Aanei C et al. (November 2011)
Experimental cell research 317 18 2616--29
Focal adhesion protein abnormalities in myelodysplastic mesenchymal stromal cells.
Direct cell-cell contact between haematopoietic progenitor cells (HPCs) and their cellular microenvironment is essential to maintain 'stemness'. In cancer biology, focal adhesion (FA) proteins are involved in survival signal transduction in a wide variety of human tumours. To define the role of FA proteins in the haematopoietic microenvironment of myelodysplastic syndromes (MDS), CD73-positive mesenchymal stromal cells (MSCs) were immunostained for paxillin, pFAK [Y(397)], and HSP90α/β and p130CAS, and analysed for reactivity, intensity and cellular localisation. Immunofluorescence microscopy allowed us to identify qualitative and quantitative differences, and subcellular localisation analysis revealed that in pathological MSCs, paxillin, pFAK [Y(397)], and HSP90α/β formed nuclear molecular complexes. Increased expression of paxillin, pFAK [Y(397)], and HSP90α/β and enhanced nuclear co-localisation of these proteins correlated with a consistent proliferative advantage in MSCs from patients with refractory anaemia with excess blasts (RAEB) and negatively impacted clonogenicity of HPCs. These results suggest that signalling via FA proteins could be implicated in HPC-MSC interactions. Further, because FAK is an HSP90α/β client protein, these results suggest the utility of HSP90α/β inhibition as a target for adjuvant therapy for myelodysplasia.
MesenCult™ MSC Basal Medium (Human)
MesenCult™ MSC Stimulatory Supplement (Human)
MesenCult™ Proliferation Kit (Human)
MesenCult™-ACF Dissociation Kit
Abdelwahab S et al. (December 2003)
Proceedings of the National Academy of Sciences of the United States of America 100 25 15006--10
HIV-1-suppressive factors are secreted by CD4+ T cells during primary immune responses.
CD4+ T cells are required for immunity against many viral infections, including HIV-1 where a positive correlation has been observed between strong recall responses and low HIV-1 viral loads. Some HIV-1-specific CD4+ T cells are preferentially infected with HIV-1, whereas others escape infection by unknown mechanisms. One possibility is that some CD4+ T cells are protected from infection by the secretion of soluble HIV-suppressive factors, although it is not known whether these factors are produced during primary antigen-specific responses. Here, we show that soluble suppressive factors are produced against CXCR4 and CCR5 isolates of HIV-1 during the primary immune response of human CD4+ T cells. This activity requires antigenic stimulation of na ive CD4+ T cells. One anti-CXCR4 factor is macrophage-derived chemokine (chemokine ligand 22 CCL22) and anti-CCR5 factors include macrophage inflammatory protein-1 alpha (CCL3) macrophage inflammatory protein-1 beta (CCL4) and RANTES (regulated upon activation of normal T cells expressed and secreted) (CCL5). Intracellular staining confirms that CD3+CD4+ T cells are the source of the prototype HIV-1-inhibiting chemokines CCL22 and CCL4. These results show that CD4+ T cells secrete an evolving HIV-1-suppressive activity during the primary immune response and that this activity is comprised primarily of CC chemokines. The data also suggest that production of such factors should be considered in the design of vaccines against HIV-1 and as a mechanism whereby the host can control infections with this virus."
BIT 9500 Serum Substitute
EasySep™ Human Naïve CD4+ T Cell Enrichment Kit
Abe J et al. (May 2005)
Journal of immunology (Baltimore, Md. : 1950) 174 9 5837--45
Gene expression profiling of the effect of high-dose intravenous Ig in patients with Kawasaki disease.
Kawasaki disease (KD) is an acute vasculitis of infants and young children, preferentially affecting the coronary arteries. Intravenous infusion of high dose Ig (IVIG) effectively reduces systemic inflammation and prevents coronary artery lesions in KD. To investigate the mechanisms underlying the therapeutic effects of IVIG, we examined gene expression profiles of PBMC and purified monocytes obtained from acute patients before and after IVIG therapy. The results suggest that IVIG suppresses activated monocytes and macrophages by altering various functional aspects of the genes of KD patients. Among the 18 commonly decreased transcripts in both PBMC and purified monocytes, we selected six genes, FCGR1A, FCGR3A, CCR2, ADM, S100A9, and S100A12, and confirmed the microarray results by real-time RT-PCR. Moreover, the expressions of FcgammaRI and FcgammaRIII on monocytes were reduced after IVIG. Plasma S100A8/A9 heterocomplex, but not S100A9, levels were elevated in patients with acute KD compared with those in febrile controls. Furthermore, S100A8/A9 was rapidly down-regulated in response to IVIG therapy. Persistent elevation of S100A8/A9 after IVIG was found in patients who later developed coronary aneurysms. These results indicate that the effects of IVIG in KD may be mediated by suppression of an array of immune activation genes in monocytes, including those activating FcgammaRs and the S100A8/A9 heterocomplex.
RosetteSep™ Human T Cell Enrichment Cocktail
RosetteSep™ Human Monocyte Enrichment Cocktail
Abilez O et al. (September 2011)
Biophysical journal 101 6 1326--34
Multiscale computational models for optogenetic control of cardiac function.
The ability to stimulate mammalian cells with light has significantly changed our understanding of electrically excitable tissues in health and disease, paving the way toward various novel therapeutic applications. Here, we demonstrate the potential of optogenetic control in cardiac cells using a hybrid experimental/computational technique. Experimentally, we introduced channelrhodopsin-2 into undifferentiated human embryonic stem cells via a lentiviral vector, and sorted and expanded the genetically engineered cells. Via directed differentiation, we created channelrhodopsin-expressing cardiomyocytes, which we subjected to optical stimulation. To quantify the impact of photostimulation, we assessed electrical, biochemical, and mechanical signals using patch-clamping, multielectrode array recordings, and video microscopy. Computationally, we introduced channelrhodopsin-2 into a classic autorhythmic cardiac cell model via an additional photocurrent governed by a light-sensitive gating variable. Upon optical stimulation, the channel opens and allows sodium ions to enter the cell, inducing a fast upstroke of the transmembrane potential. We calibrated the channelrhodopsin-expressing cell model using single action potential readings for different photostimulation amplitudes, pulse widths, and frequencies. To illustrate the potential of the proposed approach, we virtually injected channelrhodopsin-expressing cells into different locations of a human heart, and explored its activation sequences upon optical stimulation. Our experimentally calibrated computational toolbox allows us to virtually probe landscapes of process parameters, and identify optimal photostimulation sequences toward pacing hearts with light.
Abuljadayel I (January 2003)
Current medical research and opinion 19 5 355--75
Induction of stem cell-like plasticity in mononuclear cells derived from unmobilised adult human peripheral blood.
Undifferentiated pluripotent stem cells with flexible developmental potentials are not normally found in peripheral blood. However, such cells have recently been reported to reside in the bone marrow. Herein are reported methods of inducing pluripotency in cells derived from unmobilised adult human peripheral blood. In response to the inclusion of purified CR3/43 monoclonal antibody (mAb) to well-established culture conditions, mononuclear cells (MNC) obtained from a single blood donor are converted into pluripotent haematopoietic, neuronal and cardiomyogenic progenitor stem cells or undifferentiated stem cells. The haematopoietic stem cells are CD34+, clonogenic and have been shown to repopulate non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The neuronal precursors transcribe the primitive stem cell markers OCT-4 and nestin, and on maturation, differentially stain positive for neuronal, glial or oligodendrocyte-specific antigens. The cardiomyogenic progenitor stem cells form large bodies of asynchronously beating cells and differentiate into mature cardiomyocytes which transcribe GATA-4. The undifferentiated stem cells do not express haematopoietic-associated markers, are negative for major histocompatibility complex (MHC) class I and II antigens, transcribe high levels of OCT-4 and form embryoid body (EB)-like structures. This induction of stem cell-like plasticity in MNC may have proceeded by a process of retrodifferentiation but, in any case, could have profound clinical and pharmacological implications. Finally, the flexibility and the speed by which a variety of stem cell classes can be generated ex vivo from donor blood could potentially transfer this novel process into a less invasive automated clinical procedure.
MethoCult™ H4434 Classic
Addo M et al. (February 2003)
Journal of virology 77 3 2081--92
Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load.
Cellular immune responses play a critical role in the control of human immunodeficiency virus type 1 (HIV-1); however, the breadth of these responses at the single-epitope level has not been comprehensively assessed. We therefore screened peripheral blood mononuclear cells (PBMC) from 57 individuals at different stages of HIV-1 infection for virus-specific T-cell responses using a matrix of 504 overlapping peptides spanning all expressed HIV-1 proteins in a gamma interferon-enzyme-linked immunospot (Elispot) assay. HIV-1-specific T-cell responses were detectable in all study subjects, with a median of 14 individual epitopic regions targeted per person (range, 2 to 42), and all 14 HIV-1 protein subunits were recognized. HIV-1 p24-Gag and Nef contained the highest epitope density and were also the most frequently recognized HIV-1 proteins. The total magnitude of the HIV-1-specific response ranged from 280 to 25,860 spot-forming cells (SFC)/10(6) PBMC (median, 4,245) among all study participants. However, the number of epitopic regions targeted, the protein subunits recognized, and the total magnitude of HIV-1-specific responses varied significantly among the tested individuals, with the strongest and broadest responses detectable in individuals with untreated chronic HIV-1 infection. Neither the breadth nor the magnitude of the total HIV-1-specific CD8+-T-cell responses correlated with plasma viral load. We conclude that a peptide matrix-based Elispot assay allows for rapid, sensitive, specific, and efficient assessment of cellular immune responses directed against the entire expressed HIV-1 genome. These data also suggest that the impact of T-cell responses on control of viral replication cannot be explained by the mere quantification of the magnitude and breadth of the CD8+-T-cell response, even if a comprehensive pan-genome screening approach is applied.
RosetteSep™ Human CD4+ T Cell Enrichment Cocktail
RosetteSep™ Human CD8+ T Cell Enrichment Cocktail
Agarwal S et al. (March 2010)
Nature 464 7286 292--6
Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients.
Patients with dyskeratosis congenita (DC), a disorder of telomere maintenance, suffer degeneration of multiple tissues. Patient-specific induced pluripotent stem (iPS) cells represent invaluable in vitro models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity, which is accompanied by induction of the telomerase reverse transcriptase gene (TERT). We investigated whether defects in telomerase function would limit derivation and maintenance of iPS cells from patients with DC. Here we show that reprogrammed DC cells overcome a critical limitation in telomerase RNA component (TERC) levels to restore telomere maintenance and self-renewal. We discovered that TERC upregulation is a feature of the pluripotent state, that several telomerase components are targeted by pluripotency-associated transcription factors, and that in autosomal dominant DC, transcriptional silencing accompanies a 3' deletion at the TERC locus. Our results demonstrate that reprogramming restores telomere elongation in DC cells despite genetic lesions affecting telomerase, and show that strategies to increase TERC expression may be therapeutically beneficial in DC patients.
Agerstam H et al. (September 2010)
Blood 116 12 2103--11
Modeling the human 8p11-myeloproliferative syndrome in immunodeficient mice.
The 8p11 myeloproliferative syndrome (EMS), also referred to as stem cell leukemia/lymphoma, is a chronic myeloproliferative disorder that rapidly progresses into acute leukemia. Molecularly, EMS is characterized by fusion of various partner genes to the FGFR1 gene, resulting in constitutive activation of the tyrosine kinases in FGFR1. To date, no previous study has addressed the functional consequences of ectopic FGFR1 expression in the potentially most relevant cellular context, that of normal primary human hematopoietic cells. Herein, we report that expression of ZMYM2/FGFR1 (previously known as ZNF198/FGFR1) or BCR/FGFR1 in normal human CD34(+) cells from umbilical-cord blood leads to increased cellular proliferation and differentiation toward the erythroid lineage in vitro. In immunodeficient mice, expression of ZMYM2/FGFR1 or BCR/FGFR1 in human cells induces several features of human EMS, including expansion of several myeloid cell lineages and accumulation of blasts in bone marrow. Moreover, bone marrow fibrosis together with increased extramedullary hematopoiesis is observed. This study suggests that FGFR1 fusion oncogenes, by themselves, are capable of initiating an EMS-like disorder, and provides the first humanized model of a myeloproliferative disorder transforming into acute leukemia in mice. The established in vivo EMS model should provide a valuable tool for future studies of this disorder.
Agosti V et al. (March 2004)
The Journal of experimental medicine 199 6 867--78
Critical role for Kit-mediated Src kinase but not PI 3-kinase signaling in pro T and pro B cell development.
The Kit receptor functions in hematopoiesis, lymphocyte development, gastrointestinal tract motility, melanogenesis, and gametogenesis. To investigate the roles of different Kit signaling pathways in vivo, we have generated knock-in mice in which docking sites for PI 3-kinase (KitY719) or Src kinase (KitY567) have been mutated. Whereas steady-state hematopoiesis is normal in KitY719F/Y719F and KitY567F/Y567F mice, lymphopoiesis is affected differentially. The KitY567F mutation, but not the KitY719F mutation, blocks pro T cell and pro B cell development in an age-dependent manner. Thus, the Src family kinase, but not the PI 3-kinase docking site in Kit, mediates a critical signal for lymphocyte development. In agreement with these results, treatment of normal mice with the Kit tyrosine kinase inhibitor imatinib (Gleevec) leads to deficits in pro T and pro B cell development, similar to those seen in KitY567F/Y567F and KitW/W mice. The two mutations do not affect embryonic gametogenesis but the KitY719F mutation blocks spermatogenesis at the spermatogonial stages and in contrast the KitY567F mutation does not affect this process. Therefore, Kit-mediated PI 3-kinase signaling and Src kinase family signaling is highly specific for different cellular contexts in vivo.
Agostini M et al. (December 2010)
Biochemical and biophysical research communications 403 1 13--7
p73 regulates maintenance of neural stem cell.
p73, a member of the p53 family, is a transcription factor that plays a key role in many biological processes. In the present study, we show that TAp73 is expressed in neural stem cells (NSC) and its expression increases following their differentiation. NSC from p73 null mice have a reduced proliferative potential, together with reduced expression of members of the Sox-2 and Notch gene families known to be important for NSC proliferation. In parallel with this in vitro data, the width of the neurogenic areas was reduced in the brains of embryonic and adult p73-/- mice. These data suggest that p73, and in particular TAp73, is important for maintenance of the NSC pool.
EGF, Human, Recombinant
NeuroCult™ Basal Medium (Mouse)
NeuroCult™ Proliferation Supplement (Mouse)
NeuroCult™ Proliferation Kit (Mouse)
NeuroCult™ Differentiation Supplement (Mouse)
NeuroCult™ Differentiation Kit (Mouse)
NeuroCult™ Chemical Dissociation Kit (Mouse)
Agrawal B et al. (September 1998)
Cancer research 58 18 4079--81
Expression of MUC1 mucin on activated human T cells: implications for a role of MUC1 in normal immune regulation.
MUC1 mucin is expressed by normal and malignant epithelial cells and is thought to function through cell-cell interactions and transmembrane signal transduction events. Secreted cancer-associated MUC1 is immunosuppressive and inhibits human T-cell proliferation. We report here that newly synthesized MUC1 is expressed on the surface of mitogen-activated human T cells and is also found in soluble form in the supernatants from cultures of mitogen-activated human T cells. After removal of the mitogenic stimulus from the T-cell cultures, MUC1 expression is downregulated. The addition of anti-MUC1 monoclonal antibody to mitogen-activated cultures partially inhibits the T-cell proliferative response. These data suggest that MUC1 serves an immunodulatory function for human T lymphocytes.
Anti-Human MUC1 (CD227) Antibody, Clone 214D4
Aguila J et al. (July 2011)
Blood 118 3 576--85
SALL4 is a robust stimulator for the expansion of hematopoietic stem cells.
HSCs are rare cells that have the unique ability to self-renew and differentiate into cells of all hematopoietic lineages. The lack of donors and current inability to rapidly and efficiently expand HSCs are roadblocks in the development of successful cell therapies. Thus, the challenge of ex vivo human HSC expansion remains a fertile and critically important area of investigation. Here, we show that either SALL4A- or SALL4B-transduced human HSCs obtained from the mobilized peripheral blood are capable of rapid and efficient expansion ex vivo by >10 000-fold for both CD34(+)/CD38(-) and CD34(+)/CD38(+) cells in the presence of appropriate cytokines. We found that these cells retained hematopoietic precursor cell immunophenotypes and morphology as well as normal in vitro or vivo potential for differentiation. The SALL4-mediated expansion was associated with enhanced stem cell engraftment and long-term repopulation capacity in vivo. Also, we demonstrated that constitutive expression of SALL4 inhibited granulocytic differentiation and permitted expansion of undifferentiated cells in 32D myeloid progenitors. Furthermore, a TAT-SALL4B fusion rapidly expanded CD34(+) cells, and it is thus feasible to translate this study into the clinical setting. Our findings provide a new avenue for investigating mechanisms of stem cell self-renewal and achieving clinically significant expansion of human HSCs.