Data-Driven Phenotypic Dissection of AML Reveals Progenitor-like Cells that Correlate with Prognosis.

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.

IL-1ß Inhibition Partially Negates the Beneficial Effects of Diet-Induced Atherosclerosis Regression in Mice.

BACKGROUND: Thromboembolic events secondary to rupture or erosion of advanced atherosclerotic lesions is the global leading cause of death. The most common and effective means to reduce these major adverse cardiovascular events, including myocardial infarction and stroke, is aggressive lipid lowering via a combination of drugs and dietary modifications. However, we know little regarding the effects of reducing dietary lipids on the composition and stability of advanced atherosclerotic lesions, the mechanisms that regulate these processes, and what therapeutic approaches might augment the benefits of lipid lowering. METHODS: Smooth muscle cell lineage-tracing Apoe-/- mice were fed a high-cholesterol Western diet for 18 weeks and then a zero-cholesterol standard laboratory diet for 12 weeks before treating them with an IL (interleukin)-1ß or control antibody for 8 weeks. We assessed lesion size and remodeling indices, as well as the cellular composition of aortic and brachiocephalic artery lesions, indices of plaque stability, overall plaque burden, and phenotypic transitions of smooth muscle cell and other lesion cells by smooth muscle cell lineage tracing combined with single-cell RNA sequencing, cytometry by time-of-flight, and immunostaining plus high-resolution confocal microscopic z-stack analysis. RESULTS: Lipid lowering by switching Apoe-/- mice from a Western diet to a standard laboratory diet reduced LDL cholesterol levels by 70% and resulted in multiple beneficial effects including reduced overall aortic plaque burden, as well as reduced intraplaque hemorrhage and necrotic core area. However, contrary to expectations, IL-1ß antibody treatment after diet-induced reductions in lipids resulted in multiple detrimental changes including increased plaque burden and brachiocephalic artery lesion size, as well as increasedintraplaque hemorrhage, necrotic core area, and senescence as compared with IgG control antibody-treated mice. Furthermore, IL-1ß antibody treatment upregulated neutrophil degranulation pathways but downregulated smooth muscle cell extracellular matrix pathways likely important for the protective fibrous cap. CONCLUSIONS: Taken together, IL-1ß appears to be required for the maintenance of standard laboratory diet-induced reductions in plaque burden and increases in multiple indices of plaque stability.

Sexual dimorphism in the dorsal root ganglia of neonatal mice identified by protein expression profiling with single-cell mass cytometry.

Development of neuronal and glial populations in the dorsal root ganglia (DRG) is required for detection of touch, body position, temperature, and noxious stimuli. While female-male differences in somatosensory perception have been previously reported, no study has examined global sex differences in the abundance of DRG cell types, and the developmental origin of these differences has not been characterized. To investigate whether sex-specific differences in neuronal and glial cell types arise in the DRG during development, we performed single-cell mass cytometry analysis on sex-separated DRGs from 4 separate litter replicates of postnatal day 0 (P0) C57/BL6 mouse pups. In this analysis, we observed that females had a higher abundance of total neurons (p = 0.0266), as well as an increased abundance of TrkB+ (p = 0.031) and TrkC+ (p = 0.04) neurons for mechanoreception and proprioception, while males had a higher abundance of TrkA+ (p = 0.025) neurons for thermoreception and nociception. Pseudotime comparison of the female and male datasets indicates that male neurons are more mature and differentiated than female neurons at P0. These findings warrant further studies to determine whether these differences are maintained across development, and their impact on somatosensory perception.

Stem Cell Pluripotency Genes Klf4 and Oct4 Regulate Complex SMC Phenotypic Changes Critical in Late-Stage Atherosclerotic Lesion Pathogenesis.

BACKGROUND: Rupture and erosion of advanced atherosclerotic lesions with a resultant myocardial infarction or stroke are the leading worldwide cause of death. However, we have a limited understanding of the identity, origin, and function of many cells that make up late-stage atherosclerotic lesions, as well as the mechanisms by which they control plaque stability. METHODS: We conducted a comprehensive single-cell RNA sequencing of advanced human carotid endarterectomy samples and compared these with single-cell RNA sequencing from murine microdissected advanced atherosclerotic lesions with smooth muscle cell (SMC) and endothelial lineage tracing to survey all plaque cell types and rigorously determine their origin. We further used chromatin immunoprecipitation sequencing (ChIP-seq), bulk RNA sequencing, and an innovative dual lineage tracing mouse to understand the mechanism by which SMC phenotypic transitions affect lesion pathogenesis. RESULTS: We provide evidence that SMC-specific Klf4- versus Oct4-knockout showed virtually opposite genomic signatures, and their putative target genes play an important role regulating SMC phenotypic changes. Single-cell RNA sequencing revealed remarkable similarity of transcriptomic clusters between mouse and human lesions and extensive plasticity of SMC- and endothelial cell-derived cells including 7 distinct clusters, most negative for traditional markers. In particular, SMC contributed to a Myh11-, Lgals3+ population with a chondrocyte-like gene signature that was markedly reduced with SMC-Klf4 knockout. We observed that SMCs that activate Lgals3 compose up to two thirds of all SMC in lesions. However, initial activation of Lgals3 in these cells does not represent conversion to a terminally differentiated state, but rather represents transition of these cells to a unique stem cell marker gene-positive, extracellular matrix-remodeling, "pioneer" cell phenotype that is the first to invest within lesions and subsequently gives rise to at least 3 other SMC phenotypes within advanced lesions, including Klf4-dependent osteogenic phenotypes likely to contribute to plaque calcification and plaque destabilization. CONCLUSIONS: Taken together, these results provide evidence that SMC-derived cells within advanced mouse and human atherosclerotic lesions exhibit far greater phenotypic plasticity than generally believed, with Klf4 regulating transition to multiple phenotypes including Lgals3+ osteogenic cells likely to be detrimental for late-stage atherosclerosis plaque pathogenesis.

Early reprogramming regulators identified by prospective isolation and mass cytometry.

In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of non-productive and staggered productive intermediates arise at different reprogramming time points. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells, prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that, during reprogramming, cells progressively lose donor cell identity and gradually acquire iPS cell properties. Here we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen, we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200, that are absent in both fibroblasts and iPS cells. Single-cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic, reprogramming phase. Expression profiling reveals early upregulation of the transcriptional regulators Nr0b1 and Etv5 in this reprogramming state, preceding activation of key pluripotency regulators such as Rex1 (also known as Zfp42), Dppa2, Nanog and Sox2. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus represent some of the earliest known regulators of iPS cell induction. Our study deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition.

RUNX3 levels in human hematopoietic progenitors are regulated by aging and dictate erythroid-myeloid balance.

Healthy bone marrow progenitors yield a co-ordinated balance of hematopoietic lineages. This balance shifts with aging toward enhanced granulopoiesis with diminished erythropoiesis and lymphopoiesis, changes which likely contribute to the development of bone marrow disorders in the elderly. In this study, RUNX3 was identified as a hematopoietic stem and progenitor cell factor whose levels decline with aging in humans and mice. This decline is exaggerated in hematopoietic stem and progenitor cells from subjects diagnosed with unexplained anemia of the elderly. Hematopoietic stem cells from elderly unexplained anemia patients had diminished erythroid but unaffected granulocytic colony forming potential. Knockdown studies revealed human hematopoietic stem and progenitor cells to be strongly influenced by RUNX3 levels, with modest deficiencies abrogating erythroid differentiation at multiple steps while retaining capacity for granulopoiesis. Transcriptome profiling indicated control by RUNX3 of key erythroid transcription factors, including KLF1 and GATA1 These findings thus implicate RUNX3 as a participant in hematopoietic stem and progenitor cell aging, and a key determinant of erythroid-myeloid lineage balance.

Cutting Edge: Critical Roles for Microbiota-Mediated Regulation of the Immune System in a Prenatal Immune Activation Model of Autism.

Recent studies suggest that autism is often associated with dysregulated immune responses and altered microbiota composition. This has led to growing speculation about potential roles for hyperactive immune responses and the microbiome in autism. Yet how microbiome-immune cross-talk contributes to neurodevelopmental disorders currently remains poorly understood. In this study, we report critical roles for prenatal microbiota composition in the development of behavioral abnormalities in a murine maternal immune activation (MIA) model of autism that is driven by the viral mimetic polyinosinic-polycytidylic acid. We show that preconception microbiota transplantation can transfer susceptibility to MIA-associated neurodevelopmental disease and that this is associated with modulation of the maternal immune response. Furthermore, we find that ablation of IL-17a signaling provides protection against the development of neurodevelopmental abnormalities in MIA offspring. Our findings suggest that microbiota landscape can influence MIA-induced neurodevelopmental disease pathogenesis and that this occurs as a result of microflora-associated calibration of gestational IL-17a responses.

Highly multiplexed simultaneous detection of RNAs and proteins in single cells.

To enable the detection of expression signatures specific to individual cells, we developed PLAYR (proximity ligation assay for RNA), a method for highly multiplexed transcript quantification by flow and mass cytometry that is compatible with standard antibody staining. When used with mass cytometry, PLAYR allowed for the simultaneous quantification of more than 40 different mRNAs and proteins. In primary cells, we quantified multiple transcripts, with the identity and functional state of each analyzed cell defined on the basis of the expression of a separate set of transcripts or proteins. By expanding high-throughput deep phenotyping of cells beyond protein epitopes to include RNA expression, PLAYR opens a new avenue for the characterization of cellular metabolism.

Atomic mass tag of bismuth-209 for increasing the immunoassay multiplexing capacity of mass cytometry.

Mass cytometry (or CyTOF) is an atomic mass spectrometry-based single-cell immunoassay technology, which has provided an increasingly systematic and sophisticated view in basic biological and clinical studies. Using elemental reporters composed of stable heavy metal isotopes, more than 50 cellular parameters are measured simultaneously. However, this current multiplexing does not meet the theoretical capability of CyTOF instrumentation with 135 detectable channels, primarily due to the limitation of available chemistries for conjugating elemental mass tags to affinity reagents. To address this issue, we develop herein additional metallic mass tag based on bismuth-209 (209 Bi) for efficient conjugation to monoclonal antibody. This enables the use of an addtional channel m/z = 209 of CyTOF for single-cell immunoassays. Bismuth has nearly the same charge-to-radius ratio as lanthanide elements; thus, bismuth(III) cations (209 Bi3+ ) could coordinate with DTPA chelators in the same geometry of O- and N-donor groups as that of lanthanide. In this report, the coordination chemistry of 209 Bi3+ with DTPA chelators and Maxpar® X8 polymers were investigated in details. Accordingly, the protocols of conjugating antibody with bismuth mass tag were provided. A method based on UV-Vis absorbance at 280 nm of 209 Bi3+ -labeling DTPA complexes was developed to evaluate the stoichiometric ratio of 209 Bi3+ cations to the conjugated antibody. Side-by-side single-cell analysis experiments with bismuth- and lanthanide-tagged antibodies were carried out to compare the analytical sensitivities. The measurement accuracy of bismuth-tagged antibody was validated within in vitro assay using primary human natural killer cells. Furthermore, bismuth-tagged antibodies were successfully employed in cell cycle measurements and high-dimensional phenotyping immunoassays. © 2017 International Society for Advancement of Cytometry.

Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining.

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.

Direct comparison of mass cytometry and single-cell RNA sequencing of human peripheral blood mononuclear cells.

Single-cell methods offer a high-resolution approach for characterizing cell populations. Many studies rely on single-cell transcriptomics to draw conclusions regarding cell state and behavior, with the underlying assumption that transcriptomic readouts largely parallel their protein counterparts and subsequent activity. However, the relationship between transcriptomic and proteomic measurements is imprecise, and thus datasets that probe the extent of their concordance will be useful to refine such conclusions. Additionally, novel single-cell analysis tools often lack appropriate gold standard datasets for the purposes of assessment. Integrative (combining the two data modalities) and predictive (using one modality to improve results from the other) approaches in particular, would benefit from transcriptomic and proteomic data from the same sample of cells. For these reasons, we performed single-cell RNA sequencing, mass cytometry, and flow cytometry on a split-sample of human peripheral blood mononuclear cells. We directly compare the proportions of specific cell types resolved by each technique, and further describe the extent to which protein and mRNA measurements correlate within distinct cell types.

The good, the bald, and the hairy: A mechanosensor meets its fate at the target.

In this issue of Developmental Cell, Koutsioumpa et al. (2023) investigate the maturation of low-threshold mechanoreceptor nerve endings in both hairy and glabrous skin types and discover a critical role for target-derived BMP in the development of Meissner corpuscles in glabrous (i.e., hairless) skin.

A hyperparameter-randomized ensemble approach for robust clustering across diverse datasets.

Clustering analysis is widely used to group objects by similarity, but for complex datasets such as those produced by single-cell analysis, the currently available clustering methods are limited by accuracy, robustness, ease of use, and interpretability. To address these limitations, we developed an ensemble clustering method with hyperparameter randomization that outperforms other methods across a broad range of single-cell and synthetic datasets, without the need for manual hyperparameter selection. In addition to hard cluster labels, it also outputs soft cluster memberships to characterize continuum-like regions and per cell overlap scores to quantify the uncertainty in cluster assignment. We demonstrate the improved clustering interpretability from these features by tracing the intermediate stages between handwritten digits in the MNIST dataset, and between tanycyte subpopulations in the hypothalamus. This approach improves the quality of clustering and subsequent downstream analyses for single-cell datasets, and may also prove useful in other fields of data analysis.

IL-1ß inhibition partially negates the beneficial effects of diet-induced lipid lowering.

Background: Thromboembolic events secondary to rupture or erosion of advanced atherosclerotic lesions are the leading cause of death in the world. The most common and effective means to reduce these major adverse cardiovascular events (MACE), including myocardial infarction (MI) and stroke, is aggressive lipid lowering via a combination of drugs and dietary modifications. However, little is known regarding the effects of reducing dietary lipids on the composition and stability of advanced atherosclerotic lesions, the mechanisms that regulate these processes, and what therapeutic approaches might augment the benefits of lipid lowering. Methods: Smooth muscle cell (SMC)-lineage tracing Apoe-/- mice were fed a Western diet (WD) for 18 weeks and then switched to a low-fat chow diet for 12 weeks. We assessed lesion size and remodeling indices, as well as the cellular composition of aortic and brachiocephalic artery (BCA) lesions, indices of plaque stability, overall plaque burden, and phenotypic transitions of SMC, and other lesion cells by SMC-lineage tracing combined with scRNA-seq, CyTOF, and immunostaining plus high resolution confocal microscopic z-stack analysis. In addition, to determine if treatment with a potent inhibitor of inflammation could augment the benefits of chow diet-induced reductions in LDL-cholesterol, SMC-lineage tracing Apoe-/- mice were fed a WD for 18 weeks and then chow diet for 12 weeks prior to treating them with an IL-1ß or control antibody (Ab) for 8-weeks. Results: Lipid-lowering by switching Apoe-/- mice from a WD to a chow diet reduced LDL-cholesterol levels by 70% and resulted in multiple beneficial effects including reduced overall aortic plaque burden as well as reduced intraplaque hemorrhage and necrotic core area. However, contrary to expectations, IL-1ß Ab treatment resulted in multiple detrimental changes including increased plaque burden, BCA lesion size, as well as increased cholesterol crystal accumulation, intra-plaque hemorrhage, necrotic core area, and senescence as compared to IgG control Ab treated mice. Furthermore, IL-1ß Ab treatment upregulated neutrophil degranulation pathways but down-regulated SMC extracellular matrix pathways likely important for the protective fibrous cap. Conclusions: Taken together, IL-1ß appears to be required for chow diet-induced reductions in plaque burden and increases in multiple indices of plaque stability.

A developmental atlas of somatosensory diversification and maturation in the dorsal root ganglia by single-cell mass cytometry.

Precisely controlled development of the somatosensory system is essential for detecting pain, itch, temperature, mechanical touch and body position. To investigate the protein-level changes that occur during somatosensory development, we performed single-cell mass cytometry on dorsal root ganglia from C57/BL6 mice of both sexes, with litter replicates collected daily from embryonic day 11.5 to postnatal day 4. Measuring nearly 3 million cells, we quantified 30 molecularly distinct somatosensory glial and 41 distinct neuronal states across all timepoints. Analysis of differentiation trajectories revealed rare cells that co-express two or more Trk receptors and over-express stem cell markers, suggesting that these neurotrophic factor receptors play a role in cell fate specification. Comparison to previous RNA-based studies identified substantial differences between many protein-mRNA pairs, demonstrating the importance of protein-level measurements to identify functional cell states. Overall, this study demonstrates that mass cytometry is a high-throughput, scalable platform to rapidly phenotype somatosensory tissues.

Current evidence on potential of adipose derived stem cells to enhance bone regeneration and future projection.

Injuries to the postnatal skeleton are naturally repaired through successive steps involving specific cell types in a process collectively termed "bone regeneration". Although complex, bone regeneration occurs through a series of well-orchestrated stages wherein endogenous bone stem cells play a central role. In most situations, bone regeneration is successful; however, there are instances when it fails and creates non-healing injuries or fracture nonunion requiring surgical or therapeutic interventions. Transplantation of adult or mesenchymal stem cells (MSCs) defined by the International Society for Cell and Gene Therapy (ISCT) as CD105+CD90+CD73+CD45-CD34-CD14orCD11b-CD79αorCD19-HLA-DR- is being investigated as an attractive therapy for bone regeneration throughout the world. MSCs isolated from adipose tissue, adipose-derived stem cells (ADSCs), are gaining increasing attention since this is the most abundant source of adult stem cells and the isolation process for ADSCs is straightforward. Currently, there is not a single Food and Drug Administration (FDA) approved ADSCs product for bone regeneration. Although the safety of ADSCs is established from their usage in numerous clinical trials, the bone-forming potential of ADSCs and MSCs, in general, is highly controversial. Growing evidence suggests that the ISCT defined phenotype may not represent bona fide osteoprogenitors. Transplantation of both ADSCs and the CD105- sub-population of ADSCs has been reported to induce bone regeneration. Most notably, cells expressing other markers such as CD146, AlphaV, CD200, PDPN, CD164, CXCR4, and PDGFRα have been shown to represent osteogenic sub-population within ADSCs. Amongst other strategies to improve the bone-forming ability of ADSCs, modulation of VEGF, TGF-ß1 and BMP signaling pathways of ADSCs has shown promising results. The U.S. FDA reveals that 73% of Investigational New Drug applications for stem cell-based products rely on CD105 expression as the "positive" marker for adult stem cells. A concerted effort involving the scientific community, clinicians, industries, and regulatory bodies to redefine ADSCs using powerful selection markers and strategies to modulate signaling pathways of ADSCs will speed up the therapeutic use of ADSCs for bone regeneration.

Identification of human immune cell subtypes most responsive to IL-1ß-induced inflammatory signaling using mass cytometry.

IL-1ß is a key mediator of the cytokine storm linked to high morbidity and mortality from COVID-19, and IL-1ß blockade with anakinra and canakinumab during COVID-19 infection has entered clinical trials. Using mass cytometry of human peripheral blood mononuclear cells, we identified effector memory CD4+ T cells and CD4-CD8low/-CD161+ T cells, specifically those positive for the chemokine receptor CCR6, as the circulating immune subtypes with the greatest response to IL-1ß. This response manifested as increased phosphorylation and, thus, activation of the proinflammatory transcription factor NF-κB and was also seen in other subsets, including CD11c+ myeloid dendritic cells, classical monocytes, two subsets of natural killer cells (CD16-CD56brightCD161- and CD16-CD56dimCD161+), and lineage- (Lin-) cells expressing CD161 and CD25. IL-1ß also induced a rapid but less robust increase in the phosphorylation of the kinase p38 as compared to that of NF-κB in most of these immune cell subsets. Prolonged IL-1ß stimulation increased the phosphorylation of the transcription factor STAT3 and to a lesser extent that of STAT1 and STAT5 across various immune cell types. IL-1ß-induced production of IL-6 likely led to the activation of STAT1 and STAT3 at later time points. Interindividual heterogeneity and inhibition of STAT activation by anakinra raise the possibility that assays measuring NF-κB phosphorylation in response to IL-1ß in CCR6+ T cell subtypes could identify those patients at higher risk of cytokine storm and most likely to benefit from IL-1ß-neutralizing therapies.

FLOW-MAP: a graph-based, force-directed layout algorithm for trajectory mapping in single-cell time course datasets.

High-dimensional single-cell technologies present new opportunities for biological discovery, but the complex nature of the resulting datasets makes it challenging to perform comprehensive analysis. One particular challenge is the analysis of single-cell time course datasets: how to identify unique cell populations and track how they change across time points. To facilitate this analysis, we developed FLOW-MAP, a graphical user interface (GUI)-based software tool that uses graph layout analysis with sequential time ordering to visualize cellular trajectories in high-dimensional single-cell datasets obtained from flow cytometry, mass cytometry or single-cell RNA sequencing (scRNAseq) experiments. Here we provide a detailed description of the FLOW-MAP algorithm and how to use the open-source R package FLOWMAPR via its GUI or with text-based commands. This approach can be applied to many dynamic processes, including in vitro stem cell differentiation, in vivo development, oncogenesis, the emergence of drug resistance and cell signaling dynamics. To demonstrate our approach, we perform a step-by-step analysis of a single-cell mass cytometry time course dataset from mouse embryonic stem cells differentiating into the three germ layers: endoderm, mesoderm and ectoderm. In addition, we demonstrate FLOW-MAP analysis of a previously published scRNAseq dataset. Using both synthetic and experimental datasets for comparison, we perform FLOW-MAP analysis side by side with other single-cell analysis methods, to illustrate when it is advantageous to use the FLOW-MAP approach. The protocol takes between 30 min and 1.5 h to complete.

AN UPDATED DEBARCODING TOOL FOR MASS CYTOMETRY WITH CELL TYPE-SPECIFIC AND CELL SAMPLE-SPECIFIC STRINGENCY ADJUSTMENT.

Pooled sample analysis by mass cytometry barcoding carries many advantages: reduced antibody consumption, increased sample throughput, removal of cell doublets, reduction of cross-contamination by sample carryover, and the elimination of tube-to-tube-variability in antibody staining. A single-cell debarcoding algorithm was previously developed to improve the accuracy and yield of sample deconvolution, but this method was limited to using fixed parameters for debarcoding stringency filtering, which could introduce cell-specific or sample-specific bias to cell yield in scenarios where barcode staining intensity and variance are not uniform across the pooled samples. To address this issue, we have updated the algorithm to output debarcoding parameters for every cell in the sample-assigned FCS files, which allows for visualization and analysis of these parameters via flow cytometry analysis software. This strategy can be used to detect cell type-specific and sample-specific effects on the underlying cell data that arise during the debarcoding process. An additional benefit to this strategy is the decoupling of barcode stringency filtering from the debarcoding and sample assignment process. This is accomplished by removing the stringency filters during sample assignment, and then filtering after the fact with 1- and 2-dimensional gating on the debarcoding parameters which are output with the FCS files. These data exploration strategies serve as an important quality check for barcoded mass cytometry datasets, and allow cell type and sample-specific stringency adjustment that can remove bias in cell yield introduced during the debarcoding process.

A continuous molecular roadmap to iPSC reprogramming through progression analysis of single-cell mass cytometry.

To analyze cellular reprogramming at the single-cell level, mass cytometry was used to simultaneously measure markers of pluripotency, differentiation, cell-cycle status, and cellular signaling throughout the reprogramming process. Time-resolved progression analysis of the resulting data sets was used to construct a continuous molecular roadmap for three independent reprogramming systems. Although these systems varied substantially in Oct4, Sox2, Klf4, and c-Myc stoichiometry, they presented a common set of reprogramming landmarks. Early in the reprogramming process, Oct4(high)Klf4(high) cells transitioned to a CD73(high)CD104(high)CD54(low) partially reprogrammed state. Ki67(low) cells from this intermediate population reverted to a MEF-like phenotype, but Ki67(high) cells advanced through the M-E-T and then bifurcated into two distinct populations: an ESC-like Nanog(high)Sox2(high)CD54(high) population and a mesendoderm-like Nanog(low)Sox2(low)Lin28(high)CD24(high)PDGFR-α(high) population. The methods developed here for time-resolved, single-cell progression analysis may be used for the study of additional complex and dynamic systems, such as cancer progression and embryonic development.