Article

Single-Cell Mass Cytometry of Differential Immune and Drug Responses Across a Human Hematopoietic Continuum

Baxter Laboratory in Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA.
Science (Impact Factor: 31.48). 05/2011; 332(6030):687-96. DOI: 10.1126/science.1198704
Source: PubMed

ABSTRACT 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.

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    • "However, this approach has many problems and limitations, including variability between different analysts, non-standardization and non-reproducibility of results, an unrealistic assumption that biological relationships between the markers exist only in the projected low-dimensional space and, most importantly, non-scalability to high-dimensional analysis, especially involving a large number of samples. With the advancement of technology, modern day flow cytometers allow simultaneous measurements of many markers on millions of cells, with some latest revolutionary mass cytometers capable of extending this up to 100 simultaneous parameters [1] [2]. As the number of markers increases, the number of bivariate projections increase rapidly. "
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    • "Thus CyTOF studies can combine ~40 labels in a sample. CyTOF has recently been employed to characterize peripheral blood cells in detail (Bendall et al., 2011) as well as NK cells (Horowitz et al., 2013), γδ cells in Celiac disease (Han et al., 2013), responding phenotypes in cancer (Irish and Doxie, 2014), and even holds the promise of examining solid tumors (Giesen et al., 2014). "
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    • "This method has been dubbed " mass cytometry " because fluorophore-tagged antibody reporters are replaced with isotopically tagged ones, and depends on the use of an inductively coupled-plasma time-of-flight mass spectrometer, which can resolve up to 100 different rare-earth-metal-labeled antibody probes with single-cell sensitivity (Bandura et al. 2009; Zivanovic et al. 2013). This technology has been used successfully to define the state of 31 different proteins in particular cell types of the hematopoietic lineage in bone marrow (Bendall et al. 2011), examine the effect of 27 small-molecule protein kinase inhibitors on 14 different phosphorylation sites in human peripheral blood mononuclear cells from eight donors (Bodenmiller et al. 2012), and identify T cells specific for particular epitopes among 77 candidate rotaviral antigens (Newell et al. 2013). However, the sophisticated instrumentation necessary is expensive and not widely available. "
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