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1.  Palladium-based Mass-Tag Cell Barcoding with a Doublet-Filtering Scheme and Single Cell Deconvolution Algorithm 
Nature protocols  2015;10(2):316-333.
SUMMARY
Mass-tag cell barcoding (MCB) labels individual cell samples with unique combinatorial barcodes, after which they are pooled for processing and measurement as a single multiplexed sample. The MCB method eliminates variability between samples in antibody staining and instrument sensitivity, reduces antibody consumption, and shortens instrument measurement time. Here, we present an optimized MCB protocol with several improvements over previously described methods. The use of palladium-based labeling reagents expands the number of measurement channels available for mass cytometry and reduces interference with lanthanide-based antibody measurement. An error-detecting combinatorial barcoding scheme allows cell doublets to be identified and removed from the analysis. A debarcoding algorithm that is single cell-based rather than population-based improves the accuracy and efficiency of sample deconvolution. This debarcoding algorithm has been packaged into software that allows rapid and unbiased sample deconvolution. The MCB procedure takes 3–4 h, not including sample acquisition time of ~1 h per million cells.
doi:10.1038/nprot.2015.020
PMCID: PMC4347881  PMID: 25612231
2.  Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining1 
Fluorescent cellular barcoding and mass-tag cellular barcoding are cytometric methods that enable high sample throughput, minimize inter-sample variation, and reduce reagent consumption. Previously employed barcoding protocols require that barcoding be performed after surface marker staining, complicating combining the technique with measurement of alcohol-sensitive surface epitopes. This report describes a method of barcoding fixed cells after a transient partial permeabilization with 0.02% saponin that results in efficient and consistent barcode staining with fluorescent or mass-tagged reagents while preserving surface marker staining. This approach simplifies barcoding protocols and allows direct comparison of surface marker staining of multiple samples without concern for variations in the antibody cocktail volume, antigen-antibody ratio, or machine sensitivity. Using this protocol, cellular barcoding can be used to reliably detect subtle differences in surface marker expression.
doi:10.1002/cyto.a.22573
PMCID: PMC4361015  PMID: 25274027
Fluorescent Cellular Barcoding; Mass-tag Cellular Barcoding; Mass Cytometry; Permeabilization; Saponin
3.  Single Cell Mass Cytometry for Analysis of Immune System Functional States 
Current opinion in immunology  2013;25(4):10.1016/j.coi.2013.07.004.
Single cell mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on cell populations at single-cell resolution. Datasets are generated with antibody panels (upwards of 40) in which each antibody is conjugated to a polymer chelated with a stable metal isotope, usually in the Lanthanide series of the periodic table. Isotope labelled antibodies recognize surface markers to delineate cell types and intracellular signaling molecules to provide a measure of the network state—and thereby demarcating multiple cell state functions such as apoptosis, DNA damage and cell cycle. By measuring all these parameters simultaneously, the signaling state of an individual cell can be measured at its network state. This review will cover the basics of mass cytometry as well as outline steps already taken to allow it to stand aside traditional fluorescence based cytometry in the immunologist’s analytical arsenal in their study of immune states during infection.
doi:10.1016/j.coi.2013.07.004
PMCID: PMC3835664  PMID: 23999316
Mass cytometry; flow cytometry; drug discovery; pathogens; animal models
4.  The T cell STAT signaling network is reprogrammed within hours of bacteremia via secondary signals1 
The delicate balance between protective immunity and inflammatory disease is challenged during sepsis, a pathologic state characterized by aspects of both a hyper-active immune response and immunosuppression. The events driven by systemic infection by bacterial pathogens on the T cell signaling network that likely control these responses have not been illustrated in great detail. We characterized how intracellular signaling within the immune compartment is reprogrammed at the single cell level when the host is challenged with a high levels of pathogen. To accomplish this, we applied flow cytometry to measure the phosphorylation potential of key signal transduction proteins during acute bacterial challenge. We modeled the onset of sepsis by intravenous administration of avirulent strains of Listeria and E. coli to mice. Within six hours of bacterial challenge, T cells were globally restricted in their ability to respond to specific cytokine stimulations as determined by assessing the extent of STAT protein phosphorylation. Mechanisms by which this negative feedback response occurred included SOCS1 and SOCS3 gene up regulation and IL-6 induced endocystosis of the IL-6 receptor. In addition, macrophages were partially tolerized in their ability to respond to TLR agonists. Thus, in contrast to the view that there is a wholesale immune activation during sepsis, one immediate host response to blood borne bacteria was induction of a refractory period during which leukocyte activation by specific stimulations was attenuated.
doi:10.4049/jimmunol.0803666
PMCID: PMC4136495  PMID: 19494279
Bacteria; T cell; macrophage; cytokine; signal transduction
5.  A platinum-based covalent viability reagent for single cell mass cytometry 
In fluorescence-based flow cytometry, cellular viability is determined with membrane-impermeable fluorescent reagents that specifically enter and label plasma membrane-compromised non-viable cells. A recent technological advance in flow cytometry uses antibodies conjugated to elemental metal isotopes, rather than to fluorophores, to allow signal detection by atomic mass spectrometry. Unhampered by the limitations of overlapping emission fluorescence, mass cytometry increases the number of parameters that can be measured in single cells. However, mass cytometry is unable to take advantage of current fluorescent viability dyes. An alternative methodology was therefore developed here in which the platinum-containing chemotherapy drug cisplatin was used to label cells for mass cytometry determinations of live/dead ratios. In a one-minute incubation step, cisplatin preferentially labeled non-viable cells, from both adherent and suspension cultures, resulting in a platinum signal quantifiable by mass cytometry. This protocol was compatible with established sample processing steps for cytometry. Furthermore, the live/dead ratios were comparable between mass and fluorescence based cytometry. Importantly, although cisplatin is a known DNA-damaging agent, a one-minute “pulse” of cisplatin did not induce observable DNA damage or apoptotic responses even within 6 hours post-exposure. Cisplatin can therefore be used as a viability reagent for a wide range of mass cytometry protocols.
doi:10.1002/cyto.a.22067
PMCID: PMC3808967  PMID: 22577098
Mass cytometry; cisplatin; viability reagent
6.  Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators 
Nature biotechnology  2012;30(9):858-867.
The ability to comprehensively explore the impact of bio-active molecules on human samples at the single-cell level can provide great insight for biomedical research. Mass cytometry enables quantitative single-cell analysis with deep dimensionality, but currently lacks high-throughput capability. Here we report a method termed mass-tag cellular barcoding (MCB) that increases mass cytometry throughput by sample multiplexing. 96-well format MCB was used to characterize human peripheral blood mononuclear cell (PBMC) signaling dynamics, cell-to-cell communication, the signaling variability between 8 donors, and to define the impact of 27 inhibitors on this system. For each compound, 14 phosphorylation sites were measured in 14 PBMC types, resulting in 18,816 quantified phosphorylation levels from each multiplexed sample. This high-dimensional systems-level inquiry allowed analysis across cell-type and signaling space, reclassified inhibitors, and revealed off-target effects. MCB enables high-content, high-throughput screening, with potential applications for drug discovery, pre-clinical testing, and mechanistic investigation of human disease.
doi:10.1038/nbt.2317
PMCID: PMC3627543  PMID: 22902532
7.  Tyramide signal amplification for analysis of kinase activity by intracellular flow cytometry 
Intracellular flow cytometry permits quantitation of diverse molecular targets at the single-cell level. However, limitations in detection sensitivity inherently restrict the method, sometimes resulting in the inability to measure proteins of very low abundance or to differentiate cells expressing subtly different protein concentrations. To improve these measurements, an enzymatic amplification approach called tyramide signal amplification (TSA) was optimized for assessment of intracellular kinase cascades. First, Pacific Blue, Pacific Orange, and Alexa Fluor 488 tyramide reporters were shown to exhibit low non-specific binding in permeabilized cells. Next, the effects of antibody concentration, tyramide concentration, and reaction time on assay resolution were characterized. Use of optimized TSA resulted in a 10-fold or greater improvement in measurement resolution of endogenous Erk and Stat cell signaling pathways relative to standard, non-amplified detection. TSA also enhanced assay sensitivity and, in conjunction with fluorescent cell barcoding, improved assay performance according to a metric used to evaluate high-throughput drug screens. TSA was used to profile Stat1 phosphorylation in primary immune system cells, which revealed heterogeneity in various populations, including CD4+ FoxP3+ regulatory T cells. We anticipate the approach will be broadly applicable to intracellular flow cytometry assays with low signal-to-noise ratios.
doi:10.1002/cyto.a.20970
PMCID: PMC3036012  PMID: 20824632
flow cytometry; intracellular flow cytometry; phospho flow; tyramide signal amplification; catalyzed reporter deposition; enzymatic amplification staining; cell signaling; signal transduction; cell-based assay; background
8.  ALTERNATE MECHANISMS OF INITIAL PATTERN RECOGNITION DRIVE DIFFERENTIAL IMMUNE RESPONSES TO RELATED POXVIRUSES 
Cell host & microbe  2010;8(2):174-185.
Summary
Although vaccinia virus infection results in induction of a robust immunizing response, many closely related poxviruses such as variola (smallpox) and ectromelia (mousepox) are highly pathogenic in their natural hosts. We developed a strategy to map the activation of key signaling networks in vivo and applied this approach to define and compare the earliest signaling events elicited by poxvirus infections in mice. Vaccinia induced rapid TLR2-dependent responses leading to IL-6 production, which then initiated STAT3 signaling in dendritic cells and T cells. In contrast, ectromelia did not induce TLR2 activation and profound mouse strain-dependent responses were observed. In resistant C57BL/6 mice, the STAT1 and STAT3 pathways were rapidly activated, whereas in susceptible BALB/c mice, IL-6-dependent STAT3 activation did not occur. These results indicate that vaccination with vaccinia is dependent on rapid TLR2 and IL-6 driven responses and link the earliest immune signaling events to the outcome of infection.
doi:10.1016/j.chom.2010.07.008
PMCID: PMC2940993  PMID: 20709294
9.  WebFlow: A Software Package for High-Throughput Analysis of Flow Cytometry Data 
Abstract
Flow cytometry has emerged as a powerful tool for quantitative, single-cell analysis of both surface markers and intracellular antigens, including phosphoproteins and kinase signaling cascades, with the flexibility to process hundreds of samples in multiwell plate format. Quantitative flow cytometric analysis is being applied in many areas of biology, from the study of immunology in animal models or human patients to high-content drug screening of pharmacologically active compounds. However, these experiments generate thousands of data points per sample, each with multiple measured parameters, leading to data management and analysis challenges. We developed WebFlow (http://webflow.stanford.edu), a web server-based software package to manage, analyze, and visualize data from flow cytometry experiments. WebFlow is accessible via standard web browsers and does not require users to install software on their personal computers. The software enables plate-based annotation of large data sets, which provides the basis for exploratory data analysis tools and rapid visualization of multiple different parameters. These tools include custom user-defined statistics to normalize data to other wells or other channels, as well as interactive, user-selectable heat maps for viewing the underlying single-cell data. The web-based approach of WebFlow allows for sharing of data with collaborators or the general public. WebFlow provides a novel platform for quantitative analysis of flow cytometric data from high-throughput drug screening or disease profiling experiments.
doi:10.1089/adt.2008.174
PMCID: PMC2956679  PMID: 19187010
10.  Single Cell Mass Cytometry Analysis of Human Tonsil T Cell Remodeling by Varicella Zoster Virus 
Cell reports  2014;8(2):633-645.
Summary
Although pathogens must infect differentiated host cells that exhibit substantial diversity, documenting the consequences of infection against this heterogeneity is challenging. Single cell mass cytometry permits deep profiling based on combinatorial expression of surface and intracellular proteins. We used this method to investigate varicella-zoster virus (VZV) infection of tonsil T cells, which mediate viral transport to skin. Our results indicate that VZV induces a continuum of changes regardless of basal phenotypic and functional T cell characteristics. Contrary to the premise that VZV selectively infects T cells with skin trafficking profiles, VZV infection altered T cell surface proteins to enhance or induce these properties. Zap70 and Akt signaling pathways that trigger such surface changes were activated in VZV-infected naïve and memory cells by a T cell receptor (TCR)-independent process. Single cell mass cytometry is likely to be broadly relevant for demonstrating how intracellular pathogens modulate differentiated cells to support pathogenesis in the natural host.
doi:10.1016/j.celrep.2014.06.024
PMCID: PMC4127309  PMID: 25043183
11.  Reversibility of Defective Hematopoiesis Caused by Telomere Shortening in Telomerase Knockout Mice 
PLoS ONE  2015;10(7):e0131722.
Telomere shortening is common in bone marrow failure syndromes such as dyskeratosis congenita (DC), aplastic anemia (AA) and myelodysplastic syndromes (MDS). However, improved knowledge of the lineage-specific consequences of telomere erosion and restoration of telomere length in hematopoietic progenitors is required to advance therapeutic approaches. We have employed a reversible murine model of telomerase deficiency to compare the dependence of erythroid and myeloid lineage differentiation on telomerase activity. Fifth generation Tert-/- (G5 Tert-/-) mice with shortened telomeres have significant anemia, decreased erythroblasts and reduced hematopoietic stem cell (HSC) populations associated with neutrophilia and increased myelopoiesis. Intracellular multiparameter analysis by mass cytometry showed significantly reduced cell proliferation and increased sensitivity to activation of DNA damage checkpoints in erythroid progenitors and in erythroid-biased CD150hi HSC, but not in myeloid progenitors. Strikingly, Cre-inducible reactivation of telomerase activity restored hematopoietic stem and progenitor cell (HSPC) proliferation, normalized the DNA damage response, and improved red cell production and hemoglobin levels. These data establish a direct link between the loss of TERT activity, telomere shortening and defective erythropoiesis and suggest that novel strategies to restore telomerase function may have an important role in the treatment of the resulting anemia.
doi:10.1371/journal.pone.0131722
PMCID: PMC4489842  PMID: 26133370
12.  Mass cytometry as a platform for the discovery of cellular biomarkers to guide effective rheumatic disease therapy 
The development of biomarkers for autoimmune diseases has been hampered by a lack of understanding of disease etiopathogenesis and of the mechanisms underlying the induction and maintenance of inflammation, which involves complex activation dynamics of diverse cell types. The heterogeneous nature and suboptimal clinical response to treatment observed in many autoimmune syndromes highlight the need to develop improved strategies to predict patient outcome to therapy and personalize patient care. Mass cytometry, using CyTOF®, is an advanced technology that facilitates multiparametric, phenotypic analysis of immune cells at single-cell resolution. In this review, we outline the capabilities of mass cytometry and illustrate the potential of this technology to enhance the discovery of cellular biomarkers for rheumatoid arthritis, a prototypical autoimmune disease.
doi:10.1186/s13075-015-0644-z
PMCID: PMC4436107  PMID: 25981462
13.  Single-Cell Trajectory Detection Uncovers Progression and Regulatory Coordination in Human B cell Development 
Cell  2014;157(3):714-725.
Summary
Tissue regeneration is an orchestrated progression of cells from an immature state to a mature one, conventionally represented as distinctive cell subsets. A continuum of transitional cell states exists between these discrete stages. We combine the depth of single-cell mass cytometry and an algorithm developed to leverage this continuum by aligning single cells of a given lineage onto a unified trajectory that accurately predicts the developmental path de novo. Applied to human B cell lymphopoiesis, the algorithm (termed Wanderlust) constructed trajectories spanning from hematopoietic stem cells through to naïve B cells. This trajectory revealed nascent fractions of B cell progenitors and aligned them with developmentally-cued regulatory signaling including IL-7/STAT5 and cellular events such as immunoglobulin rearrangement, highlighting checkpoints across which regulatory signals are rewired paralleling changes in cellular state. This study provides a comprehensive analysis of human B lymphopoiesis, laying a foundation to apply this approach to other tissues and “corrupted” developmental processes including cancer.
doi:10.1016/j.cell.2014.04.005
PMCID: PMC4045247  PMID: 24766814
14.  T-cell STAT3 is required for the maintenance of humoral immunity to LCMV 
European Journal of Immunology  2014;45(2):418-427.
STAT3 is a critical transcription factor activated downstream of cytokine signaling and is integral for the function of multiple immune cell types. Human mutations in STAT3 cause primary immunodeficiency resulting in impaired control of a variety of infections, including reactivation of latent viruses. In this study, we investigate how T-cell functions of STAT3 contribute to responses to viral infection by inducing chronic lymphocytic choriomeningitis virus (LCMV) infection in mice lacking STAT3 specifically in T cells. Although mice with conditional disruption of STAT3 in T cells were able to mount early responses to viral infection similar to control animals, including expansion of effector T cells, we found generation of T-follicular helper (Tfh) cells to be impaired. As a result, STAT3 T cell deficient mice produced attenuated germinal center reactions, and did not accumulate bone marrow virus specific IgG-secreting cells, resulting in failure to maintain levels of virus-specific IgG or mount neutralizing responses to LCMV in the serum. These effects were associated with reduced control of viral replication and prolonged infection. Our results demonstrate the importance of STAT3 in T cells for the generation of functional long-term humoral immunity to viral infections.
doi:10.1002/eji.201445060
PMCID: PMC4383653  PMID: 25393615
Chronic infection; Humoral immunity; LCMV; STAT3; T follicular helper cells
15.  Coordinated Surgical Immune Signatures Contain Correlates of Clinical Recovery 
Science translational medicine  2014;6(255):255ra131.
Delayed recovery from surgery causes personal suffering and substantial societal and economic costs. Whether immune mechanisms determine recovery after surgical trauma remains ill-defined. Single-cell mass cytometry was applied to serial whole blood samples from 32 patients undergoing hip replacement to comprehensively characterize the phenotypical and functional immune response to surgical trauma. The simultaneous analysis of 14,000 phosphorylation events in precisely phenotyped immune cell subsets revealed uniform signaling responses among patients, demarcating a surgical immune signature. When regressed against clinical parameters of surgical recovery, including functional impairment and pain, strong correlations were found with STAT3, CREB and NF-kB signaling responses in subsets of CD14+ monocytes (R=0.7–0.8, FDR < 0.01). These sentinel results demonstrate the capacity of mass cytometry to survey the human immune system in a relevant clinical context. The mechanistically derived immune correlates point to diagnostic signatures, and potential therapeutic targets, that could postoperatively improve patient recovery.
doi:10.1126/scitranslmed.3009701
PMCID: PMC4334126  PMID: 25253674
16.  Conditional Density-based Analysis of T cell Signaling in Single Cell Data 
Science (New York, N.Y.)  2014;346(6213):1250689.
Cellular circuits sense the environment, process signals, and compute decisions using networks of interacting proteins. To model such a system, the abundance of each activated protein species can be described as a stochastic function of the abundance of other proteins. High-dimensional single-cell technologies, like mass cytometry, offer an opportunity to characterize signaling circuit-wide. However, the challenge of developing and applying computational approaches to interpret such complex data remains. Here, we developed computational methods, based on established statistical concepts, to characterize signaling network relationships by quantifying the strengths of network edges and deriving signaling response functions. In comparing signaling between naïve and antigen-exposed CD4+ T-lymphocytes, we find that although these two cell subtypes had similarly-wired networks, naïve cells transmitted more information along a key signaling cascade than did antigen-exposed cells. We validated our characterization on mice lacking the extracellular-regulated MAP kinase (ERK2), which showed stronger influence of pERK on pS6 (phosphorylated-ribosomal protein S6), in naïve cells compared to antigen-exposed cells, as predicted. We demonstrate that by using cell-to-cell variation inherent in single cell data, we can algorithmically derive response functions underlying molecular circuits and drive the understanding of how cells process signals.
doi:10.1126/science.1250689
PMCID: PMC4334155  PMID: 25342659
17.  New technologies for autoimmune disease monitoring 
Purpose of review
This article will review new technologies used to characterize the immune phenotype of cells and serum for potential use in studies of autoimmunity.
Recent findings
One area of recent development in studies of immune phenotyping is the contrast between cells of the immune system at rest and following activation. This simply involves comparing these cells at rest and following ligand-induced activation and measuring signaling system activation (phosphoepitope identification) or intracellular cytokine production or activation-induced gene expression. Preliminary data using these techniques have begun to identify signatures of disease (biomarkers) that are only seen when using these activation-induced assays. One of the most exciting new technologies, cytometry by time-of-flight mass spectrometry, couples a flow cytometer to a mass spectrometer, allowing many more parameters to be analyzed per cell, and without spillover between assay reagents, compared to conventional optical flow cytometry (heavy ions, mass, replaces fluorophore readout). Another new technology to analyze soluble proteins, bead-based immunoassays, can simultaneously measure up to 75 soluble analytes in a multiplexed array. Other technologies provide similar innovations in terms of analytical content, throughput, and miniaturization.
Summary
We believe that new cellular genomic and protein-based technologies can provide key insights into autoimmune disease pathogenesis, progression, and therapy, and that these assays need to be applied in a systematic way to samples from patients with autoimmune diseases.
doi:10.1097/MED.0b013e32833ada91
PMCID: PMC4311749  PMID: 20531181
cytometry by time-of-flight mass spectrometry; intracellular cytokine assays; multiplexed immunoassays; phosphoepitope
18.  Genomic and Proteomic Analysis Reveals a Threshold Level of MYC Required for Tumor Maintenance 
Cancer research  2008;68(13):5132-5142.
MYC overexpression has been implicated in the pathogenesis of most types of human cancers. MYC is likely to contribute to tumorigenesis by its effects on global gene expression. Previously, we have shown that the loss of MYC overexpression is sufficient to reverse tumorigenesis. Here, we show that there is a precise threshold level of MYC expression required for maintaining the tumor phenotype, whereupon there is a switch from a gene expression program of proliferation to a state of proliferative arrest and apoptosis. Oligonucleotide microarray analysis and quantitative PCR were used to identify changes in expression in 3,921 genes, of which 2,348 were down-regulated and 1,573 were up-regulated. Critical changes in gene expression occurred at or near the MYC threshold, including genes implicated in the regulation of the G1-S and G2-M cell cycle checkpoints and death receptor/apoptosis signaling. Using two-dimensional protein analysis followed by mass spectrometry, phospho-flow fluorescence-activated cell sorting, and antibody arrays, we also identified changes at the protein level that contributed to MYC-dependent tumor regression. Proteins involved in mRNA translation decreased below threshold levels of MYC. Thus, at the MYC threshold, there is a loss of its ability to maintain tumorigenesis, with associated shifts in gene and protein expression that reestablish cell cycle checkpoints, halt protein translation, and promote apoptosis.
doi:10.1158/0008-5472.CAN-07-6192
PMCID: PMC4191850  PMID: 18593912
19.  Multiplexed ion beam imaging (MIBI) of human breast tumors 
Nature medicine  2014;20(4):436-442.
Immunohistochemistry (IHC) is a tool for visualizing protein expression employed as part of the diagnostic work-up for the majority of solid tissue malignancies. Existing IHC methods use antibodies tagged with fluorophores or enzyme reporters that generate colored pigments. Because these reporters exhibit spectral and spatial overlap when used simultaneously, multiplexed IHC is not routinely used in clinical settings. We have developed a method that uses secondary ion mass spectrometry to image antibodies tagged with isotopically pure elemental metal reporters. Multiplexed ion beam imaging (MIBI) is capable of analyzing up to 100 targets simultaneously over a five-log dynamic range. Here, we used MIBI to analyze formalin-fixed, paraffin-embedded (FFPE) human breast tumor tissue sections stained with ten labels simultaneously. The resulting data suggest that MIBI will provide new insights by integrating tissue microarchitecture with highly multiplexed protein expression patterns, and will be valuable for basic research, drug discovery and clinical diagnostics.
doi:10.1038/nm.3488
PMCID: PMC4110905  PMID: 24584119
20.  Novel hematopoietic progenitor populations revealed by direct assessment of GATA1 protein expression and cMPL signaling events 
Stem cells (Dayton, Ohio)  2011;29(11):1774-1782.
Hematopoietic stem cells must exhibit tight regulation of both self-renewal and differentiation in order to maintain homeostasis of the hematopoietic system, as well as to avoid aberrations in growth that may result in leukemias or other disorders. In this study, we sought to understand the molecular basis of lineage determination, with particular focus on factors that influence megakaryocyte/erythrocyte-lineage commitment, in hematopoietic stem and progenitor cells. We used intracellular flow cytometry to identify two novel hematopoietic progenitor populations within the mouse bone-marrow cKit(+) Lineage (−) Sca1(+) [KLS] Flk2 (+) compartment that differ in their protein-level expression of GATA1, a critical megakaryocyte/erythrocyte-promoting transcription factor. GATA1-high repopulating cells exhibited the cell surface phenotype KLS Flk2(+ to int), CD150(int), CD105(+), cMPL(+), and were termed ‘FSE cells’. GATA1-low progenitors were identified as KLS Flk2(+), CD150(−), cMPL(−), and were termed ‘Flk(+) CD150(−) cells’. FSE cells had increased megakaryocyte/platelet potential in culture and transplant settings and exhibited a higher clonal frequency of CFU-S activity compared to Flk(+) CD150(−) cells, suggesting functional consequences of GATA1 upregulation in promoting megakaryocyte and erythroid lineage priming. Activation of ERK and AKT signal-transduction cascades was observed by intracellular flow cytometry in long-term hematopoietic stem cells (LT-HSC) and FSE cells, but not in Flk(+) CD150(−) cells in response to stimulation with thrombopoietin (TPO), an important megakaryocyte-promoting cytokine. We provide a mechanistic rationale for megakaryocyte/erythroid bias within KLS Flk2(+) cells, and demonstrate how assessment of intracellular factors and signaling events can be used to refine our understanding of lineage commitment during early definitive hematopoiesis.
doi:10.1002/stem.719
PMCID: PMC4100980  PMID: 21898686
21.  viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia 
Nature biotechnology  2013;31(6):545-552.
High-dimensional single-cell technologies are revolutionizing the way we understand biological systems. Technologies such as mass cytometry measure dozens of parameters simultaneously in individual cells, making interpretation daunting. We developed viSNE, a tool to map high-dimensional cytometry data onto 2D while conserving high-dimensional structure. We integrated mass cytometry with viSNE to map healthy and cancerous bone marrow samples. Healthy bone marrow maps into a canonical shape that separates between immune subtypes. In leukemia, however, the shape is malformed: the maps of cancer samples are distinct from the healthy map and from each other. viSNE highlights structure in the heterogeneity of surface phenotype expression in cancer, traverses the progression from diagnosis to relapse, and identifies a rare leukemia population in minimal residual disease settings. As several new technologies raise the number of simultaneously measured parameters in each cell to the hundreds, viSNE will become a mainstay in analyzing and interpreting such experiments.
doi:10.1038/nbt.2594
PMCID: PMC4076922  PMID: 23685480
22.  Joint Modeling and Registration of Cell Populations in Cohorts of High-Dimensional Flow Cytometric Data 
PLoS ONE  2014;9(7):e100334.
In biomedical applications, an experimenter encounters different potential sources of variation in data such as individual samples, multiple experimental conditions, and multivariate responses of a panel of markers such as from a signaling network. In multiparametric cytometry, which is often used for analyzing patient samples, such issues are critical. While computational methods can identify cell populations in individual samples, without the ability to automatically match them across samples, it is difficult to compare and characterize the populations in typical experiments, such as those responding to various stimulations or distinctive of particular patients or time-points, especially when there are many samples. Joint Clustering and Matching (JCM) is a multi-level framework for simultaneous modeling and registration of populations across a cohort. JCM models every population with a robust multivariate probability distribution. Simultaneously, JCM fits a random-effects model to construct an overall batch template – used for registering populations across samples, and classifying new samples. By tackling systems-level variation, JCM supports practical biomedical applications involving large cohorts. Software for fitting the JCM models have been implemented in an R package EMMIX-JCM, available from http://www.maths.uq.edu.au/~gjm/mix_soft/EMMIX-JCM/.
doi:10.1371/journal.pone.0100334
PMCID: PMC4077578  PMID: 24983991
23.  Proceedings from the 2009 Genetic Syndromes of the Ras/MAPK Pathway: From Bedside to Bench and Back 
The RASopathies are a group of genetic syndromes caused by germline mutations in genes that encode components of the Ras/mitogen-activated protein kinase (MAPK) pathway. Some of these syndromes are neurofibromatosis type 1, Noonan syndrome, Costello syndrome, cardio-facio-cutaneous syndrome, LEOPARD syndrome and Legius syndrome. Their common underlying pathogenetic mechanism brings about significant overlap in phenotypic features and includes craniofacial dysmorphology, cardiac, cutaneous, musculoskeletal, GI and ocular abnormalities, and a predisposition to cancer. The proceedings from the symposium “Genetic Syndromes of the Ras/MAPK Pathway: From Bedside to Bench and Back” chronicle the timely and typical research symposium which brought together clinicians, basic scientists, physician-scientists, advocate leaders, trainees, students and individuals with Ras syndromes and their families. The goals, to discuss basic science and clinical issues, to set forth a solid framework for future research, to direct translational applications towards therapy and to set forth best practices for individuals with RASopathies was successfully meet with a commitment to begin to move towards clinical trials.
doi:10.1002/ajmg.a.33183
PMCID: PMC4051786  PMID: 20014119
Cardio-facio-cutaneous syndrome; clinical trial; Costello syndrome; neurofibromatosis type 1; Noonan syndrome; Legius syndrome; Ras/MAPK; signal transduction pathway; RASopathies; therapy
24.  Normalization of mass cytometry data with bead standards 
Mass cytometry uses atomic mass spectrometry combined with isotopically pure reporter elements to currently measure as many as 40 parameters per single cell. As with any quantitative technology, there is a fundamental need for quality assurance and normalization protocols. In the case of mass cytometry, the signal variation over time due to changes in instrument performance combined with intervals between scheduled maintenance must be accounted for and then normalized. Here, samples were mixed with polystyrene beads embedded with metal lanthanides, allowing monitoring of mass cytometry instrument performance over multiple days of data acquisition. The protocol described here includes simultaneous measurements of beads and cells on the mass cytometer, subsequent extraction of the bead-based signature, and the application of an algorithm enabling correction of both short- and long-term signal fluctuations. The variation in the intensity of the beads that remains after normalization may also be used to determine data quality. Application of the algorithm to a one-month longitudinal analysis of a human peripheral blood sample reduced the range of median signal fluctuation from 4.9-fold to 1.3-fold.
doi:10.1002/cyto.a.22271
PMCID: PMC3688049  PMID: 23512433
25.  Snapin, Positive Regulator of Stimulation- Induced Ca2+ Release through RyR, Is Necessary for HIV-1 Replication in T Cells 
PLoS ONE  2013;8(10):e75297.
To identify critical host factors necessary for human immunodeficiency virus 1 (HIV-1) replication, large libraries of short-peptide-aptamers were expressed retrovirally. The target of one inhibitor peptide, Pep80, identified in this screen was determined to be Snapin, a protein associated with the soluble N-ethyl maleimide sensitive factor adaptor protein receptor (SNARE) complex that is critical for calcium-dependent exocytosis during neurotransmission. Pep80 inhibited Ca2+ release from intracellular stores and blocked downstream signaling by direct interruption of the association between Snapin and an intracellular calcium release channel, the ryanodine receptor (RyR). NFAT signaling was preferentially abolished by Pep80. Expression of Snapin overcame Pep80-mediated inhibition of Ca2+/NFAT signaling and HIV-1 replication. Furthermore, Snapin induced HIV-1 replication in primary CD4+ T cells. Thus, through its interaction with RyR, Snapin is a critical regulator of Ca2+ signaling and T cell activation. Use of the genetically selected intracellular aptamer inhibitors allowed us to define unique mechanisms important to HIV-1 replication and T cell biology.
doi:10.1371/journal.pone.0075297
PMCID: PMC3794929  PMID: 24130701

Results 1-25 (56)