Honors & Awards
NIH Director's New Innovator Award, National Institutes of Health (2016)
NIH Pathway to Independence Award, NIH / NIGMS (2013)
Dale F. Frey Breakthrough Scientist, Damon Runyon Cancer Research Foundation (2012)
ISAC President's Award of Excellence, International Society for Advancement of Cytometry (2012)
Fellowship, Canadian Institute of Health Research (CIHR) (2009)
Fellowship, Damon Runyon Cancer Research Foundation (2009)
PhD, University of Western Ontario, Proteomic Analysis of Human Embryonic Stem Cell Culture (2008)
BSc, University of Victoria (2002)
Current Research and Scholarly Interests
Our goal is to understand the mechanisms regulating the development of human systems (both embryonic and adult). In particular, we are interested in clarifying the roles of both protein coding genes as well as pathobiology (disease state or pathogen) known to be uniquely human – therefore, not analogously studied in model organisms. Drawing on both pluripotent stem cell biology, hematopoiesis, and immunology, combined with novel high-content single-cell analysis (CyTOF – Mass Cytometry) and imagining (MIBI-Multiplexed Ion Beam Imaging) we are creating templates of ‘normal’ human cellular behavior. Using these we can decipher the roles of protein regulators on cellular specification as well as the influence of human-specific pathobiology on system remodeling at the single cell level. This work will enable a better understanding of how disease corrupts this process. Ultimately, our objective will be to use such approaches to not only reveal how novel regulators function in the context of complex cellular systems, but also enable the mechanistic characterization of human pathobiology in primary human tissues. In doing so we will understand how changes in related physiological or pathological systems can be more readily recognized and controlled.
In addition to the lab’s work on human hematopoiesis and pluripotent stem cell specification we are seeking collaborative partnerships surrounding problems in human immunology as well as in regenerative medicine, including efforts to exploit next generation single-cell analysis and new computational methods to create systems level models of these processes so that they may be better understood and directed.
Independent Studies (13)
- Directed Reading in Cancer Biology
CBIO 299 (Win, Spr)
- Directed Reading in Immunology
IMMUNOL 299 (Win, Spr)
- Directed Reading in Pathology
PATH 299 (Win, Spr)
- Early Clinical Experience in Immunology
IMMUNOL 280 (Aut, Win, Spr)
- Early Clinical Experience in Pathology
PATH 280 (Win, Spr)
- Graduate Research
CBIO 399 (Aut, Win, Spr)
- Graduate Research
IMMUNOL 399 (Aut, Win, Spr, Sum)
- Graduate Research
PATH 399 (Aut, Win, Spr)
- Medical Scholars Research
PATH 370 (Win, Spr)
- Teaching in Cancer Biology
CBIO 260 (Spr)
- Teaching in Immunology
IMMUNOL 290 (Aut, Win, Spr)
- Undergraduate Research
IMMUNOL 199 (Aut, Win, Spr)
- Undergraduate Research
PATH 199 (Win, Spr)
- Directed Reading in Cancer Biology
Graduate and Fellowship Programs
Data-Driven Phenotypic Dissection of AML Reveals Progenitor-like Cells that Correlate with Prognosis
2015; 162 (1): 184-197
Acute myeloid leukemia (AML) manifests as phenotypically and functionally diverse cells, often within the same patient. Intratumor phenotypic and functional heterogeneity have been linked primarily by physical sorting experiments, which assume that functionally distinct subpopulations can be prospectively isolated by surface phenotypes. This assumption has proven problematic, and we therefore developed a data-driven approach. Using mass cytometry, we profiled surface and intracellular signaling proteins simultaneously in millions of healthy and leukemic cells. We developed PhenoGraph, which algorithmically defines phenotypes in high-dimensional single-cell data. PhenoGraph revealed that the surface phenotypes of leukemic blasts do not necessarily reflect their intracellular state. Using hematopoietic progenitors, we defined a signaling-based measure of cellular phenotype, which led to isolation of a gene expression signature that was predictive of survival in independent cohorts. This study presents new methods for large-scale analysis of single-cell heterogeneity and demonstrates their utility, yielding insights into AML pathophysiology.
View details for DOI 10.1016/j.cell.2015.05.047
View details for Web of Science ID 000357542300019
View details for PubMedID 26095251
Single-Cell Trajectory Detection Uncovers Progression and Regulatory Coordination in Human B Cell Development
2014; 157 (3): 714-725
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 naive 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.
View details for DOI 10.1016/j.cell.2014.04.005
View details for Web of Science ID 000335392100019
View details for PubMedID 24766814
View details for PubMedCentralID PMC4045247
Multiplexed ion beam imaging of human breast tumors.
2014; 20 (4): 436-442
Immunohistochemistry (IHC) is a tool for visualizing protein expression that is employed as part of the diagnostic workup 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 human breast tumor tissue sections stained with ten labels simultaneously. The resulting data suggest that MIBI can provide new insights into disease pathogenesis that will be valuable for basic research, drug discovery and clinical diagnostics.
View details for DOI 10.1038/nm.3488
View details for PubMedID 24584119
Single-Cell Mass Cytometry of Differential Immune and Drug Responses Across a Human Hematopoietic Continuum
2011; 332 (6030): 687-696
Flow cytometry is an essential tool for dissecting the functional complexity of hematopoiesis. We used single-cell "mass cytometry" to examine healthy human bone marrow, measuring 34 parameters simultaneously in single cells (binding of 31 antibodies, viability, DNA content, and relative cell size). The signaling behavior of cell subsets spanning a defined hematopoietic hierarchy was monitored with 18 simultaneous markers of functional signaling states perturbed by a set of ex vivo stimuli and inhibitors. The data set allowed for an algorithmically driven assembly of related cell types defined by surface antigen expression, providing a superimposable map of cell signaling responses in combination with drug inhibition. Visualized in this manner, the analysis revealed previously unappreciated instances of both precise signaling responses that were bounded within conventionally defined cell subsets and more continuous phosphorylation responses that crossed cell population boundaries in unexpected manners yet tracked closely with cellular phenotype. Collectively, such single-cell analyses provide system-wide views of immune signaling in healthy human hematopoiesis, against which drug action and disease can be compared for mechanistic studies and pharmacologic intervention.
View details for DOI 10.1126/science.1198704
View details for Web of Science ID 000290265800035
View details for PubMedID 21551058
High-resolution myogenic lineage mapping by single-cell mass cytometry
NATURE CELL BIOLOGY
2017; 19 (5): 558-?
Muscle regeneration is a dynamic process during which cell state and identity change over time. A major roadblock has been a lack of tools to resolve a myogenic progression in vivo. Here we capitalize on a transformative technology, single-cell mass cytometry (CyTOF), to identify in vivo skeletal muscle stem cell and previously unrecognized progenitor populations that precede differentiation. We discovered two cell surface markers, CD9 and CD104, whose combined expression enabled in vivo identification and prospective isolation of stem and progenitor cells. Data analysis using the X-shift algorithm paired with single-cell force-directed layout visualization defined a molecular signature of the activated stem cell state (CD44(+)/CD98(+)/MyoD(+)) and delineated a myogenic trajectory during recovery from acute muscle injury. Our studies uncover the dynamics of skeletal muscle regeneration in vivo and pave the way for the elucidation of the regulatory networks that underlie cell-state transitions in muscle diseases and ageing.
View details for DOI 10.1038/ncb3507
View details for Web of Science ID 000400376100019
View details for PubMedID 28414312
- Assessing basophil activation by using flow cytometry and mass cytometry in blood stored 24 hours before analysis JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY 2017; 139 (3): 889-?
Systemic Immunity Is Required for Effective Cancer Immunotherapy.
2017; 168 (3): 487-502 e15
Immune responses involve coordination across cell types and tissues. However, studies in cancer immunotherapy have focused heavily on local immune responses in the tumor microenvironment. To investigate immune activity more broadly, we performed an organism-wide study in genetically engineered cancer models using mass cytometry. We analyzed immune responses in several tissues after immunotherapy by developing intuitive models for visualizing single-cell data with statistical inference. Immune activation was evident in the tumor and systemically shortly after effective therapy was administered. However, during tumor rejection, only peripheral immune cells sustained their proliferation. This systemic response was coordinated across tissues and required for tumor eradication in several immunotherapy models. An emergent population of peripheral CD4 T cells conferred protection against new tumors and was significantly expanded in patients responding to immunotherapy. These studies demonstrate the critical impact of systemic immune responses that drive tumor rejection.
View details for DOI 10.1016/j.cell.2016.12.022
View details for PubMedID 28111070
View details for PubMedCentralID PMC5312823
Distinct signaling programs control human hematopoietic stem cell survival and proliferation.
2017; 129 (3): 307-318
Several growth factors (GFs) that together promote quiescent human hematopoietic stem cell (HSC) expansion ex vivo have been identified; however, the molecular mechanisms by which these GFs regulate the survival, proliferation. and differentiation of human HSCs remain poorly understood. We now describe experiments in which we used mass cytometry to simultaneously measure multiple surface markers, transcription factors, active signaling intermediates, viability, and cell-cycle indicators in single CD34(+) cord blood cells before and up to 2 hours after their stimulation with stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor (5 GFs) either alone or combined. Cells with a CD34(+)CD38(-)CD45RA(-)CD90(+)CD49f(+) (CD49f(+)) phenotype (∼10% HSCs with >6-month repopulating activity in immunodeficient mice) displayed rapid increases in activated STAT1/3/5, extracellular signal-regulated kinase 1/2, AKT, CREB, and S6 by 1 or more of these GFs, and β-catenin only when the 5 GFs were combined. Certain minority subsets within the CD49f(+) compartment were poorly GF-responsive and, among the more GF-responsive subsets of CD49f(+) cells, different signaling intermediates correlated with the levels of the myeloid- and lymphoid-associated transcription factors measured. Phenotypically similar, but CD90(-)CD49f(-) cells (MPPs) contained lower baseline levels of multiple signaling intermediates than the CD90(+)CD49f(+) cells, but showed similar response amplitudes to the same GFs. Importantly, we found activation or inhibition of AKT and β-catenin directly altered immediate CD49f(+) cell survival and proliferation. These findings identify rapid signaling events that 5 GFs elicit directly in the most primitive human hematopoietic cell types to promote their survival and proliferation.
View details for DOI 10.1182/blood-2016-09-740654
View details for PubMedID 27827829
View details for PubMedCentralID PMC5271174
Mutant IDH1 Downregulates ATM and Alters DNA Repair and Sensitivity to DNA Damage Independent of TET2.
2016; 30 (2): 337-348
Mutations in the isocitrate dehydrogenase-1 gene (IDH1) are common drivers of acute myeloid leukemia (AML) but their mechanism is not fully understood. It is thought that IDH1 mutants act by inhibiting TET2 to alter DNA methylation, but there are significant unexplained clinical differences between IDH1- and TET2-mutant diseases. We have discovered that mice expressing endogenous mutant IDH1 have reduced numbers of hematopoietic stem cells (HSCs), in contrast to Tet2 knockout (TET2-KO) mice. Mutant IDH1 downregulates the DNA damage (DD) sensor ATM by altering histone methylation, leading to impaired DNA repair, increased sensitivity to DD, and reduced HSC self-renewal, independent of TET2. ATM expression is also decreased in human IDH1-mutated AML. These findings may have implications for treatment of IDH-mutant leukemia.
View details for DOI 10.1016/j.ccell.2016.05.018
View details for PubMedID 27424808
View details for PubMedCentralID PMC5022794
Assessing basophil activation by using flow cytometry and mass cytometry in blood stored 24 hours before analysis.
journal of allergy and clinical immunology
Basophil activation tests (BATs) have promise for research and for clinical monitoring of patients with allergies. However, BAT protocols vary in blood anticoagulant used and temperature and time of storage before testing, complicating comparisons of results from various studies.We attempted to establish a BAT protocol that would permit analysis of blood within 24 hours of obtaining the sample.Blood from 46 healthy donors and 120 patients with peanut allergy was collected into EDTA or heparin tubes, and samples were stored at 4°C or room temperature for 4 or 24 hours before performing BATs.Stimulation with anti-IgE or IL-3 resulted in strong upregulation of basophil CD203c in samples collected in EDTA or heparin, stored at 4°C, and analyzed 24 hours after sample collection. However, a CD63(hi) population of basophils was not observed in any conditions in EDTA-treated samples unless exogenous calcium/magnesium was added at the time of anti-IgE stimulation. By contrast, blood samples collected in heparin tubes were adequate for quantification of upregulation of basophil CD203c and identification of a population of CD63(hi) basophils, irrespective of whether the specimens were analyzed by means of conventional flow cytometry or cytometry by time-of-flight mass spectrometry, and such tests could be performed after blood was stored for 24 hours at 4°C.BATs to measure upregulation of basophil CD203c and induction of a CD63(hi) basophil population can be conducted with blood obtained in heparin tubes and stored at 4°C for 24 hours.
View details for DOI 10.1016/j.jaci.2016.04.060
View details for PubMedID 27527263
View details for PubMedCentralID PMC5237629
Visualization and cellular hierarchy inference of single-cell data using SPADE.
2016; 11 (7): 1264-1279
High-throughput single-cell technologies provide an unprecedented view into cellular heterogeneity, yet they pose new challenges in data analysis and interpretation. In this protocol, we describe the use of Spanning-tree Progression Analysis of Density-normalized Events (SPADE), a density-based algorithm for visualizing single-cell data and enabling cellular hierarchy inference among subpopulations of similar cells. It was initially developed for flow and mass cytometry single-cell data. We describe SPADE's implementation and application using an open-source R package that runs on Mac OS X, Linux and Windows systems. A typical SPADE analysis on a 2.27-GHz processor laptop takes ∼5 min. We demonstrate the applicability of SPADE to single-cell RNA-seq data. We compare SPADE with recently developed single-cell visualization approaches based on the t-distribution stochastic neighborhood embedding (t-SNE) algorithm. We contrast the implementation and outputs of these methods for normal and malignant hematopoietic cells analyzed by mass cytometry and provide recommendations for appropriate use. Finally, we provide an integrative strategy that combines the strengths of t-SNE and SPADE to infer cellular hierarchy from high-dimensional single-cell data.
View details for DOI 10.1038/nprot.2016.066
View details for PubMedID 27310265
Wishbone identifies bifurcating developmental trajectories from single-cell data
2016; 34 (6): 637-645
Recent single-cell analysis technologies offer an unprecedented opportunity to elucidate developmental pathways. Here we present Wishbone, an algorithm for positioning single cells along bifurcating developmental trajectories with high resolution. Wishbone uses multi-dimensional single-cell data, such as mass cytometry or RNA-Seq data, as input and orders cells according to their developmental progression, and it pinpoints bifurcation points by labeling each cell as pre-bifurcation or as one of two post-bifurcation cell fates. Using 30-channel mass cytometry data, we show that Wishbone accurately recovers the known stages of T-cell development in the mouse thymus, including the bifurcation point. We also apply the algorithm to mouse myeloid differentiation and demonstrate its generalization to additional lineages. A comparison of Wishbone to diffusion maps, SCUBA and Monocle shows that it outperforms these methods both in the accuracy of ordering cells and in the correct identification of branch points.
View details for DOI 10.1038/nbt.3569
View details for Web of Science ID 000377846400031
View details for PubMedID 27136076
View details for PubMedCentralID PMC4900897
Pacific Symposium on Biocomputing. Pacific Symposium on Biocomputing
2016; 22: 557-563
Recent technological developments allow gathering single-cell measurements across different domains (genomic, transcriptomics, proteomics, imaging etc). Sophisticated computational algorithms are required in order to harness the power of single-cell data. This session is dedicated to computational methods for single-cell analysis in various biological domains, modelling of population heterogeneity, as well as translational applications of single cell data.
View details for PubMedID 27897006
Single-cell systems-level analysis of human Toll-like receptor activation defines a chemokine signature in patients with systemic lupus erythematosus
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY
2015; 136 (5): 1326-1336
Activation of Toll-like receptors (TLRs) induces inflammatory responses involved in immunity to pathogens and autoimmune pathogenesis, such as in patients with systemic lupus erythematosus (SLE). Although TLRs are differentially expressed across the immune system, a comprehensive analysis of how multiple immune cell subsets respond in a system-wide manner has not been described.We sought to characterize TLR activation across multiple immune cell subsets and subjects, with the goal of establishing a reference framework against which to compare pathologic processes.Peripheral whole-blood samples were stimulated with TLR ligands and analyzed by means of mass cytometry simultaneously for surface marker expression, activation states of intracellular signaling proteins, and cytokine production. We developed a novel data visualization tool to provide an integrated view of TLR signaling networks with single-cell resolution. We studied 17 healthy volunteer donors and 8 patients with newly diagnosed and untreated SLE.Our data revealed the diversity of TLR-induced responses within cell types, with TLR ligand specificity. Subsets of natural killer cells and T cells selectively induced nuclear factor κ light chain enhancer of activated B cells in response to TLR2 ligands. CD14(hi) monocytes exhibited the most polyfunctional cytokine expression patterns, with more than 80 distinct cytokine combinations. Monocytic TLR-induced cytokine patterns were shared among a group of healthy donors, with minimal intraindividual and interindividual variability. Furthermore, autoimmune disease altered baseline cytokine production; newly diagnosed untreated SLE patients shared a distinct monocytic chemokine signature, despite clinical heterogeneity.Mass cytometry defined a systems-level reference framework for human TLR activation, which can be applied to study perturbations in patients with inflammatory diseases, such as SLE.
View details for DOI 10.1016/j.jaci.2015.04.008
View details for Web of Science ID 000364787200023
View details for PubMedID 26037552
Synthetically Modified Viral Capsids as Versatile Carriers for Use in Antibody-Based Cell Targeting.
2015; 26 (8): 1590-1596
The present study describes an efficient and reliable method for the preparation of MS2 viral capsids that are synthetically modified with antibodies using a rapid oxidative coupling strategy. The overall protocol delivers conjugates in high yields and recoveries, requires a minimal excess of antibody to achieve modification of more than 95% of capsids, and can be completed in a short period of time. Antibody-capsid conjugates targeting extracellular receptors on human breast cancer cell lines were prepared and characterized. Notably, conjugation to the capsid did not significantly perturb the binding of the antibodies, as indicated by binding affinities similar to those obtained for the parent antibodies. An array of conjugates was synthesized with various reporters on the interior surface of the capsids to be used in cell studies, including fluorescence-based flow cytometry, confocal microscopy, and mass cytometry. The results of these studies lay the foundation for further exploration of these constructs in the context of clinically relevant applications, including drug delivery and in vivo diagnostics.
View details for DOI 10.1021/acs.bioconjchem.5b00226
View details for PubMedID 26076186
- Synthetically Modified Viral Capsids as Versatile Carriers for Use in Antibody-Based Cell Targeting BIOCONJUGATE CHEMISTRY 2015; 26 (8): 1590-1596
- An interactive reference framework for modeling a dynamic immune system SCIENCE 2015; 349 (6244): 155-?
IMMUNOLOGY. An interactive reference framework for modeling a dynamic immune system.
2015; 349 (6244)
Immune cells function in an interacting hierarchy that coordinates the activities of various cell types according to genetic and environmental contexts. We developed graphical approaches to construct an extensible immune reference map from mass cytometry data of cells from different organs, incorporating landmark cell populations as flags on the map to compare cells from distinct samples. The maps recapitulated canonical cellular phenotypes and revealed reproducible, tissue-specific deviations. The approach revealed influences of genetic variation and circadian rhythms on immune system structure, enabled direct comparisons of murine and human blood cell phenotypes, and even enabled archival fluorescence-based flow cytometry data to be mapped onto the reference framework. This foundational reference map provides a working definition of systemic immune organization to which new data can be integrated to reveal deviations driven by genetics, environment, or pathology.
View details for DOI 10.1126/science.1259425
View details for PubMedID 26160952
View details for PubMedCentralID PMC4537647
- Conditional density-based analysis of T cell signaling in single-cell data SCIENCE 2014; 346 (6213): 1079-?
NRASG12V oncogene facilitates self-renewal in a murine model of acute myelogenous leukemia.
2014; 124 (22): 3274-3283
Mutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. To elucidate the downstream functions of activated NRAS in AML, we used a murine model that harbors Mll-AF9 and a tetracycline-repressible, activated NRAS (NRAS(G12V)). Using computational approaches to explore our gene-expression data sets, we found that NRAS(G12V) enforced the leukemia self-renewal gene-expression signature and was required to maintain an MLL-AF9- and Myb-dependent leukemia self-renewal gene-expression program. NRAS(G12V) was required for leukemia self-renewal independent of its effects on growth and survival. Analysis of the gene-expression patterns of leukemic subpopulations revealed that the NRAS(G12V)-mediated leukemia self-renewal signature is preferentially expressed in the leukemia stem cell-enriched subpopulation. In a multiplexed analysis of RAS-dependent signaling, Mac-1(Low) cells, which harbor leukemia stem cells, were preferentially sensitive to NRAS(G12V) withdrawal. NRAS(G12V) maintained leukemia self-renewal through mTOR and MEK pathway activation, implicating these pathways as potential targets for cancer stem cell-specific therapies. Together, these experimental results define a RAS oncogene-driven function that is critical for leukemia maintenance and represents a novel mechanism of oncogene addiction.
View details for DOI 10.1182/blood-2013-08-521708
View details for PubMedID 25316678
- NRAS9(G12V) oncogene facilitates self-renewal in a murine model of acute myelogenous leukemia BLOOD 2014; 124 (22): 3274-3283
- Single-cell mass cytometry of TCR signaling: Amplification of small initial differences results in low ERK activation in NOD mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2014; 111 (46): 16466-16471
- The Split Virus Influenza Vaccine rapidly activates immune cells through Fc gamma receptors VACCINE 2014; 32 (45): 5989-5997
- Clinical recovery from surgery correlates with single-cell immune signatures SCIENCE TRANSLATIONAL MEDICINE 2014; 6 (255)
Clinical recovery from surgery correlates with single-cell immune signatures.
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 phenotypic 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 (signal transducer and activator of transcription), CREB (adenosine 3',5'-monophosphate response element-binding protein), and NF-κB (nuclear factor κB) signaling responses in subsets of CD14(+) monocytes (R = 0.7 to 0.8, false discovery rate <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.
View details for DOI 10.1126/scitranslmed.3009701
View details for PubMedID 25253674
Single-cell mass cytometry analysis of human tonsil T cell remodeling by varicella zoster virus.
2014; 8 (2): 633-645
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 naive 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.
View details for DOI 10.1016/j.celrep.2014.06.024
View details for PubMedID 25043183
- Single-Cell Mass Cytometry Analysis of Human Tonsil T Cell Remodeling by Varicella Zoster Virus CELL REPORTS 2014; 8 (2): 632-644
Antigen-Dependent Integration of Opposing Proximal TCR-Signaling Cascades Determines the Functional Fate of T Lymphocytes
JOURNAL OF IMMUNOLOGY
2014; 192 (5): 2109-2119
T cell anergy is a key tolerance mechanism to mitigate unwanted T cell activation against self by rendering lymphocytes functionally inactive following Ag encounter. Ag plays an important role in anergy induction where high supraoptimal doses lead to the unresponsive phenotype. How T cells "measure" Ag dose and how this determines functional output to a given antigenic dose remain unclear. Using multiparametric phospho-flow and mass cytometry, we measured the intracellular phosphorylation-dependent signaling events at a single-cell resolution and studied the phosphorylation levels of key proximal human TCR activation- and inhibition-signaling molecules. We show that the intracellular balance and signal integration between these opposing signaling cascades serve as the molecular switch gauging Ag dose. An Ag density of 100 peptide-MHC complexes/cell was found to be the transition point between dominant activation and inhibition cascades, whereas higher Ag doses induced an anergic functional state. Finally, the neutralization of key inhibitory molecules reversed T cell unresponsiveness and enabled maximal T cell functions, even in the presence of very high Ag doses. This mechanism permits T cells to make integrated "measurements" of Ag dose that determine subsequent functional outcomes.
View details for DOI 10.4049/jimmunol.1301142
View details for Web of Science ID 000332701400015
View details for PubMedID 24489091
viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia.
2013; 31 (6): 545-552
New high-dimensional, single-cell technologies offer unprecedented resolution in the analysis of heterogeneous tissues. However, because these technologies can measure dozens of parameters simultaneously in individual cells, data interpretation can be challenging. Here we present viSNE, a tool that allows one to map high-dimensional cytometry data onto two dimensions, yet conserve the high-dimensional structure of the data. viSNE plots individual cells in a visual similar to a scatter plot, while using all pairwise distances in high dimension to determine each cell's location in the plot. We integrated mass cytometry with viSNE to map healthy and cancerous bone marrow samples. Healthy bone marrow automatically maps into a consistent shape, whereas leukemia samples map into malformed shapes that are distinct from healthy bone marrow and from each other. We also use viSNE and mass cytometry to compare leukemia diagnosis and relapse samples, and to identify a rare leukemia population reminiscent of minimal residual disease. viSNE can be applied to any multi-dimensional single-cell technology.
View details for DOI 10.1038/nbt.2594
View details for PubMedID 23685480
The transcriptional landscape of aß T cell differentiation.
2013; 14 (6): 619-632
The differentiation of αβT cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited the breadth of expression data sets from the Immunological Genome Project to analyze how the differentiation of thymic precursors gives rise to mature T cell transcriptomes. We found that early T cell commitment was driven by unexpectedly gradual changes. In contrast, transit through the CD4(+)CD8(+) stage involved a global shutdown of housekeeping genes that is rare among cells of the immune system and correlated tightly with expression of the transcription factor c-Myc. Selection driven by major histocompatibility complex (MHC) molecules promoted a large-scale transcriptional reactivation. We identified distinct signatures that marked cells destined for positive selection versus apoptotic deletion. Differences in the expression of unexpectedly few genes accompanied commitment to the CD4(+) or CD8(+) lineage, a similarity that carried through to peripheral T cells and their activation, demonstrated by mass cytometry phosphoproteomics. The transcripts newly identified as encoding candidate mediators of key transitions help define the 'known unknowns' of thymocyte differentiation.
View details for DOI 10.1038/ni.2590
View details for PubMedID 23644507
Normalization of mass cytometry data with bead standards.
Cytometry. Part A : the journal of the International Society for Analytical Cytology
2013; 83 (5): 483-494
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.
View details for DOI 10.1002/cyto.a.22271
View details for PubMedID 23512433
MASS CYTOMETRY TO COMPREHENSIVELY STUDY SINGLE CELL SIGNALING IN BIOLOGY AND DISEASE
12th Euroconference on Clinical Cell Analysis / 8th European Clinical Cytometry Course
WILEY-BLACKWELL. 2012: 376–77
View details for Web of Science ID 000310386300014
Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators
2012; 30 (9): 858-U89
Mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on human samples at single-cell resolution, but instruments process only one sample at a time. Here we describe mass-tag cellular barcoding (MCB), which increases mass cytometry throughput by using n metal ion tags to multiplex up to 2n samples. We used seven tags to multiplex an entire 96-well plate, and applied MCB to characterize human peripheral blood mononuclear cell (PBMC) signaling dynamics and cell-to-cell communication, signaling variability between PBMCs from eight human donors, and the effects of 27 inhibitors on this system. For each inhibitor, we measured 14 phosphorylation sites in 14 PBMC types at 96 conditions, 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. High-content, high-throughput screening with MCB should be useful for drug discovery, preclinical testing and mechanistic investigation of human disease.
View details for DOI 10.1038/nbt.2317
View details for Web of Science ID 000308705700020
View details for PubMedID 22902532
View details for PubMedCentralID PMC3627543
Single-cell mass cytometry adapted to measurements of the cell cycle.
Cytometry. Part A : the journal of the International Society for Analytical Cytology
2012; 81 (7): 552-566
Mass cytometry is a recently introduced technology that utilizes transition element isotope-tagged antibodies for protein detection on a single-cell basis. By circumventing the limitations of emission spectral overlap associated with fluorochromes utilized in traditional flow cytometry, mass cytometry currently allows measurement of up to 40 parameters per cell. Recently, a comprehensive mass cytometry analysis was described for the hematopoietic differentiation program in human bone marrow from a healthy donor. The current study describes approaches to delineate cell cycle stages utilizing 5-iodo-2-deoxyuridine (IdU) to mark cells in S phase, simultaneously with antibodies against cyclin B1, cyclin A, and phosphorylated histone H3 (S28) that characterize the other cell cycle phases. Protocols were developed in which an antibody against phosphorylated retinoblastoma protein (Rb) at serines 807 and 811 was used to separate cells in G0 and G1 phases of the cell cycle. This mass cytometry method yielded cell cycle distributions of both normal and cancer cell populations that were equivalent to those obtained by traditional fluorescence cytometry techniques. We applied this to map the cell cycle phases of cells spanning the hematopoietic hierarchy in healthy human bone marrow as a prelude to later studies with cancers and other disorders of this lineage.
View details for DOI 10.1002/cyto.a.22075
View details for PubMedID 22693166
- Single-cell mass cytometry adapted to measurements of the cell cycle CYTOMETRY PART A 2012; 81A (7): 552-566
From single cells to deep phenotypes in cancer
2012; 30 (7): 639-647
In recent years, major advances in single-cell measurement systems have included the introduction of high-throughput versions of traditional flow cytometry that are now capable of measuring intracellular network activity, the emergence of isotope labels that can enable the tracking of a greater variety of cell markers and the development of super-resolution microscopy techniques that allow measurement of RNA expression in single living cells. These technologies will facilitate our capacity to catalog and bring order to the inherent diversity present in cancer cell populations. Alongside these developments, new computational approaches that mine deep data sets are facilitating the visualization of the shape of the data and enabling the extraction of meaningful outputs. These applications have the potential to reveal new insights into cancer biology at the intersections of stem cell function, tumor-initiating cells and multilineage tumor development. In the clinic, they may also prove important not only in the development of new diagnostic modalities but also in understanding how the emergence of tumor cell clones harboring different sets of mutations predispose patients to relapse or disease progression.
View details for DOI 10.1038/nbt.2283
View details for Web of Science ID 000306293400023
View details for PubMedID 22781693
A deep profiler's guide to cytometry
TRENDS IN IMMUNOLOGY
2012; 33 (7): 323-332
In recent years, advances in technology have provided us with tools to quantify the expression of multiple genes in individual cells. The ability to measure simultaneously multiple genes in the same cell is necessary to resolve the great diversity of cell subsets, as well as to define their function in the host. Fluorescence-based flow cytometry is the benchmark for this; with it, we can quantify 18 proteins per cell, at >10 000 cells/s. Mass cytometry is a new technology that promises to extend these capabilities significantly. Immunophenotyping by mass spectrometry provides the ability to measure >36 proteins at a rate of 1000 cells/s. We review these cytometric technologies, capable of high-content, high-throughput single-cell assays.
View details for DOI 10.1016/j.it.2012.02.010
View details for Web of Science ID 000306639500001
View details for PubMedID 22476049
Cytometry by Time-of-Flight Shows Combinatorial Cytokine Expression and Virus-Specific Cell Niches within a Continuum of CD8(+) T Cell Phenotypes
2012; 36 (1): 142-152
Cytotoxic CD8(+) T lymphocytes directly kill infected or aberrant cells and secrete proinflammatory cytokines. By using metal-labeled probes and mass spectrometric analysis (cytometry by time-of-flight, or CyTOF) of human CD8(+) T cells, we analyzed the expression of many more proteins than previously possible with fluorescent labels, including surface markers, cytokines, and antigen specificity with modified peptide-MHC tetramers. With 3-dimensional principal component analysis (3D-PCA) to display phenotypic diversity, we observed a relatively uniform pattern of variation in all subjects tested, highlighting the interrelatedness of previously described subsets and the continuous nature of CD8(+) T cell differentiation. These data also showed much greater complexity in the CD8(+) T cell compartment than previously appreciated, including a nearly combinatorial pattern of cytokine expression, with distinct niches occupied by virus-specific cells. This large degree of functional diversity even between cells with the same specificity gives CD8(+) T cells a remarkable degree of flexibility in responding to pathogens.
View details for DOI 10.1016/j.immuni.2012.01.002
View details for Web of Science ID 000299766000017
View details for PubMedID 22265676
Extracting a cellular hierarchy from high-dimensional cytometry data with SPADE
2011; 29 (10): 886-U181
The ability to analyze multiple single-cell parameters is critical for understanding cellular heterogeneity. Despite recent advances in measurement technology, methods for analyzing high-dimensional single-cell data are often subjective, labor intensive and require prior knowledge of the biological system. To objectively uncover cellular heterogeneity from single-cell measurements, we present a versatile computational approach, spanning-tree progression analysis of density-normalized events (SPADE). We applied SPADE to flow cytometry data of mouse bone marrow and to mass cytometry data of human bone marrow. In both cases, SPADE organized cells in a hierarchy of related phenotypes that partially recapitulated well-described patterns of hematopoiesis. We demonstrate that SPADE is robust to measurement noise and to the choice of cellular markers. SPADE facilitates the analysis of cellular heterogeneity, the identification of cell types and comparison of functional markers in response to perturbations.
View details for DOI 10.1038/nbt.1991
View details for Web of Science ID 000296273000015
View details for PubMedID 21964415
Clonal tracking of hESCs reveals differential contribution to functional assays
2010; 7 (11): 917-U75
Human embryonic stem cells (hESCs) have unique self-renewal and differentiation properties, which are experimentally measured using functional assays. hESC cultures are known to be heterogeneous, but whether subsets of cells contribute differently to functional assays has yet to be examined. Here, using clonal tracking by retroviral integration, we analyzed in situ the propensity of individual hESCs to contribute to different functional assays. We observed different clonal distributions in teratomas versus in vitro differentiation assays. Some hESC subsets apparently contributed substantially to lineage-specific embryoid body differentiation and lacked clonogenic capacity, although they had self-renewal ability. In contrast, other subsets of self-renewing hESCs with clonogenic ability contributed to teratoma formation but were less frequently observed after in vitro differentiation. Our study suggests that assays used to measure pluripotency may detect distinct subsets of hESCs. These findings have direct implications for hESC-based therapies that may be optimized based on such functional assays.
View details for DOI 10.1038/nmeth.1519
View details for Web of Science ID 000283586600014
View details for PubMedID 20953174
A HUPO test sample study reveals common problems in mass spectrometry-based proteomics
2009; 6 (6): 423-U40
We performed a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in liquid chromatography-mass spectrometry-based proteomics. We distributed an equimolar test sample, comprising 20 highly purified recombinant human proteins, to 27 laboratories. Each protein contained one or more unique tryptic peptides of 1,250 Da to test for ion selection and sampling in the mass spectrometer. Of the 27 labs, members of only 7 labs initially reported all 20 proteins correctly, and members of only 1 lab reported all tryptic peptides of 1,250 Da. Centralized analysis of the raw data, however, revealed that all 20 proteins and most of the 1,250 Da peptides had been detected in all 27 labs. Our centralized analysis determined missed identifications (false negatives), environmental contamination, database matching and curation of protein identifications as sources of problems. Improved search engines and databases are needed for mass spectrometry-based proteomics.
View details for DOI 10.1038/NMETH.1333
View details for Web of Science ID 000266493300012
View details for PubMedID 19448641
An Enhanced Mass Spectrometry Approach Reveals Human Embryonic Stem Cell Growth Factors in Culture
MOLECULAR & CELLULAR PROTEOMICS
2009; 8 (3): 421-432
The derivation and long-term maintenance of human embryonic stem cells (hESCs) has been established in culture formats that are both dependent and independent of support (feeder) cells. However, the factors responsible for preserving the viability of hESCs in a nascent state remain unknown. We describe a mass spectrometry-based method for probing the secretome of the hESC culture microenvironment to identify potential regulating protein factors that are in low abundance. Individual samples were analyzed several times, using successive mass (m/z) and retention time-directed exclusion, without sampling the same peptide ion twice. This iterative exclusion -mass spectrometry (IE-MS) approach more than doubled protein and peptide metrics in comparison to a simple repeat analysis method on the same instrument, even after extensive sample pre-fractionation. Furthermore, implementation of the IE-MS approach was shown to enhance the performance of an older quadrupole time of flight (Q-ToF) MS. The resulting number of identified peptides approached that of a parallel repeat analysis on a newer LTQ-Orbitrap MS. The combination of the results of both instruments proved to be superior to that achieved by a single instrument in the identification of additional proteins. Using the IE-MS strategy, combined with complementary gel- and solution-based fractionation methods, the hESC culture microenvironment was extensively probed. Over 10 to 12 times more extracellular proteins were observed compared with previously published surveys. The detection of previously undetectable growth factors, present at concentrations ranging from 10(-9) to 10(-11) g/ml, highlights the depth of our profiling. The IE-MS approach provides a simple and reliable technique that greatly enhances instrument performance by increasing the effective depth of MS-based proteomic profiling. This approach should be widely applicable to any LC-MS/MS instrument platform or biological system.
View details for DOI 10.1074/mcp.M800190-MCP200
View details for Web of Science ID 000264240000003
View details for PubMedID 18936058
Prevention of amino acid conversion in SILAC experiments with embryonic stem cells
MOLECULAR & CELLULAR PROTEOMICS
2008; 7 (9): 1587-1597
Recent studies using stable isotope labeling with amino acids in culture (SILAC) in quantitative proteomics have made mention of the problematic conversion of isotope-coded arginine to proline in cells. The resulting converted proline peptide divides the heavy peptide ion signal causing inaccuracy when compared with the light peptide ion signal. This is of particular concern as it can effect up to half of all peptides in a proteomic experiment. Strategies to both compensate for and limit the inadvertent conversion have been demonstrated, but none have been shown to prevent it. Additionally, these methods combined with SILAC labeling in general have proven problematic in their large scale application to sensitive cell types including embryonic stem cells (ESCs) from the mouse and human. Here, we show that by providing as little as 200 mg/liter L-proline in SILAC media, the conversion of arginine to proline can be rendered completely undetectable. At the same time, there was no compromise in labeling with isotope-coded arginine, indicating there is no observable back conversion from the proline supplement. As a result, when supplemented with proline, correct interpretation of "light" and "heavy" peptide ratios could be achieved even in the worst cases of conversion. By extending these principles to ESC culture protocols and reagents we were able to routinely SILAC label both mouse and human ESCs in the absence of feeder cells and without compromising the pluripotent phenotype. This study provides the simplest protocol to prevent proline artifacts in SILAC labeling experiments with arginine. Moreover, it presents a robust, feeder cell-free, protocol for performing SILAC experiments on ESCs from both the mouse and the human.
View details for DOI 10.1074/mcp.M800113-MCP200
View details for Web of Science ID 000259154800001
View details for PubMedID 18487603
Deconstructing human embryonic stem cell cultures: niche regulation of self-renewal and pluripotency
JOURNAL OF MOLECULAR MEDICINE-JMM
2008; 86 (8): 875-886
The factors and signaling pathways controlling pluripotent human cell properties, both embryonic and induced, have not been fully investigated. Failure to account for functional heterogeneity within human embryonic stem cell (hESC) cultures has led to inconclusive results in previous work examining extrinsic influences governing hESC fate (self renewal vs. differentiation vs. death). Here, we attempt to reconcile these inconsistencies with recent reports demonstrating that an autologously produced in vitro niche regulates hESCs. Moreover, we focus on the reciprocal paracrine signals within the in vitro hESC niche allowing for the maintenance and/or expansion of the hESC colony-initiating cell (CIC). Based on this, it is clear that separation of hESC-CICs, apart from their differentiated derivatives, will be essential in future studies involving their molecular regulation. Understanding how extrinsic factors control hESC self-renewal and differentiation will allow us to culture and differentiate these pluripotent cells with higher efficiency. This knowledge will be essential for clinical applications using human pluripotent cells in regenerative medicine.
View details for DOI 10.1007/s00109-008-0356-9
View details for Web of Science ID 000257945800003
View details for PubMedID 18521556
Human embryonic stem cells: lessons from stem cell niches in vivo
2008; 3 (3): 365-376
In vivo the stem cell niche is an essential component in controlling and maintaining the stem cells' ability to survive and respond to injury. Human embryonic stem cells (hESCs) appear to be an exception to this rule as they can be removed from their blastocytic microenvironment and maintained indefinitely in vitro. However, recent observations reveal the existence of an autonomously derived in vitro hESC niche. This provides a previously unappreciated mechanism to control hESC expansion and differentiation. Recognizing this, it may now be possible to take aspects of in vivo stem cell niches, namely extracellular matrices, paracrine signals and accessory cell types, and exploit them in order to gain fidelity in directed hESC differentiation. In doing so, routine customization of hESC lines and their application in regenerative therapies may be further enhanced using unique hESC niche-based approaches.
View details for DOI 10.2217/174607184.108.40.2065
View details for Web of Science ID 000257995200020
View details for PubMedID 18462059
IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro
2007; 448 (7157): 1015-U3
Distinctive properties of stem cells are not autonomously achieved, and recent evidence points to a level of external control from the microenvironment. Here, we demonstrate that self-renewal and pluripotent properties of human embryonic stem (ES) cells depend on a dynamic interplay between human ES cells and autologously derived human ES cell fibroblast-like cells (hdFs). Human ES cells and hdFs are uniquely defined by insulin-like growth factor (IGF)- and fibroblast growth factor (FGF)-dependence. IGF 1 receptor (IGF1R) expression was exclusive to the human ES cells, whereas FGF receptor 1 (FGFR1) expression was restricted to surrounding hdFs. Blocking the IGF-II/IGF1R pathway reduced survival and clonogenicity of human ES cells, whereas inhibition of the FGF pathway indirectly caused differentiation. IGF-II is expressed by hdFs in response to FGF, and alone was sufficient in maintaining human ES cell cultures. Our study demonstrates a direct role of the IGF-II/IGF1R axis on human ES cell physiology and establishes that hdFs produced by human ES cells themselves define the stem cell niche of pluripotent human stem cells.
View details for DOI 10.1038/nature06027
View details for Web of Science ID 000249097600030
View details for PubMedID 17625568
Proteomic analysis of pluripotent stem cells.
Current protocols in stem cell biology
2007; Chapter 1: Unit 1B 1-?
Mass spectrometry (MS)-based proteomics has become one of the most powerful tools for identifying expressed proteins, providing quick insights into molecular and cellular biology. Traditionally, proteins isolated by either one- or two-dimensional gel electrophoresis are digested with a site specific protease. The resulting peptides are subject to one of two forms of analysis: (1) matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, where a "mass fingerprint" of all the peptides in a sample is generated, or (2) electrospray ionization tandem MS (ESI-MS/MS), where a mass fragmentation spectra is generated for each peptide in a sample. The resulting mass information is then compared to that of a theoretical database created with available genomic sequence information. This unit provides protocols for this type of assessment in embryonic stem cells (ESCs).
View details for DOI 10.1002/9780470151808.sc01b01s2
View details for PubMedID 18785159
Clonal isolation of hESCs reveals heterogeneity within the pluripotent stem cell compartment
2006; 3 (10): 807-815
Human embryonic stem cell (hESC) lines are known to be morphologically and phenotypically heterogeneous. The functional nature and relationship of cells residing within hESC cultures, however, has not been evaluated because isolation of single hESCs is limited to drug or manual selection. Here we provide a quantitative method using flow cytometry to isolate and clonally expand hESCs based on undifferentiated markers, alone or in combination with a fluorescent reporter. This method allowed for isolation of stage-specific embryonic antigen-3-positive (SSEA-3+) and SSEA-3- cells from hESC cultures. Although both SSEA-3+ and SSEA-3- cells could initiate pluripotent hESC cultures, we show that they possess distinct cell-cycle properties, clonogenic capacity and expression of ESC transcription factors. Our study provides formal evidence for heterogeneity among self-renewing pluripotent hESCs, illustrating that this isolation technique will be instrumental in further dissecting the biology of hESC lines.
View details for DOI 10.1038/nmeth939
View details for Web of Science ID 000240942600015
View details for PubMedID 16990813
- Complement targeting of nonhuman sialic acid does not mediate cell death of human embryonic stem cells NATURE MEDICINE 2006; 12 (10): 1113-1114