CD117-positive Cells of the Heart: Progenitor Cells or Mast Cells?

Human cardiac stem/progenitor cells and their potential for repair of heart injury are a current hot topic of research. CD117 has been used frequently as a marker for identification of stem/progenitor cells in the heart. However, cardiac mast cells, which are also CD117⁺, have not been excluded by credible means when selecting putative cardiac progenitors by using CD117 as a marker. We evaluated the relationship between CD117⁺ cells and mast cells in the left ventricle of human hearts (n=5 patients, ages 1 week–75 years) with the well-established mast cell markers tryptase, toluidine blue, and thionine. A large number (85–100%) of CD117⁺ cells in the human heart were specifically identified as mast cells. In addition, mast cells showed weak or moderate CD45 immunostaining signals. These results indicate that the majority of CD117⁺ cells in the heart are mast cells and that these cells are distinctly positive for CD45, although staining was weak or moderate. These results strongly suggest that the newly reported CD117⁺/CD45^dim/moderate putative cardiac progenitor cells are mast cells. The significance of this observation in stem cell research of the heart is discussed. (J Histochem Cytochem 58:309–316, 2010)     

Inhibition by α-Amanitin of Adenovirus 12 Replication in Human Embryo Kidney Cells and of Adenovirus Transformation of Hamster Cells

ADENOVIRUS infection of human embryonic kidney (HEK) cultures seems to induce cellular RNA synthesis, which is preceded by a transient increase in the activities of the Mg²⁺-activated and Mn²⁺-(NH₄)₂SO₄-activated DNA dependent RNA polymerases and in the rate of histone acetylation¹. The two polymerase reactions, assayed in isolated cell nuclei, apparently reflect the activities of distinct nucleolar and nucleo-plasmic RNA polymerases^2,3. We were therefore prompted to test the effect of a specific inhibitor of the mammalian DNA-dependent RNA polymerase function, α-amanitin, on the multiplication of adenovirus. α-Amanitin is a bicyclic octapeptide isolated from the poisonous mushroom Amanita phalloides⁴ and which blocks RNA synthesis in intact animals^5,6. Nuclei isolated from the livers of such animals show a reduced activity of the RNA polymerases associated with nucleoplasm^5,6 and the nucleolus⁶.     

Excretion of 5′-Nucleotides from Bacterial Cells

Incubating the dried cells of Brevibacterium sojae No. 425-40 in alkaline buffer, the excretion of 5′-nucleotides accompanying with the decrease of intracellular RNA was observed. Then the determination of the optimum condition of the excretion and the investigation on the enzyme responsible for the degradation of endogenous RNA were carried out.

In the experiments using sonicate and disrupted cells, it appeared that orthophos-phate and Mg⁺⁺ might be accelerative or essential for the degradation of endogenous RNA and, in addition to four 5′-nucleotides (AMP, GMP, UMP and CMP), each nucleoside 5′-diphosphate was also contained in its degraded products. Nucleoside 2′- or 3′-monophos-phates were not detected. Although it was not clear whether phosphodiesterase concerned with the degradation of intracellular RNA or not, it was suggested that polynucleotide phos-phorylase acted mainly on the degradation.

The maximal excretion of 5′-nucleotides from dried cells was obtained by suspending 1 to 2% of dried cells in 0.05 M carbonate-bicarbonate buffer (pH 10) and incubating it at 60°C for two to three hours. Orthophosphate and Mg⁺⁺ were not required for the excretion.     

Endocytosis of KATP Channels Drives Glucose-Stimulated Excitation of Pancreatic β Cells

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Resveratrol Ameliorates the Maturation Process of β-Cell-Like Cells Obtained from an Optimized Differentiation Protocol of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) retain the extraordinary capacity to differentiate into different cell types of an adult organism, including pancreatic β-cells. For this particular lineage, although a lot of effort has been made in the last ten years to achieve an efficient and reproducible differentiation protocol, it was not until recently that this aim was roughly accomplished. Besides, several studies evidenced the impact of resveratrol (RSV) on insulin secretion, even though the mechanism by which this polyphenol potentiates glucose-stimulated insulin secretion (GSIS) is still not clear. The aim of this study was to optimize an efficient differentiation protocol that mimics in vivo pancreatic organogenesis and to investigate whether RSV may improve the final maturation step to obtain functional insulin-secreting cells. Our results indicate that treatment of hESCs (HS-181) with activin-A induced definitive endoderm differentiation as detected by the expression of SOX17 and FOXA2. Addition of retinoic acid (RA), Noggin and Cyclopamine promoted pancreatic differentiation as indicated by the expression of the early pancreatic progenitor markers ISL1, NGN3 and PDX1. Moreover, during maturation in suspension culture, differentiating cells assembled in islet-like clusters, which expressed specific endocrine markers such as PDX1, SST, GCG and INS. Similar results were confirmed with the human induced Pluripotent Stem Cell (hiPSC) line MSUH-001. Finally, differentiation protocols incorporating RSV treatment yielded numerous insulin-positive cells, induced significantly higher PDX1 expression and were able to transiently normalize glycaemia when transplanted in streptozotocin (STZ) induced diabetic mice thus promoting its survival. In conclusion, our strategy allows the efficient differentiation of hESCs into pancreatic endoderm capable of generating β-cell-like cells and demonstrates that RSV improves the maturation process.     

Calcineurin Inhibitors Suppress Cytokine Production from Memory T Cells and Differentiation of Na?ve T Cells into Cytokine-Producing Mature T Cells

T cells have been classified as belonging to the Th1 or Th2 subsets according to the production of defining cytokines such as IFN-γ and IL-4. The discovery of the Th17 lineage and regulatory T cells shifted the simple concept of the Th1/Th2 balance into a 4-way mechanistic pathway of local and systemic immunological activity. Clinically, the blockage of cytokine signals or non-specific suppression of cytokine predominance by immunosuppressants is the first-line treatment for inflammatory T cell-mediated disorders. Cyclosporine A (CsA) and Tacrolimus (Tac) are commonly used immunosuppressants for the treatment of autoimmune disease, psoriasis, and atopic disorders. Many studies have shown that these compounds suppress the activation of the calcium-dependent phosphatase calcineurin, thereby inhibiting T-cell activation. Although CsA and Tac are frequently utilized, their pharmacological mechanisms have not yet been fully elucidated.In the present study, we focused on the effects of CsA and Tac on cytokine secretion from purified human memory CD4⁺T cells and the differentiation of naïve T cells into cytokine-producing memory T cells. CsA or Tac significantly inhibited IFN-γ, IL-4, and IL-17 production from memory T cells. These compounds also inhibited T cell differentiation into the Th1, Th2, and Th17 subsets, even when used at a low concentration. This study provided critical information regarding the clinical efficacies of CsA and Tac as immunosuppressants.     

HIV Infection of Monocytes-Derived Dendritic Cells Inhibits Vγ9Vδ2 T Cells Functions

DCs act as sentinel cells against incoming pathogens and represent the most potent antigen presenting cells, having the unique capability to prime naïve T cells. In addition to their role in induction of adaptive immune responses, DC are also able to activate innate cells as γδ T cells; in particular, a reciprocal crosstalk between DC and γδ T cells was demonstrated. However, whether HIV infection may alter DC-Vγ9Vδ2 T cells cross-talk was not yet described. To clarify this issue, we cultured activated Vγ9Vδ2 T cells with HIV infected monocyte derived DC (MoDC). After 5 days we evaluated MoDC phenotype, and Vγ9Vδ2 T cells activation and proliferation. In our model, Vγ9Vδ2 T cells were not able to proliferate in response to HIV-infected MoDC, although an up-regulation of CD69 was observed. Upon phosphoantigens stimulation, Vγ9Vδ2 T cells proliferation and cytokine production were inhibited when cultured with HIV-infected MoDC in a cell-contact dependent way. Moreover, HIV-infected MoDC are not able to up-regulate CD86 molecules when cultured with activated Vγ9Vδ2 T cells, compared with uninfected MoDC. Further, activated Vγ9Vδ2 T cells are not able to induce HLA DR up-regulation and CCR5 down-regulation on HIV-infected MoDC. These data indicate that HIV-infected DC alter the capacity of Vγ9Vδ2 T cells to respond to their antigens, pointing out a new mechanisms of induction of Vγ9Vδ2 T cells anergy carried out by HIV, that could contribute to immune evasion.     

Why Don't CD8+ T Cells Reduce the Lifespan of SIV-Infected Cells In Vivo?

In January 2010 two groups independently published the observation that the depletion of CD8+ cells in SIV-infected macaques had no detectable impact on the lifespan of productively infected cells. This unexpected observation led the authors to suggest that CD8+ T cells control SIV viraemia via non-lytic mechanisms. However, a number of alternative plausible explanations, compatible with a lytic model of CD8+ T cell control, were proposed. This left the field with no consensus on how to interpret these experiments and no clear indication whether CD8+ T cells operated primarily via a lytic or a non-lytic mechanism. The aim of this work was to investigate why CD8+ T cells do not appear to reduce the lifespan of SIV-infected cells in vivo.     

Chronic TNFα Exposure Induces Robust Proliferation of Olfactory Ensheathing Cells,but not Schwann Cells

TNFα is persistently elevated in many injury and disease conditions. Previous reports of cytotoxicity of TNFα for oligodendrocytes and their progenitors suggest that the poor endogenous remyelination in patients with traumatic injury or multiple sclerosis may be due in part to persistent inflammation. Understanding the effects of inflammatory cytokines on potential cell therapy candidates is therefore important for evaluating the feasibility of their use. In this study, we assessed the effects of long term exposure to TNFα on viability, proliferation, migration and TNFα receptor expression of cultured rat olfactory ensheathing cells (OECs) and Schwann cells (SCs). Although OECs and SCs transplanted into the CNS produce similar myelinating phenotypes, and might be expected to have similar therapeutic uses, we report that they have very different sensitivities to TNFα. OECs exhibited positive proliferative responses to TNFα over a much broader range of concentrations than SCs. Low TNFα concentrations increased proliferation and migration of both OECs and SCs, but SC number declined in the presence of 100 ng/ml or higher concentrations of TNFα. In contrast, OECs exhibited enhanced proliferation even at high TNFα concentrations (up to 1 µg/ml) and showed no evidence of TNF cytotoxicity even at 4 weeks post-treatment. Furthermore, while both OECs and SCs expressed TNFαR1 and TNFαR2, TNFα receptor levels were downregulated in OECs after exposure to100 ng/ml TNFα for 5–7 days, but were either elevated or unchanged in SCs. These results imply that OECs may be a more suitable cell therapy candidate if transplanted into areas with persistent inflammation.     

Redistribution of Surface Antigens– a General Property of Animal Cells?

MEMBRANE antigens of animal cells are reported to be localized either in discontinuous discrete areas (H-2^1,2, HL-A³, organ⁴ and immunoglobulin (Ig)^5–9 or continuously (species⁴ and blood group^9,10). These results have been obtained by immunofluorescence (IFL) and electron microscopy performed by double layer principles except in the case of Ig where direct IFL technique has been used. The influence on the localization pattern of marker molecules by variables such as Ig concentration of the reagents used, effects of interactions between Ig molecules in the double layer and temperature dependence of the reaction steps have not been investigated. Redistribution of lymphocyte surface Ig molecules induced by anti-Ig antibody has been described and this was observed to be temperature dependent occurring at 20° C but not at 0° C^11,12. Here I discuss the distribution and localization of different surface antigens (blood group substance A and B, species, H-2 and organ) on several cell types (monkey kidney, human lymphoid cells, HeLa, human thyroid cells, RK13 and L-cells of C3H/K) as recorded by direct and indirect IFL technique. I show that the temperature and Ig concentration used exert an influence on the distribution of these antigens. The cells used in IFL tests were cultured and harvested as described before¹⁰. IFL staining was performed according to Möller1³. The stained cells were examined with a Leitz ‘Orthoplan’ ultraviolet microscope. Direct IFL technique was applied to two test systems. A FITC-conjugated rabbit antiserum to blood group substance A was used on monkey (Macaca fascicularis) kidney cells known to be A-positive¹⁰ and a FITC-conjugated horse anti-human thymocyte globulin were tested on a human lymphoid cell line Robinson (obtained from Dr Moore, Buffalo). In both test systems the staining pattern was completely confluent at all temperatures used: 4° C, 20° C and 37° C. The staining pattern was not related to antibody concentration.     

Ligand-Mediated Friction Determines Morphodynamics of Spreading T?Cells

Spreading of T cells on antigen presenting cells is a crucial initial step in immune response. Spreading occurs through rapid morphological changes concomitant with the reorganization of surface receptors and of the cytoskeleton. Ligand mobility and frictional coupling of receptors to the cytoskeleton were separately recognized as important factors but a systematic study to explore their biophysical role in spreading was hitherto missing. To explore the impact of ligand mobility, we prepared chemically identical substrates on which molecules of anti-CD3 (capable of binding and activating the T cell receptor complex), were either immobilized or able to diffuse. We quantified the T cell spreading area and cell edge dynamics using quantitative reflection interference contrast microscopy, and imaged the actin distribution. On mobile ligands, as compared to fixed ligands, the cells spread much less, the actin is centrally, rather than peripherally distributed and the edge dynamics is largely altered. Blocking myosin-II or adding molecules of ICAM1 on the substrate largely abrogates these differences. We explain these observations by building a model based on the balance of forces between activation-dependent actin polymerization and actomyosin-generated tension on one hand, and on the frictional coupling of the ligand-receptor complexes with the actin cytoskeleton, the membrane and the substrate, on the other hand. Introducing the measured edge velocities in the model, we estimate the coefficient of frictional coupling between T Cell receptors or LFA-1 and the actin cytoskeleton. Our results provide for the first time, to our knowledge, a quantitative framework bridging T cell-specific biology with concepts developed for integrin-based mechanisms of spreading.     

Islet Endothelial Cells Induce Glycosylation and Increase Cell-surface Expression of Integrin β1 in β Cells

The co-culturing of insulinoma and islet-derived endothelial cell (iEC) lines results in the spontaneous formation of free-floating pseudoislets (PIs). We previously showed that iEC-induced PIs display improved insulin expression and secretion in response to glucose stimulation. This improvement was associated with a de novo deposition of extracellular matrix (ECM) proteins by iECs in and around the PIs. Here, iEC-induced PIs were used to study the expression and posttranslational modification of the ECM receptor integrin β1. A wide array of integrin β subunits was detected in βTC3 and NIT-1 insulinomas as well as in primary islets, with integrin β1 mRNA and protein detected in all three cell types. Interestingly, the formation of iEC-induced PIs altered the glycosylation patterns of integrin β1, resulting in a higher molecular weight form of the receptor. This form was found in native pancreas but was completely absent in monolayer β-cells. Fluorescence-activated cell sorting analysis of monolayers and PIs revealed a higher expression of integrin β1 in PIs. Antibody-mediated blocking of integrin β1 led to alterations in β-cell morphology, reduced insulin gene expression, and enhanced glucose secretion under baseline conditions. These results suggest that iEC-induced PI formation may alter integrin β1 expression and posttranslational modification by enhancing glycosylation, thereby providing a more physiological culture system for studying integrin-ECM interactions in β cells.     

Repair of DNA–Protein Cross-links in Mammalian Cells

DNA–protein cross-links are generated by both endogenous and exogenous DNA damaging agents, as intermediates during normal DNA metabolism, and during abortive base excision repair. Cross-links are relatively common lesions that are lethal when they block progression of DNA polymerases. DNA–protein cross-links may be broadly categorized into four groups by the DNA and protein chemistries near the cross-link and by the source of the cross-link: DNA–protein cross-links may be found (1) in nicked DNA at the 3' end of one strand (topo I), (2) in nicked DNA at the 5' end of one strand (pol beta), (3) at the 5' ends of both strands adjacent to nicks in close proximity (topo II; Spo 11), and (4) in one strand of duplex DNA (UV irradiation; bifunctional carcinogens and chemotherapeutic agents). Repair mechanisms are reasonably well-defined for groups 1 and 3, and suggested for groups 2 and 4. Our work is focused on the recognition and removal of DNA–protein cross-links in duplex DNA (group 4).     

Three Forms of α-Glucosidase from Suspension-cultured Rice Cells

Three forms of α-glucosidase (EC 3.2.1.20), designated as I, II, and III, have been isolated from suspension-cultured rice cells by a procedure including fractionation with ammonium sulfate, CM-cellulose column chromatography, and preparative disc gel electrophoresis. The three enzymes were homogeneous by Polyacrylamide disc gel electrophoresis. α-Glucosidase I was secreted in the culture medium during growth, α-glucosidase II was readily extracted from rice cells with the buffer alone, and α-glucosidase III required NaCl to be solubilized. The molecular weights of the three enzymes were 96,000 (I), 84,000 (II), and 58,000 (III). The three enzymes readily hydrolyzed maltose, maltotriose, maltotetraose, amylose, and soluble starch. α-Glucosidase I possessed strong isomaltose-hydrolyzing activity and hydrolyzed isomaltose about three times as rapidly as α-glucosidase III. The three enzymes produced panose as the main α-glucosyltransfer product from maltose. Half the maltose-hydrolyzing activities of the three enzymes were inhibited by 11.25 ng of castanospermine. The inhibition was competitive.     

What is the purpose of launching World Journal of Stem Cells?

The first issue of World Journal of Stem Cells (WJSC), whose preparatory work was initiated on September 27, 2008, will be published on December 31, 2009. The WJSC Editorial Board has now been established and consists of 281 distinguished experts from 28 countries. Our purpose of launching WJSC is to publish peer-reviewed, high-quality articles via an open-access online publishing model, thereby acting as a platform for communication between peers and the wider public, and maximizing the benefits to editorial board members, authors and readers.