技术资料

Cell therapy is a novel promising option for treatment of ischemic diseases. Administered endothelial progenitor cells (EPCs) are recruited to ischemic regions and improve neovascularization. However,the number of cells that home to ischemic tissues is restricted. The GTPase Rap1 plays an important role in the regulation of adhesion and chemotaxis. We investigated whether pharmacologic activation of Epac1,a nucleotide exchange protein for Rap1,which is directly activated by cAMP,can improve the adhesive and migratory capacity of distinct progenitor cell populations. Stimulation of Epac by a cAMP-analog increased Rap1 activity and stimulated the adhesion of human EPCs,CD34(+) hematopoietic progenitor cells,and mesenchymal stem cells (MSCs). Specifically,short-term stimulation with a specific Epac activator increased the beta2-integrin-dependent adhesion of EPCs to endothelial cell monolayers,and of EPC and CD34(+) cells to ICAM-1. Furthermore,the Epac activator enhanced the beta1-integrin-dependent adhesion of EPCs and MSCs to the matrix protein fibronectin. In addition,Epac1 activation induced the beta1- and beta2-integrin-dependent migration of EPCs on fibronectin and fibrinogen. Interestingly,activation of Epac rapidly increased lateral mobility of beta1- and beta2-integrins,thereby inducing integrin polarization,and stimulated beta1-integrin affinity,whereas the beta2-integrin affinity was not increased. Furthermore,prestimulation of EPCs with the Epac activator increased homing to ischemic muscles and neovascularization-promoting capacity of intravenously injected EPCs in the model of hind limb ischemia. These data demonstrate that activation of Epac1 increases integrin activity and integrin-dependent homing functions of progenitor cells and enhances their in vivo therapeutic potential. These results may provide a platform for the development of novel therapeutic approaches to improve progenitor cell homing. View Publication

Human bone marrow multipotent mesenchymal stromal cells are progenitor cells that can be expanded in vitro and differentiate into various cells of mesodermal origin. They contribute to the bone marrow reticular niche,where mature B cells and long-lived plasma cells are maintained. Multipotent mesenchymal stromal cells were recently shown to modulate T- and B-cell proliferation and differentiation,dendritic cell maturation,and natural killer activity. These immunoregulatory properties encouraged a possible use of these cells to modulate autoimmune responses in humans. We studied the influence of bone marrow mesenchymal stem cells on highly purified B-cell subsets isolated from healthy donors and total B cells from pediatric systemic lupus erythematosus patients. Bone marrow mesenchymal stem cells promoted proliferation and differentiation into immunoglobulin-secreting cells of transitional and naive B cells stimulated with an agonist of Toll-like receptor 9,in the absence of B cell receptor triggering. They strongly enhanced proliferation and differentiation into plasma cells of memory B-cell populations. A similar effect was observed in response to polyclonal stimulation of B cells isolated from pediatric patients with systemic lupus erythematosus. This study casts important questions on bone marrow mesenchymal stem cells as a therapeutic tool in autoimmune diseases in which B-cell activation is crucially implicated in the pathogenesis of the disease. View Publication

Implanted materials,such as medical devices,provoke the body to initiate an inflammatory reaction,known as the foreign body reaction (FBR),which causes several complications for example in hip prostheses,silicone implants,peritoneal dialysis catheters and left ventricular assist devices. FBR is initiated by macrophage adherence and results in granulation tissue formation. The early immunobiology and development of this tissue is not completely understood,but there are indications from related myofibroblast-forming diseases such as vascular repair and fibrosis that primitive stem cells also play a role in the formation of FBR-tissue. To investigate this,acellular photo-oxidized bovine pericardium patches were implanted intraperitoneally in rats and retrieved at time-points ranging from 6h to 7 days. A significant fraction of Sca-1(+) (6h-2 days),c-kit(+),CD34(+) and CD271(+) (2-3 days) stem/progenitor cells were detected. Colony-forming and differentiation capacity of the primitive stem cells into adipo-,osteo-,and myofibroblasts were shown. The presence of these primitive cells and their myofibroblastic differentiation potential were also confirmed at RNA level. The identification of specific primitive cells during FBR may have important implications for the inflammatory responses to inert materials and their use in tissue prostheses. View Publication

Recently,a number of clinical trials used either mesenchymal stem cells (MSCs) or natural killer (NK) cells in an attempt to improve the effectiveness of hematopoietic stem cell transplantation (HSCT). In view of the relevant role of both MSCs and NK cells in HSCT,we have recently explored the result of possible interactions between the 2 cell types. We found that activated NK cells could kill MSCs,whereas MSCs strongly inhibited interleukin-2 (IL-2)-induced NK-cell proliferation. In this study,we further analyzed the inhibitory effect exerted by MSCs on NK cells. We show that MSCs not only inhibit the cytokine-induced proliferation of freshly isolated NK cells but also prevent the induction of effector functions,such as cytotoxic activity and cytokine production. Moreover,we show that this inhibitory effect is related to a sharp down-regulation of the surface expression of the activating NK receptors NKp30,NKp44,and NKG2D. Finally,we demonstrate that indoleamine 2,3-dioxygenase and prostaglandin E2 represent key mediators of the MSC-induced inhibition of NK cells. View Publication

Mesenchymal stem cells (MSC) establish close interactions with bone marrow sinusoids in a putative perivascular niche. These vessels contain a large storage pool of mature nonproliferating neutrophils. Here,we have investigated the effects of human bone marrow MSC on neutrophil survival and effector functions. MSC from healthy donors,at very low MSC:neutrophil ratios (up to 1:500),significantly inhibited apoptosis of resting and interleukin (IL)-8-activated neutrophils and dampened N-formyl-l-methionin-l-leucyl-l-phenylalanine (f-MLP)-induced respiratory burst. The antiapoptotic activity of MSC did not require cell-to-cell contact,as shown by transwell experiments. Antibody neutralization experiments demonstrated that the key MSC-derived soluble factor responsible for neutrophil protection from apoptosis was IL-6,which signaled by activating STAT-3 transcription factor. Furthermore,IL-6 expression was detected in MSC by real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Finally,recombinant IL-6 was found to protect neutrophils from apoptosis in a dose-dependent manner. MSC had no effect on neutrophil phagocytosis,expression of adhesion molecules,and chemotaxis in response to IL-8,f-MLP,or C5a. These results support the following conclusions: (a) in the bone marrow niche,MSC likely protect neutrophils of the storage pool from apoptosis,preserving their effector functions and preventing the excessive or inappropriate activation of the oxidative metabolism,and (b) a novel mechanism whereby the inflammatory potential of activated neutrophils is harnessed by inhibition of apoptosis and reactive oxygen species production without impairing phagocytosis and chemotaxis has been identified. View Publication

Mesenchymal stromal cells (MSC) provide a supportive cellular microenvironment and are able to maintain the self-renewal capacity of hematopoietic progenitor cells (HPC). Isolation procedures for MSC vary extensively,and this may influence their biologic properties. In this study,we have compared human MSC isolated from bone marrow (BM) using two culture conditions,from cord blood (CB),and from adipose tissue (AT). The ability to maintain long-term culture-initiating cell frequency and a primitive CD34(+)CD38(-) immunophenotype was significantly higher for MSC derived from BM and CB compared with those from AT. These results were in line with a significantly higher adhesion of HPC to MSC from BM and CB versus MSC from AT. We have compared the cytokine production of MSC by cytokine antibody arrays,enzyme-linked immunosorbent assay,and a cytometric bead array. There were reproducible differences in the chemokine secretion profiles of various MSC preparations,but there was no clear concordance with differences in their potential to maintain primitive function of HPC. Global gene expression profiles of MSC preparations were analyzed and showed that adhesion proteins including cadherin-11,N-cadherin,vascular cell adhesion molecule 1,neural cell adhesion molecule 1,and integrins were highly expressed in MSC preparations derived from BM and CB. Thus,MSC from BM and CB are superior to MSC from AT for maintenance of primitive HPC. The latter property is associated with specific molecular profiles indicating the significance of cell-cell junctions but not with secretory profiles. Disclosure of potential conflicts of interest is found at the end of this article. View Publication

The aims of our study were to verify whether it was possible to generate in vitro,from different adult human tissues,a population of cells that behaved,in culture,as multipotent stem cells and if these latter shared common properties. To this purpose,we grew and cloned finite cell lines obtained from adult human liver,heart,and bone marrow and named them human multipotent adult stem cells (hMASCs). Cloned hMASCs,obtained from the 3 different tissues,expressed the pluripotent state-specific transcription factors Oct-4,NANOG,and REX1,displayed telomerase activity,and exhibited a wide range of differentiation potential,as shown both at a morphologic and functional level. hMASCs maintained a human diploid DNA content,and shared a common gene expression signature,compared with several somatic cell lines and irrespectively of the tissue of isolation. In particular,the pathways regulating stem cell self-renewal/maintenance,such as Wnt,Hedgehog,and Notch,were transcriptionally active. Our findings demonstrate that we have optimized an in vitro protocol to generate and expand cells from multiple organs that could be induced to acquire morphologic and functional features of mature cells even embryologically not related to the tissue of origin. View Publication

Mesenchymal stem cells (MSC) are part of the bone marrow that provides signals supporting survival and growth of bystander hematopoietic stem cells (HSC). MSC modulate also the immune response,as they inhibit proliferation of lymphocytes. In order to investigate whether MSC can support survival of T cells,we investigated MSC capacity of rescuing T lymphocytes from cell death induced by different mechanisms. We observed that MSC prolong survival of unstimulated T cells and apoptosis-prone thymocytes cultured under starving conditions. MSC rescued T cells from activation induced cell death (AICD) by downregulation of Fas receptor and Fas ligand on T cell surface and inhibition of endogenous proteases involved in cell death. MSC dampened also Fas receptor mediated apoptosis of CD95 expressing Jurkat leukemic T cells. In contrast,rescue from AICD was not associated with a significant change of Bcl-2,an inhibitor of apoptosis induced by cell stress. Accordingly,MSC exhibited a minimal capacity of rescuing Jurkat cells from chemically induced apoptosis,a process disrupting the mitochondrial membrane potential regulated by Bcl-2. These results suggest that MSC interfere with the Fas receptor regulated process of programmed cell death. Overall,MSC can inhibit proliferation of activated T cells while supporting their survival in a quiescent state,providing a model of their activity inside the HSC niche. Disclosure of potential conflicts of interest is found at the end of this article. View Publication

Umbilical cord blood (UCB) has been used as a potential source of various kinds of stem cells,including hematopoietic stem cells,mesenchymal stem cells,and endothelial progenitor cells (EPCs),for a variety of cell therapies. Recently,EPCs were introduced for restoring vascularization in ischemic tissues. An appropriate procedure for isolating EPCs from UCB is a key issue for improving therapeutic efficacy and eliminating the unexpected expansion of nonessential cells. Here we report a novel method for isolating EPCs from UCB by a combination of negative immunoselection and cell culture techniques. In addition,we divided EPCs into 2 subpopulations according to the aldehyde dehydrogenase (ALDH) activity. We found that EPCs with low ALDH activity (Alde-Low) possess a greater ability to proliferate and migrate compared to those with high ALDH activity (Alde-High). Moreover,hypoxia-inducible factor proteins are up-regulated and VEGF,CXCR4,and GLUT-1 mRNAs are increased in Alde-Low EPCs under hypoxic conditions,while the response was not significant in Alde-High EPCs. In fact,the introduction of Alde-Low EPCs significantly reduced tissue damage in ischemia in a mouse flap model. Thus,the introduction of Alde-Low EPCs may be a potential strategy for inducing rapid neovascularization and subsequent regeneration of ischemic tissues. View Publication

AIM: The recently published REPAIR-AMI and ASTAMI trial showed differences in contractile recovery of left ventricular function after infusion of bone marrow-derived cells in acute myocardial infarction. Since the trials used different protocols for cell isolation and storage (REPAIR-AMI: Ficoll,storage in X-vivo 10 medium plus serum; ASTAMI: Lymphoprep,storage in NaCl plus plasma),we compared the functional activity of BMC isolated by the two different protocols. METHODS AND RESULTS: The recovery of total cell number,colony-forming units (CFU),and the number of mesenchymal stem cells were significantly reduced to 77 +/- 4%,83 +/- 16%,and 65 +/- 15%,respectively,when using the ASTAMI protocol compared with the REPAIR protocol. The capacity of the isolated BMC to migrate in response to stromal cell-derived factor 1 (SDF-1) was profoundly reduced when using the ASTAMI cell isolation procedure (42 +/- 8% and 78 +/- 3% reduction in healthy and CAD-patient cells,respectively). Finally,infusion of BMC into a hindlimb ischaemia model demonstrated a significantly blunted blood-flow-recovery by BMC isolated with the ASTAMI protocol (54 +/- 6% of the effect obtained by REPAIR cells). Comparison of the individual steps identified the use of NaCl and plasma for cell storage as major factors for functional impairment of the BMC. CONCLUSION: Cell isolation protocols have a major impact on the functional activity of bone marrow-derived progenitor cells. The assessment of cell number and viability may not entirely reflect the functional capacity of cells in vivo. Additional functional testing appears to be mandatory to assure proper cell function before embarking on clinical cell therapy trials. View Publication

Stem cells exhibit a promiscuous gene expression pattern. We show herein that the early embryo and adult MSCs express B-cell receptor component mRNAs. To examine possible bearings of these genes on the expressing cells,we studied immunoglobulin mu chain-deficient mice. Pregnant mu chain-deficient females were found to produce a higher percentage of defective morulae compared with control females. Structure analysis indicated that the mu mRNA species found in embryos and in mesenchyme consist of the constant region of the mu heavy chain that encodes a recombinant 50-kDa protein. In situ hybridization localized the constant mu gene expression to loose mesenchymal tissues within the day-12.5 embryo proper and the yolk sac. In early embryo and in adult mesenchyme from mu-deficient mice,delta replaced mu chain,implying a possible requirement of these alternative molecules for embryo development and mesenchymal functions. Indeed,overexpression of the mesenchymal-truncated mu heavy chain in 293T cells resulted in specific subcellular localization and in G(1) growth arrest. The lack of such occurrence following overexpression of a complete,rearranged form of mu chain suggests that the mesenchymal version of this mRNA may possess unique functions. View Publication

In previous reports,we have demonstrated that only direct cell-cell contact with stromal cells,such as the murine stromal cell line AFT024,was able to alter the cell division kinetics and self-renewing capacity of hematopoietic progenitor cells (HPC). Because beta(1)-integrins were shown to be crucial for the interaction of HPC with the bone marrow microenvironment,we have studied the role of beta(1)-integrins in the regulation of self-renewing cell divisions. For this purpose,we used primary human mesenchymal stromal (MS) cells as in vitro surrogate niche and monitored the division history and subsequent functional fate of individually plated CD34(+)133(+) cells in the absence or presence of an anti-beta(1)-integrin blocking antibody by time-lapse microscopy and subsequent long-term culture-initiating cell (LTC-IC) assays. beta(1)-Integrin-mediated contact with MS cells significantly increased the proportion of asymmetrically dividing cells and led to a substantial increase of LTC-IC. Provided that beta(1)-integrin-mediated contact was available within the first 72 hours,human MS cells were able to recruit HPC into cell cycle and accelerate their division kinetics without loss of stem cell function. Activation of beta(1)-integrins by ligands alone (e.g.,fibronectin and vascular cell adhesion molecule-1) was not sufficient to alter the cell division symmetry and promote self-renewal of HPC,thus indicating an indirect effect. These results have provided evidence that primary human MS cells are able to induce self-renewing divisions of HPC by a beta(1)-integrin-dependent mechanism. View Publication