measure for the adhesion strength of the studied cell population on the substrate, the critical shear force τ50 is determined which is required to remove 50% of the initially adherent cell fraction.

Induced Pluripotent Stem Cells to Model Blood Diseases

Our group is developing induced pluripotent stem cell (iPSC) models of myeloid malignancies, including MDS, MPN and AML. We are generating iPSCs from the bone marrow or blood of patients, which can be maintained indefinitely as pluripotent cell lines and, upon in vitro differentiation along hematopoietic lineages, exhibit hallmark features of these diseases. By integrating mutational analyses with cell reprogramming we can derive iPSCs capturing dominant clones, subclones and normal cells from the same patient and thus have established a collection of iPSC lines representing distinct disease stages along the spectrum of myeloid transformation: predisposition syndromes/preleukemic cells/clonal hematopoiesis; low risk MDS; high risk MDS; and MDS/AML. In parallel, we are using the CRISPR/Cas9 system to introduce or correct mutations in normal or malignant iPSCs, respectively, in isogenic settings and sequential CRISPR gene editing to model mutational cooperation. We recently reported that iPSC lines derived from patients with AML re-establish upon differentiation a leukemic phenotype characterized by extensive proliferation of immature myeloid cells that serially transplant a lethal leukemia into NSG mice (Kotini et al. Cell Stem Cell 2017). Strikingly, we observed that the AML-iPSC-derived hematopoietic stem/progenitor cells (HSPCs) contain two morphologically and immunophenotypically distinct cell subpopulations: a cell fraction (adherent, A) exhibiting adherent growth and containing immature cells with an HSC immunophenotype (CD34+/CD38-/CD90+/CD45RA-/CD49f+); and a non-adherent fraction (suspension, S) of more differentiated cells. Fate-tracking experiments revealed a hierarchical organization, with the A cells renewing themselves and continuously giving rise to the S cells through symmetric and asymmetric divisions. The NSG engraftment potential was largely contained within the adherent cell fraction. Thus, AML-iPSCs exhibit the hallmarks of a leukemia stem cell (LSC) model, namely phenotypic and functional heterogeneity and hierarchical organization, with the A fraction containing LSCs that serially transplant leukemia and give rise to more differentiated cells (S fraction) without engraftment potential. LSCs are believed to be a prominent source of AML relapse, but their rarity and the unavailability of universal and specific immunophenotypic markers prohibits their prospective isolation and makes the study of their properties challenging. This new iPSC-based AML-LSC model enables us for the first time to prospectively obtain large numbers of genetically clonal human LSCs and perform genome-wide integrative molecular analyses and large-scale screening to identify key molecular mechanisms sustaining the properties of LSCs as potential new therapeutic targets. Using this model we characterized the effects of previously proposed compounds with LSC selectivity in self-renewal vs differentiation of LSCs. We also screened a small molecule library of 1280 compounds in the A and S cells in parallel to identify compounds with selectivity for the former as candidates for LSC-specific targeting. DisclosuresNo relevant conflicts of interest to declare.

Adhesion structures in leukemia cells and their regulation by Src family kinases

ABSTRACTInteraction of leukemia blasts with the bone marrow extracellular matrix often results in protection of leukemia cells from chemotherapy and in persistence of the residual disease which is on the basis of subsequent relapses. The adhesion signaling pathways have been extensively studied in adherent cells as well as in mature haematopoietic cells, but the adhesion structures and signaling in haematopoietic stem and progenitor cells, either normal or malignant, are much less explored. We analyzed the interaction of leukemia cells with fibronectin (FN) using interference reflection microscopy, immunofluorescence, measurement of adherent cell fraction, real-time microimpedance measurement and live cell imaging. We found that leukemia cells form very dynamic adhesion structures similar to early stages of focal adhesions. In contrast to adherent cells, where Src family kinases (SFK) belong to important regulators of focal adhesion dynamics, we observed only minor effects of SFK inhibitor dasatinib on leukemia cell binding to FN. The relatively weak involvement of SFK in adhesion structure regulation might be associated with the lack of cytoskeletal mechanical tension in leukemia cells. On the other hand, active Lyn kinase was found to specifically localize to leukemia cell adhesion structures and a less firm cell attachment to FN was often associated with higher Lyn activity (this unexpectedly occurred also after cell treatment with the inhibitor SKI-1). Lyn thus may be important for signaling from integrin-associated complexes to other processes in leukemia cells.

Human Umbilical Cord Mesenchymal Stromal Cells Support Viability of Umbilical Cord Blood Hematopoietic Stem Cells but not the "Stemness" of Their Progeny in Co-Culture.

Cell-cell interactions and the ability of mesenchymal stromal cells to support the expansion of hematopoietic progenitor cells were studied in co-culture of human umbilical cord tissue-derived mesenchymal stromal cells and nucleated umbilical cord blood cells. It was found that hematopoietic stem cells from the umbilical cord blood are capable to adhere to mesenchymal stromal cells and proliferate during 3-4 weeks in co-culture. However, despite the formation of hematopoietic foci and accumulation of CD34+ and CD133+ cells in the adherent cell fraction, the ability of newly generated blood cells to form colonies in semi-solid culture medium was appreciably reduced. These findings suggest that human umbilical cord tissue-derived mesenchymal stromal cells display a weak capability to support the "stemness" of hematopoietic stem cell progeny despite long-term maintenance of their viability and proliferation.

Late Adherent Subpopulation in Umbilical Cord Blood Has the Same Characteristics and Hematopoiesis-Supporting Capacity As Mesenchymal Stromal/Stem Cells

Mesenchymal stromal/stem cells (MSCs) are a major source of cell for cell therapy. MSCs derived from bone marrow (BMMSCs) have been mostly used in clinical applications. BMMSCs can be easily isolated as a cell population that adheres to plastic culture dishes within 1 week of culture. A recent report has demonstrated that cells that remain in suspension and fail to form adherent colonies contain a fraction of late adherent cells that resembles BMMSCs (Biomed Res Int, 2013; 2013: 790842). Umbilical cord blood (UCB) is as accessible as bone marrow for the isolation of MSCs. In this study, we identified a late adherent subpopulation in UCB and determined its hematopoiesis-supporting activity.Forty-five UCB units, which were not matched to the eligibility criterion defined in the Japan UCB donation program, were collected after delivery of placenta. Written informed consent was obtained before delivery from all pregnant women who participated in the study. The study protocol was approved by the ethics committee of the Kyoto University Graduate School of Medicine.Mononuclear cells were isolated from UCB by the density gradient centrifugation method with (n = 19) and without (n = 18) subsequent separation of CD34 negative cells using anti-CD34 immunomagnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Nucleated cells were separated by the hydroxyethyl starch sedimentation method from the other eight UCB units. The cells were then seeded into a culture flask and cultured in alpha minimal essential medium supplemented with 15% FBS (Culture 1; C1). After 1 week of culture, non-adherent cells in C1 supernatant were collected and re-seeded into a new flask (C2). The attached cells in C1 were cultured until adherent colonies emerged, after which they were detached using trypsin/EDTA and twice passaged to obtain a sufficient number of cells (C1 cells). In the same way, after 1 week of culture, non-adherent cells in C2 supernatant were collected and re-seeded into a new flask (C3). The attached cells in C2 were cultured to obtain C2 cells. Afterwards, re-seeding and culture (C4, C5c) were repeated until no new colonies were formed. Collected cells were cryopreserved and thawed when required in experiments. BMMSCs were isolated from human bone marrow cells purchased from AllCells (Emeryville, CA).C1 cells, the so-called UCBMSCs, were successfully isolated from 18 units (40 %). Adherent cells isolated from C2 and later were defined as elate adherent cellsf and, were obtained from 9 units: these cells were referred to as C2 cells (from 9 units), C3 cells (from 9 units), C4 cells (from 6 units) and C5 cells (from 2 units). The interval from seeding to the first colony formation in C1 was shorter in these 9 units than that in the other 9 units that contained only C1 cells: 10.8 } 1.4 vs 15.9 } 4.5 days, p < 0.01. The volume of the former 9 units tended to be large compared to the latter 9 units: 49.6 } 10.5 vs 33.7 } 21.0 mL, p = 0.07. These findings indicated that UCB containing late adherent cells was suitable for a cell source of MSCs. Next, we examined whether these late adherent cells (C2 and C3 cells) had properties consistent with those of MSCs. Both C2 and C3 cells showed spindle-shaped fibroblast-like morphology and the same immunophenotype as C1 cells: positive for CD73, CD90 and CD105, and negative for CD34, CD45 and HLA-DR. They had osteogenic, adipogenic and chondrogenic differentiation potentials in vitro. These findings are the minimal criteria for MSCs (Cytotherapy, 2006; 8:315). Finally, we evaluated the hematopoiesis-supporting activity of these cells in vitro and in vivo. CD45-positive hematopoietic cells were expanded when co-cultured of CD34-positive hematopoietic progenitor cells (6 ~ 102 cells) with C2 or C3 cells (2 ~ 104 cells) in vitro as much as when co-cultured with C1 cells (Figure A). In vivo analysis was conducted by using subcutaneous transplantation of MSCs on NOD/SCID mice (Int J Hematol, 2015; 102: 218). C2 cells induced trabecular bone formation and bone marrow hematopoiesis as well as C1 cells, however, C3 cells did not induce hematopoiesis (Figure B).In conclusion, we demonstrated that UCB contains a late adherent cell subpopulation with the same characteristics and hematopoiesis-supporting activity as those of UCBMSCs isolated using the conventional method. The continuance of cell culture without discarding suspension cells could improve the efficiency of isolation of MSCs from UCB. [Display omitted] DisclosuresHirai:Kyowa Hakko Kirin: Research Funding; Novartis Pharma: Research Funding. Maekawa:Bristol-Myers K.K.: Research Funding.

Quantifying adhesion mechanisms and dynamics of human hematopoietic stem and progenitor cells.

Using planar lipid membranes with precisely defined concentrations of specific ligands, we have determined the binding strength between human hematopoietic stem cells (HSC) and the bone marrow niche. The relative significance of HSC adhesion to the surrogate niche models via SDF1α-CXCR4 or N-cadherin axes was quantified by (a) the fraction of adherent cells, (b) the area of tight adhesion, and (c) the critical pressure for cell detachment. We have demonstrated that the binding of HSC to the niche model is a cooperative process, and the adhesion mediated by the CXCR4- SDF1α axis is stronger than that by homophilic N-cadherin binding. The statistical image analysis of stochastic morphological dynamics unraveled that HSC dissipated energy by undergoing oscillatory deformation. The combination of an in vitro niche model and novel physical tools has enabled us to quantitatively determine the relative significance of binding mechanisms between normal HSC versus leukemia blasts to the bone marrow niche.

Umbilical cord blood as a source of nerve and stem cells

The aim of the study was to assess proliferative ability of the stem cells in the umbilical cord blood and their potential to differentiate in in vitro culture. The material consisted of 14 samples of umbilical cord blood collected from the umbilical cord vein. Mononuclear cells were isolated using the method of density gradient medium. Next, CD34 cells were isolated from the interphase with the use of the VarioMACS sorter and anti-CD34 antibodies. Long-term cultures were conducted on Iscove's modified Dulbecco medium (IMDM) with addition of granulocyte-macrophage colony stimulating factor (GM-CSF) and nerve growth factor (NGF). Qualitative identification was performed using the May-Grunwald-Giemsy staining method, taking photographs with a confocal microscope, and with the immunoenzymatic method. In our research, CD34+ stem cells constituted 1.16% of the mononuclear cells, and after centrifugation in medium 0.37% of leukocytes in whole umbilical cord blood. Even after 60 days of culture without addition of the growth factors, CD34+ cells were present in the fraction of adherent cells. After stimulation with GM-CSF and NGF a part of the umbilical cord blood cells were transformed into nerve cells (presence of neuron-specific enolase was shown) and into cells morphologically similar to fibroblast and dendritic cells. After stimulation with GM-CSF and NGF cytokines, the umbilical cord blood cells proliferate in long-term medium, transform into nerve cells and into cells similar to fibroblast and dendritic cells.

Abstract 2539: Clinical prospective study of PepTivator® WT1-pulsed DC vaccines with assessment of immunological responses in solid tumors

Abstract Wilms' tumor gene 1 (WT1) is reported to be expressed in various types of malignancies and be one of promising target antigens for cancer immunotherapy. However, the synthetic short peptide vaccines based on the WT1 sequences limit a candidate patient by its HLA restriction. In this study, we performed the clinical study for dendritic cell (DC) vaccines using peptide pools consisting mainly of 15-mer sequences with 11 amino acids overlaps of full-length WT1 protein (PepTivator® WT1). We previously reported that copulsing tumor antigen-pulsed DC with zoledronate (Zol) could efficiently enhance expansion of tumor antigen specific CD8+ T cells by activation of gamma-delta T cells. In the present study, we studied the immunological response induced by PepTivator® WT1-pulsed Zol-DC vaccines in solid tumors, and also investigated the feasibility and safety of PepTivator® WT1-pulsed Zol-DC vaccines. Five patients with WT1-positive solid tumor (ovary: 4, lung: 1) were enrolled. PBMCs were collected by leukapheresis from the patients and the adherent cell fraction was differentiated into DCs by conventional method. In this vaccine therapy, PepTivator® WT1-pulsed Zol-DC (more than 5x106 cells per injection) was administered to patients subcutaneously for six times every two weeks. In order to examine the specific immune responses to WT1 antigen, PBMCs before therapy (pre-PBMC) and PBMCs after therapy (post-PBMC) were in vitro restimulated with PepTivator® WT1-pulsed Zol-DCs for 14 days and the expanded cells were separated into CD8+ cells and CD4+ cells by MACS, and the antigen specific IFN-gamma- producing cells were detected by ELISpot assay. As a result, no serious adverse events were seen in three patients who had completed all DC vaccines (Patient ID: #1, #2 and #5). DTH reaction was observed in all three patients. During in vitro restimulation and expansion, there was more than three-fold higher expansion of T cells responding to PepTivator® WT1-pulsed Zol-DC in post-PBMC compared to pre-PBMC among two of three patients (#1, #2). In these two patients, the antigen specific IFN-gamma-producing cells were detected in CD8+ T cells from post-PBMC, but not from pre-PBMC. As for CD4+ T cells, however, the antigen specific IFN-gamma-producing cells were not detected, neither in pre-PBMC, nor in post-PBMC. We found that PepTivator® WT1-pulsed Zol-DC vaccines are feasible and safe, and can induce antigen specific CD8+ T cells. We need more extensive investigation for the immunological monitoring and clinical outcome. Citation Format: Shoko Saiwaki, Shigenori Goto, Masashi Takahara, Haruka Matsushita, Takashige Kondo, Hermann Bohnenkamp, Ryuji Maekawa, Takashi Kamigaki. Clinical prospective study of PepTivator® WT1-pulsed DC vaccines with assessment of immunological responses in solid tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2539. doi:10.1158/1538-7445.AM2014-2539

Roles of adherent myogenic cells and dynamic culture in engineered muscle function and maintenance of satellite cells

Highly functional engineered skeletal muscle constructs could serve as physiological models of muscle function and regeneration and have utility in therapeutic replacement of damaged or diseased muscle tissue. In this study, we examined the roles of different myogenic cell fractions and culturing conditions in the generation of highly functional engineered muscle. Fibrin-based muscle bundles were fabricated using either freshly-isolated myogenic cells or their adherent fraction pre-cultured for 36 h. Muscle bundles made of these cells were cultured in both static and dynamic conditions and systematically characterized with respect to early myogenic events and contractile function. Following 2 weeks of culture, we observed both individual and synergistic benefits of using the adherent cell fraction and dynamic culture on muscle formation and function. In particular, optimal culture conditions resulted in significant increase in the total cross-sectional muscle area (∼3-fold), myofiber size (∼1.6-fold), myonuclei density (∼1.2-fold), and force generation (∼9-fold) compared to traditional use of freshly-isolated cells and static culture. Curiously, we observed that only a simultaneous use of the adherent cell fraction and dynamic culture resulted in accelerated formation of differentiated myofibers which were critical for providing a niche-like environment for maintenance of a satellite cell pool early during culture. Our study identifies key parameters for engineering large-size, highly functional skeletal muscle tissues with improved ability for retention of functional satellite cells.

Purified Dendritic Cell-Tumor Fusion Hybrids Supplemented with Non-Adherent Dendritic Cells Fraction Are Superior Activators of Antitumor Immunity

BackgroundStrong evidence supports the DC-tumor fusion hybrid vaccination strategy, but the best fusion product components to use remains controversial. Fusion products contain DC-tumor fusion hybrids, unfused DCs and unfused tumor cells. Various fractions have been used in previous studies, including purified hybrids, the adherent cell fraction or the whole fusion mixture. The extent to which the hybrids themselves or other components are responsible for antitumor immunity or which components should be used to maximize the antitumor immunity remains unknown.MethodsPatient-derived breast tumor cells and DCs were electro-fused and purified. The antitumor immune responses induced by the purified hybrids and the other components were compared.ResultsExcept for DC-tumor hybrids, the non-adherent cell fraction containing mainly unfused DCs also contributed a lot in antitumor immunity. Purified hybrids supplemented with the non-adherent cell population elicited the most powerful antitumor immune response. After irradiation and electro-fusion, tumor cells underwent necrosis, and the unfused DCs phagocytosed the necrotic tumor cells or tumor debris, which resulted in significant DC maturation. This may be the immunogenicity mechanism of the non-adherent unfused DCs fraction.ConclusionsThe non-adherent cell fraction (containing mainly unfused DCs) from total DC/tumor fusion products had enhanced immunogenicity that resulted from apoptotic/necrotic tumor cell phagocytosis and increased DC maturation. Purified fusion hybrids supplemented with the non-adherent cell population enhanced the antitumor immune responses, avoiding unnecessary use of the tumor cell fraction, which has many drawbacks. Purified hybrids supplemented with the non-adherent cell fraction may represent a better approach to the DC-tumor fusion hybrid vaccination strategy.

Non-Adherent Mesenchymal Progenitors from Adipose Tissue Stromal Vascular Fraction

In primary human bone marrow cultures, the initial adherent cell fraction has been shown to provide a microenvironment for self-renewal of primitive non-adherent mesenchymal progenitors (non-adherent progenitors of bone marrow stroma [BM-NAMP]), with increased differentiation potential compared to adherent colony-forming units-fibroblast (CFU-f). The present study investigates whether NAMP exist also in cultures of stromal vascular fraction (SVF) cells derived from human adipose tissue. Adipose-tissue NAMP (AT-NAMP) were shown to be stably non-adherent and their number correlated with the number of the initial adhering CFU-f. Unlike BM-NAMP, AT-NAMP did not propagate in suspension in serial replating experiments and the number of colonies steadily decreased with each replating step. However, when AT-NAMP were kept on the initially adhering SVF cells, they could significantly expand without loss of clonogenic, proliferation, and differentiation potential. Although AT-NAMP progeny differentiated into mesodermal lineages similar to that of adherent CFU-f, it was enriched in early mesenchymal progenitor populations, characterized by increased expression of SSEA-4 and CD146. Furthermore, FGF-2 supported AT-NAMP survival and could not be replaced by another mitogenic factor, such as platelet derived growth factor BB. In conclusion, these data suggest that the SVF adherent fraction provides niche signals that regulate the expansion of adipose non-adherent mesenchymal progenitors with the maintenance of their potency. The biological differences described between BM- and AT-NAMP further qualify the properties of the stroma from different tissues and will be relevant for the selection of a cell source for specific regeneration strategies.

Streptozotocin-induced diabetes in rats diminishes the size of the osteoprogenitor pool in bone marrow

AimsBone formation is reduced in animals and humans with type 1 diabetes, leading to lower bone mass and inferior osseous healing. Since bone formation greatly depends on the recruitment of osteoblasts from their bone marrow precursors, we tested whether experimental type 1 diabetes in rats diminishes the number of bone marrow osteoprogenitors. MethodsDiabetes was induced by 65mg/kg streptozotocin and after 4 weeks, femoral bone marrow cells were extracted and cultured. Tibia and femur were frozen for further analysis. ResultsThe size of the osteoprogenitor pool in bone marrow of diabetic rats was significantly reduced, as evidenced by (1) lower (∼35%) fraction of adherent stromal cells (at 24h of culture); (2) lower (20–25%) alkaline phosphatase activity at 10 days of culture; and (3) lower (∼40%) mineralized nodule formation at 21 days of culture. Administration of insulin to hyperglycemic rats normalized glycemia and abrogated most of the decline in ex vivo mineralized nodule formation. Apoptotic cells in tibial bone marrow were more numerous in hyperglycemic rats. Also, the levels of malondialdehyde (indicator of oxidative stress) were significantly elevated in bone marrow of diabetic animals. ConclusionsExperimental type 1 diabetes diminishes the osteoprogenitor population in bone marrow, possibly due to increased apoptosis via Oxidative Stress. Reduced number of osteoprogenitors is likely to impair osteoblastogenesis, bone formation, and bone healing in diabetic animals.

Integrity and Cell Cycle Of Leukemic Cells Are Modified By Mesenchymal Stem Cells From Healthy Individuals and AML Patients

IntroductionThe contribution of the stromal cell compartment to leukemogenesis remains poorly understood. Several studies have described abnormalities involving this compartment in acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS) including proliferative defect of mesenchymal stem/stromal cells (MSCs) as reported by our group in AML patients (Domenech et al., Haematologica 2012, EHA meeting, abstr. 37). Recently, evidence of an involvement of MSCs in the leukemic process has been provided in murine models (Walkley et al., Cell 2007; Raaijmakers et al. Nature 2010). In the present study, we investigated potential modifications of human AML blast cell biology induced in vitro by MSCs from healthy individuals and AML patients. MethodsBone marrow MSCs from 6 AML patients (3 M0, 3 M1 [FAB classification]) were compared to those from 6 normal individuals. All the MSCs were analyzed at the end of the second passage of culture. Capacity of MSCs to influence leukemic cell behavior was assessed by co-culture with immature leukemia cells from KG1a line and with heterologous or autologous primary blast cells from the AML patients. Apoptotic cell frequencies, cell cycle phase distributions, and presence of DNA double-strand breaks of leukemia cells with or without MSCs were performed by flow cytometry. ResultsIn co-cultures, no difference in leukemia cell adhesion were found on AML and normal MSCs. The presence of MSCs reduced apoptosis of primary blast cells (-30%, p=0.002) although no effect was observed on campthotecin-induced apoptosis of KG1a cells, as compared to MSC-free culture conditions. Cell cycle analysis revealed that G0/G1 ratios of KG1a cells were strongly increased by the presence of MSCs considering the whole cells (+128%, p=0.005) as well as the cell fraction adherent to MSCs (+138%, p=0.006). Comparable results were obtained with primary blast cells considering all the cells (+51%, p=0.117), especially within the adherent cell fraction (+229%, p=0.004). In addition, direct contact with MSCs was associated with a slight decrease in the proportion of blast cells with DNA double-strand breaks compared to MSC-free cultures (-10%, p= 0.034). No significant differences were found for all these results when co-cultures with AML and normal MSCs were compared. Likewise, no differences existed between co-cultures with M0 and M1 AML MSCs or between co-cultures of autologous and heterologous primary blast cells with AML MSCs. ConclusionsThis study shows that MSCs influence leukemic cell behavior, irrespective of their healthy or leukemic origin. In particular, they protect blast cells from apoptosis and induce their quiescence, (mainly by direct contact) which could contribute to decreased yields of DNA double-strand breaks, a source of genetic instability. Further experiments are in progress to evaluate potential changes in the capacity of AML MSCs to support normal hematopoiesis. Disclosures:Gyan:FRESENIUS KABI: Consultancy, Research Funding. Domenech:Celgene Corporation: Research Funding.

A Brucella Virulence Factor Targets Macrophages to Trigger B-cell Proliferation

Brucella spp. and Trypanosoma cruzi are two intracellular pathogens that have no evolutionary common origins but share a similar lifestyle as they establish chronic infections for which they have to circumvent the host immune response. Both pathogens have a virulence factor (prpA in Brucella and tcPrac in T. cruzi) that induces B-cell proliferation and promotes the establishment of the chronic phase of the infectious process. We show here that, even though PrpA promotes B-cell proliferation, it targets macrophages in vitro and is translocated to the cytoplasm during the intracellular replication phase. We observed that PrpA-treated macrophages induce the secretion of a soluble factor responsible for B-cell proliferation and identified nonmuscular myosin IIA (NMM-IIA) as a receptor required for binding and function of this virulence factor. Finally, we show that the Trypanosoma cruzi homologue of PrpA also targets macrophages to induce B-cell proliferation through the same receptor, indicating that this virulence strategy is conserved between a bacterial and a protozoan pathogen.

β&lt;sub&gt;2&lt;/sub&gt;-Agonist Clenbuterol Suppresses Bacterial Phagocytosis of Splenic Macrophages Expressing High Levels of Macrophage Receptor with Collagenous Structure

Splenic marginal zone macrophages expressing macrophage receptor with collagenous structure (MARCO) contribute to the clearance of blood-borne pathogens. We determined a splenic adherent cell fraction abundantly containing cells expressing a higher level of MARCO by flow cytometry, and examined the effects of daily administration of an anabolic dose of β2-agonist clenbuterol on the phagocytic capacity of the cells in mice. After 6 weeks of clenbuterol (1.0 mg/kg body weight/d) or vehicle administration to the mice, splenic adherent cells were isolated. These cells were separated into three cell-size subpopulations. Among them, the small-cell subpopulation contained abundantly the cells with markedly higher levels of MARCO and exhibited more intense phagocytic capacity against Escherichia coli, as compared with the other subpopulations. The phagocytic capacity of the small cells was significantly reduced after clenbuterol administration. These results suggest that the utilization of clenbuterol as doping drug impairs bacterial clearance in the spleen.

Differentiation and selection of hepatocyte precursors in suspension spheroid culture of transgenic murine embryonic stem cells.

Embryonic stem cell-derived hepatocyte precursor cells represent a promising model for clinical transplantations to diseased livers, as well as for establishment of in vitro systems for drug metabolism and toxicology investigations. This study aimed to establish an in vitro culture system for scalable generation of hepatic progenitor cells. We used stable transgenic clones of murine embryonic stem cells possessing a reporter/selection vector, in which the enhanced green fluorescent protein- and puromycin N-acetyltransferase-coding genes are driven by a common alpha-fetoprotein gene promoter. This allowed for “live” monitoring and puromycin selection of the desired differentiating cell type possessing the activated alpha-fetoprotein gene. A rotary culture system was established, sequentially yielding initially partially selected hepatocyte lineage-committed cells, and finally, a highly purified cell population maintained as a dynamic suspension spheroid culture, which progressively developed the hepatic gene expression phenotype. The latter was confirmed by quantitative RT-PCR analysis, which showed a progressive up-regulation of hepatic genes during spheroid culture, indicating development of a mixed hepatocyte precursor-/fetal hepatocyte-like cell population. Adherent spheroids gave rise to advanced differentiated hepatocyte-like cells expressing hepatic proteins such as albumin, alpha-1-antitrypsin, cytokeratin 18, E-cadherin, and liver-specific organic anion transporter 1, as demonstrated by fluorescent immunostaining. A fraction of adherent cells was capable of glycogen storage and of reversible up-take of indocyanine green, demonstrating their hepatocyte-like functionality. Moreover, after transplantation of spheroids into the mouse liver, the spheroid-derived cells integrated into recipient. These results demonstrate that large-scale hepatocyte precursor-/hepatocyte-like cultures can be established for use in clinical trials, as well as in in vitro screening assays.

Osteogenic- and Adipogenic- Differentiated Bone Marrow-Derived Mesenchymal Stem Cells Fail to Support Expansion of Adult Hematopoietic Stem Cells

Abstract 4810Ex vivo expansion and manipulation of primitive hematopoietic cells has become a major goal in the experimental hematology, because of its potential relevance in the development of therapeutic strategies aimed at treating a diverse group of hematologic disorders. Osteoblasts, mesenchymal stem/progenitor cells (MSC/MPC), adipocytes, reticular cells, endothelial cells and other stromal cells, have been implicated in regulation of HSC maintenance in endosteal and perivascular niches. These niches facilitate the signaling networks that control the balance between self-renewal and differentiation.In the present study, we evaluated and compared the effects of three different stromal feeder layers on expansion of HSPC derived from BM and cord blood (CB): BM mesenchymal stem cells (MSC), osteoblast-differentiated BM mesenchymal stem cells (Ost-MSC) and adipocyte-differentiated BM mesenchymal stem cells (Ad-MSC). BM-MSC cultures were established from plastic adherent BM cell fractions and analyzed for immunophenotype, frequency of colony forming units (CFU-F), frequency of osteo- (CFU-Ost) and adipo- (CFU-Ad) lineage progenitors. Cultures with similar clonogenity (CFU-F: 26,4 ± 4,5%) and progenitors frequency (CFU-Ost: 14,7 ± 4,5%; CFU-Ad: 13,3 ± 4,5%) were selected for co-culture experiments. All MSC were positive for stromal cell-associated markers (CD105, CD90, CD166, CD73) and negative for hematopoietic lineage cells markers (CD34, CD19, CD14, CD45). CD34+ cells were separared from BM and CB samples by magnetic cell sorting (MACS) and analyzed for CD34, CD38 and CD45 expression.Feeder layers (MSC, Ost-MSC, Ad-MSC) were prepared in 24-well plates prior to co-culture experiments: MSCs (4×104 cells/well) were cultured for 24 h and either used for following experiments or stimulated to differentiate into either osteoblasts or adipoctes according to standard protocols. CD34+ cells (3500-10000 cells per well) were co-cultured in Stem Span media with or without a feeder layers and in the presence of cytokines (10 ng/mL Flt3-L, 10 ng/mL SCF, 10ng/mL IL-7) for 7 days. Expanded cells were analyzed for CD34, CD38 and CD45 expression.Results are shown on figures 1 and 2. As expected, CB-derived HSPC expanded much more effectively than BM-derived HSPC. The similar levels of expansion were observed for both, the total number of HSPC, and more primitive CD34+CD38- fraction in the presence of all three feeder layers. Ost-MSC supported CB-derived HSPC slightly better than MSC and Ad-MSC which is in a good agreement with data from literature (Mishima et.al., European Journal of Haematology, 2010), but difference was not statistically significant. In contrast, whereas BM-MSC feeder facilitated CD34+CD38- fraction in BM-derived HSPC, Adipocyte-differentiated MSC and osteoblast-differentiated MSC failed to support BM-derived CD34+CD38- expansion (11,4 ±.4 folds for MSC vs 0,9 ±.0,14 for Ad-MSC, n=5, p<0,01 and 0,92 ±.0,1 for Ost-MSC, n=5, p<0,01). [Display omitted] [Display omitted] Conclusion:BM- and CB-derived CD34+CD38- cells differ in their dependence of bone marrow stroma. Coctail of growth factors facilitate CB HSPC expansion irrespective of lineage differentiation of supporting MSC feeder layer. In contrast, primitive BM CD34+CD38- HSPC were able to expand only on not differentiated MSC. Disclosures:No relevant conflicts of interest to declare.

Expression of PTH in Bone Marrow Stem Cells in Adult SD Rats

Abstract 4817 Introduction:Parathyroid hormone (PTH) is a major regulator of calcium and phosphate metabolism and is secreted by the cells of the parathyroid gland. Recent research showed that PTH treatment may regulate the hematopoietic stem cell niche, leading to beneficial effects on the HSC pool, and increasing stem cell number and the number of stem cells mobilized into the bloodstream. Many studies demonstrated that the serum PTH was decreased but maintained at the lower level suggesting persisting PTH secretion after total parathyroidectomy without autotransplantation. Some research showed that the thymus is another source of PTH. Can the bone marrow cells express PTH mRNA and release PTH and be a third source of PTH production? The aim of this study was to explore whether PTH could be expressed in bone marrow stem cells(BMSCs). Method:BMSCs were separated from adult male SD rat bone marrow and were cultured in DMEM supplemented with 10% fetal bovine serum. The total RNA was extracted using Trizol one-step method. RT-PCR was performed to examine the expression of PTH and GAPDH in BMSCs. The optical densities (ODs) value of RT-PCR product was measured using the Gel Imageware System. The signal intensity of PTH bands was normalized by corresponding GAPDH bands. The ODs in each sample were expressed as a ratio PTH over GAPDH densitometric intensities. The resultant PCR product of PTH was sequenced in both directions. Immunocytochemistry staining was utilized to examine the expression of PTH in the BMSCs from adherent cell fraction of the 2nd passage. Results:The expressions of PTH mRNA in BMSCs were detected in all 11 rats by RT-PCR. The PCR product was 193bp for PTH (Fig 1). The ratio of PTH over GAPDH ODs value ranged from 0.4407 to 3.1506, and the average ODs value (m ± SD) was 1.2617 ± 0.8953. The sequence of the PTH PCR products matched 100% with the PTH sequence in Genebank (Gene ID: 24694 Pth; gb K01268.1 RATPTH3, Rat parathyroid hormone gene, exons II and III). Some of the BMSCs were PTH positive by immunocytochemistry. The PTH positive cells were middle in size and spindle. PTH staining was localized in the cytoplasma and cell membrane of BMSCs cultured (Fig 2). Conclusion:The PTH mRNA and protein were detected in BMSCs. The results suggest that the BMSCs may be a third cell source responsible for the synthesis and secretion of PTH. It would be interesting to know what type of cells in BMSCs is secreating the PTH and to explore the role of PTH in bone marrow stem cell niche. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.