Lymphoid specification in human hematopoietic progenitors is not fully understood. To better associate lymphoid identity with protein-level cell features, we conduct a highly multiplexed single-cell proteomic screen on human bone marrow progenitors. This screen identifies terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase intrinsic to VDJ recombination, broadly expressed within CD34 progenitors prior to B/T cell emergence.
View Article and Find Full Text PDFHematopoietic stem and progenitor cell (HSPC) transplantation is an essential therapy for hematological conditions, but finer definitions of human HSPC subsets with associated function could enable better tuning of grafts and more routine, lower-risk application. To deeply phenotype HSPCs, following a screen of 328 antigens, we quantified 41 surface proteins and functional regulators on millions of CD34+ and CD34- cells, spanning four primary human hematopoietic tissues: bone marrow, mobilized peripheral blood, cord blood, and fetal liver. We propose more granular definitions of HSPC subsets and provide new, detailed differentiation trajectories of erythroid and myeloid lineages.
View Article and Find Full Text PDFMaster transcription factors (TFs) directly regulate present and future cell states by binding DNA regulatory elements and driving gene-expression programs. Their abundance influences epigenetic priming to different cell fates at the chromatin level, especially in the context of differentiation. In order to link TF protein abundance to changes in TF motif accessibility and open chromatin, we developed InTAC-seq, a method for simultaneous quantification of genome-wide chromatin accessibility and intracellular protein abundance in fixed cells.
View Article and Find Full Text PDFGranulocytes encompass diverse roles, from fighting off pathogens to regulating inflammatory processes in allergies. These roles are represented by distinct cellular phenotypes that we captured with mass cytometry (CyTOF). Our protocol enables simultaneous evaluation of human basophils, eosinophils, and neutrophils under homeostasis and upon immune activation by anti-Immunoglobulin E (anti-IgE) or interleukin-3 (IL-3).
View Article and Find Full Text PDFBasophils, the rarest granulocyte, play critical roles in parasite- and allergen-induced inflammation. We applied mass cytometry (CyTOF) to simultaneously asses 44 proteins to phenotype and functionally characterize neutrophils, eosinophils, and basophils from 19 healthy donors. There was minimal heterogeneity seen in eosinophils and neutrophils, but data-driven analyses revealed four unique subpopulations within phenotypically basophilic granulocytes (PBG; CD45HLA-DRCD123).
View Article and Find Full Text PDFB cells are capable of a wide range of effector functions including antibody secretion, antigen presentation, cytokine production, and generation of immunological memory. A consistent strategy for classifying human B cells by using surface molecules is essential to harness this functional diversity for clinical translation. We developed a highly multiplexed screen to quantify the co-expression of 351 surface molecules on millions of human B cells.
View Article and Find Full Text PDFThe advent of mass cytometry (CyTOF) has permitted simultaneous detection of more than 40 antibody parameters at the single-cell level, although a limited number of metal-labeled antibodies are commercially available. Here we present optimized and scalable protocols for conjugation of lanthanide as well as bismuth ions to immunoglobulin (Ig) using a maleimide-functionalized chelating polymer and for characterization of the conjugate. The maleimide functional group is reactive with cysteine sulfhydryl groups generated through partial reduction of the Ig Fc region.
View Article and Find Full Text PDFCellular products derived from the activity of DNA, RNA, and protein synthesis collectively control cell identity and function. Yet there is little information on how these three biosynthesis activities are coordinated during transient and sparse cellular processes, such as activation and differentiation. Here, we describe Simultaneous Overview of tri-Molecule Biosynthesis (SOMB), a molecular labeling and simultaneous detection strategy to quantify DNA, RNA, and protein synthesis in individual cells.
View Article and Find Full Text PDFSelective differentiation of naive T cells into multipotent T cells is of great interest clinically for the generation of cell-based cancer immunotherapies. Cellular differentiation depends crucially on division state and time. Here we adapt a dye dilution assay for tracking cell proliferative history through mass cytometry and uncouple division, time and regulatory protein expression in single naive human T cells during their activation and expansion in a complex ex vivo milieu.
View Article and Find Full Text PDFTransforming growth factor β (TGF-β) is well known for its important function in hematopoietic stem cell (HSC) quiescence. However, the molecular mechanism underlining this function remains obscure. Endoglin (Eng), a type III receptor for the TGF-β superfamily, has been shown to selectively mark long-term HSCs; however, its necessity in adult HSCs is unknown due to embryonic lethality.
View Article and Find Full Text PDFMechanisms of haematopoietic and cardiac patterning remain poorly understood. Here we show that the BMP and Wnt signalling pathways are integrated in an endoglin (Eng)-dependent manner in cardiac and haematopoietic lineage specification. Eng is expressed in early mesoderm and marks both haematopoietic and cardiac progenitors.
View Article and Find Full Text PDFEmbryonic stem cells (ESCs) represent an ideal model to study how lineage decisions are established during embryonic development. Using a doxycycline-inducible mouse ESC line, we have previously shown that expression of the transcriptional activator Pax3 in early mesodermal cells leads to the robust generation of paraxial mesoderm progenitors that ultimately differentiate into skeletal muscle precursors. Here, we show that the ability of this transcription factor to induce the skeletal myogenic cell fate occurs at the expenses of the cardiac lineage.
View Article and Find Full Text PDFEndoglin (Eng), an ancillary receptor of the transforming growth factor beta (TGFβ) signaling pathway superfamily, has been well recognized for its important function in vascular development and angiogenesis since its discovery more than a decade ago. Recent studies show that this receptor is also critical for the emergence of blood during embryonic development, and that at E7.5, endoglin together with Flk-1 identifies early mesoderm progenitors that are endowed with hematopoietic and endothelial potential.
View Article and Find Full Text PDFDuchenne muscular dystrophy is a progressive and incurable neuromuscular disease caused by genetic and biochemical defects of the dystrophin-glycoprotein complex. Here we show the regenerative potential of myogenic progenitors derived from corrected dystrophic induced pluripotent stem cells generated from fibroblasts of mice lacking both dystrophin and utrophin. We correct the phenotype of dystrophic induced pluripotent stem cells using a Sleeping Beauty transposon system carrying the micro-utrophin gene, differentiate these cells into skeletal muscle progenitors and transplant them back into dystrophic mice.
View Article and Find Full Text PDFIncreasing evidence points to endoglin (Eng), an accessory receptor for the transforming growth factor-β superfamily commonly associated with the endothelial lineage, as an important regulator of the hematopoietic lineage. We have shown that lack of Eng results in reduced numbers of primitive erythroid colonies as well as downregulation of key hematopoietic genes. To determine the effect of Eng overexpression in hematopoietic development, we generated a doxycycline-inducible embryonic stem cell line.
View Article and Find Full Text PDFDuring embryogenesis, the endothelial and the hematopoietic lineages first appear during gastrulation in the blood island of the yolk sac. We have previously reported that an Ets variant gene 2 (Etv2/ER71) mutant embryo lacks hematopoietic and endothelial lineages; however, the precise roles of Etv2 in yolk sac development remains unclear. In this study, we define the role of Etv2 in yolk sac blood island development using the Etv2 mutant and a novel Etv2-EYFP reporter transgenic line.
View Article and Find Full Text PDFMuch remains unknown about the signals that induce early mesoderm to initiate hematopoietic differentiation. Here, we show that endoglin (Eng), a receptor for the TGFβ superfamily, identifies all cells with hematopoietic fate in the early embryo. These arise in an Eng(+)Flk1(+) mesodermal precursor population at embryonic day 7.
View Article and Find Full Text PDFRationale: several studies demonstrate that hematopoietic tissues are a source of endothelial progenitor cells, which contribute to newly formed blood vessels during tissue repair in adults. However, it is not clear which cell type in these hematopoietic tissues gives rise to endothelial progenitor cells.
Objective: to identity the origin of endothelial progenitors within the hematopoietic hierarchy and to assess their in vivo revascularization potential.
Glioblastoma multiforme is highly aggressive and is the most common glial tumor type. Although there have been advances in treatment, the average survival expectancy is 12-15 months. Several genes have been shown to influence glioblastoma progression.
View Article and Find Full Text PDFARHGAP21 is highly expressed in the heart, which demonstrates activity over Cdc42 and interacts with proteins of the cytoskeleton and adherent junctions. The main cause of cardiac hypertrophy is mechanical stimulus; therefore we analyzed ARHGAP21 expression after acute mechanical stress in the myocardium and its association with FAK and PKCzeta. We demonstrated that ARHGAP21 is relocated to Z-lines and costameres after pressure overload, and interacts with PKCzeta and FAK in control rats (sham), rats submitted to aortic clamping and spontaneously hypertensive rats (SHR).
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