Terminal deoxynucleotidyl transferase and CD84 identify human multi-potent lymphoid progenitors.

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. While these TdT+ cells coincide with granulocyte-monocyte progenitor (GMP) immunophenotype, their accessible chromatin regions show enrichment for lymphoid-associated transcription factor (TF) motifs. TdT expression on GMPs is inversely related to the SLAM family member CD84. Prospective isolation of CD84lo GMPs demonstrates robust lymphoid potentials ex vivo, while still retaining significant myeloid differentiation capacity, akin to LMPPs. This multi-omic study identifies human bone marrow lymphoid-primed progenitors, further defining the lympho-myeloid axis in human hematopoiesis.

Unravelling human hematopoietic progenitor cell diversity through association with intrinsic regulatory factors.

Hematopoietic 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. These aspects of our revised human hematopoietic model were validated with corresponding epigenetic analysis and in vitro clonal differentiation assays. Overall, we demonstrate the utility of using molecular regulators as surrogates for cellular identity and functional potential, providing a framework for description, prospective isolation, and cross-tissue comparison of HSPCs in humans.

An Integrated Multi-omic Single-Cell Atlas of Human B Cell Identity.

B 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. We identified differentially expressed molecules and aligned their variance with isotype usage, VDJ sequence, metabolic profile, biosynthesis activity, and signaling response. Based on these analyses, we propose a classification scheme to segregate B cells from four lymphoid tissues into twelve unique subsets, including a CD45RB+CD27- early memory population, a class-switched CD39+ tonsil-resident population, and a CD19hiCD11c+ memory population that potently responds to immune activation. This classification framework and underlying datasets provide a resource for further investigations of human B cell identity and function.

Scalable Conjugation and Characterization of Immunoglobulins with Stable Mass Isotope Reporters for Single-Cell Mass Cytometry Analysis.

The 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. Incubation of Ig with polymer pre-loaded with lanthanide ions produces metal-labeled Ig without disrupting antigen specificity. Antibody recovery rates can be determined by UV spectrophotometry and frequently exceeds 60%. Each custom-conjugated antibody is validated using positive and negative cellular control populations and is titrated for optimal staining at concentrations ranging from 0.1 to 10 µg/mL. The preparation of metal-labeled antibodies can be completed in 4.5 h, and titration requires an additional 3-5 h.

Parallel analysis of tri-molecular biosynthesis with cell identity and function in single cells.

Cellular 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 (SOM3B), a molecular labeling and simultaneous detection strategy to quantify DNA, RNA, and protein synthesis in individual cells. Comprehensive interrogation of biosynthesis activities during transient cell states, such as progression through cell cycle or cellular differentiation, is achieved by partnering SOM3B with parallel quantification of select biomolecules with conjugated antibody reagents. Here, we investigate differential de novo DNA, RNA, and protein synthesis dynamics in transformed human cell lines, primary activated human immune cells, and across the healthy human hematopoietic continuum, all at a single-cell resolution.

Proliferation tracing with single-cell mass cytometry optimizes generation of stem cell memory-like T cells.

Selective 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. Using 23 markers, we defined groups of proteins controlled predominantly by division state or time and found that undivided cells account for the majority of phenotypic diversity. We next built a map of cell state changes during naive T-cell expansion. By examining cell signaling on this map, we rationally selected ibrutinib, a BTK and ITK inhibitor, and administered it before T cell activation to direct differentiation toward a T stem cell memory (TSCM)-like phenotype. This method for tracing cell fate across division states and time can be broadly applied for directing cellular differentiation.

Serial transplantation reveals a critical role for endoglin in hematopoietic stem cell quiescence.

Transforming growth factor ß (TGF-ß) 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-ß superfamily, has been shown to selectively mark long-term HSCs; however, its necessity in adult HSCs is unknown due to embryonic lethality. Using conditional deletion of Eng combined with serial transplantation, we show that this TGF-ß receptor is critical to maintain the HSC pool. Transplantation of Eng-deleted whole bone marrow or purified HSCs into lethally irradiated mice results in a profound engraftment defect in tertiary and quaternary recipients. Cell cycle analysis of primary grafts revealed decreased frequency of HSCs in G0, suggesting that lack of Eng impairs reentry of HSCs to quiescence. Using cytometry by time of flight (CyTOF) to evaluate the activity of signaling pathways in individual HSCs, we find that Eng is required within the Lin-Sca+Kit+-CD48- CD150+ fraction for canonical and noncanonical TGF-ß signaling, as indicated by decreased phosphorylation of SMAD2/3 and the p38 MAPK-activated protein kinase 2, respectively. These findings support an essential role for Eng in positively modulating TGF-ß signaling to ensure maintenance of HSC quiescence.

Expression levels of endoglin distinctively identify hematopoietic and endothelial progeny at different stages of yolk sac hematopoiesis.

Endoglin (Eng), an ancillary receptor of the transforming growth factor beta (TGFß) 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. These two lineages emerge in very close association during embryogenesis, and because they share the expression of the same surface markers, it has been difficult to distinguish the earliest hematopoietic from endothelial cells. Here, we evaluated the function of endoglin in hematopoiesis as development progresses past E7.5, and found that the hematopoietic and endothelial progenitors can be distinguished by the levels of endoglin in E9.5 yolk sacs. Whereas endothelial cells are Eng(bright), hematopoietic activity is primarily restricted to a subset of cells that display dim expression of endoglin (Eng(dim)). Molecular characterization of these subfractions showed that endoglin-mediated induction of hematopoiesis occurs in concert with BMP2/BMP4 signaling. This pathway is highly active in Eng(dim) cells but significantly downregulated in the Eng knockout. Taken together, our findings show an important function for endoglin in mediating BMP2/BMP4 signaling during yolk sac hematopoietic development and suggest that the levels of this receptor modulate TGFß versus bone morphogenetic protein (BMP) signaling.

Effect of endoglin overexpression during embryoid body development.

Increasing evidence points to endoglin (Eng), an accessory receptor for the transforming growth factor-ß 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. Our results demonstrate that induction of Eng during embryoid body differentiation leads to a significant increase in the frequency of hematopoietic progenitors, in particular, the erythroid lineage, which correlated with upregulation of Scl, Gata1, Runx1, and embryonic globin. Interestingly, activation of the hematopoietic program happened at the expense of endothelial and cardiac cells, as differentiation into these mesoderm lineages was compromised. Eng-induced enhanced erythroid activity was accompanied by high levels of Smad1 phosphorylation. This effect was attenuated by addition of a bone morphogenetic protein (BMP) signaling inhibitor to these cultures. Among the BMPs, BMP4 is well known for its role in hematopoietic specification from mesoderm by promoting expression of several hematopoietic genes, including Scl. Because Scl is considered the master regulator of the hematopoietic program, we investigated whether Scl would be capable of rescuing the defective hematopoietic phenotype observed in Eng(-/-) embryonic stem cells. Scl expression in Eng-deficient embryonic stem cells resulted in increased erythroid colony-forming activity and upregulation of Gata1 and Gata2, positioning Eng upstream of Scl. Taken together, these findings support the premise that Eng modulates the hematopoietic transcriptional network, most likely through regulation of BMP4 signaling.

Etv2 is expressed in the yolk sac hematopoietic and endothelial progenitors and regulates Lmo2 gene expression.

During 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. Both the hematopoietic and the endothelial lineages are absent in the Etv2 mutant yolk sac. In the Etv2-EYFP transgenic mouse, the EYFP reporter is activated in the nascent mesoderm, expressed in the endothelial and blood progenitors, and in the Tie2(+), c-kit(+), and CD41(+) hematopoietic population. The hematopoietic activity in the E7.75 yolk sac was exclusively localized to the Etv2-EYFP(+) population. In the Etv2 mutant yolk sac, Tie2(+) cells are present but do not express hematopoietic or endothelial markers. In addition, these cells do not form hematopoietic colonies, indicating an essential role of Etv2 in the specification of the hematopoietic lineage. Forced overexpression of Etv2 during embryoid body differentiation induces the hematopoietic and the endothelial lineages, and transcriptional profiling in this context identifies Lmo2 as a downstream target. Using electrophoretic mobility shift assay, chromatin immunoprecipitation, transcriptional assays, and mutagenesis, we demonstrate that Etv2 binds to the Lmo2 enhancer and transactivates its expression. Collectively, our studies demonstrate that Etv2 is expressed during and required for yolk sac hematoendothelial development, and that Lmo2 is one of the downstream targets of Etv2.

A critical role for endoglin in the emergence of blood during embryonic development.

Much remains unknown about the signals that induce early mesoderm to initiate hematopoietic differentiation. Here, we show that endoglin (Eng), a receptor for the TGFß superfamily, identifies all cells with hematopoietic fate in the early embryo. These arise in an Eng(+)Flk1(+) mesodermal precursor population at embryonic day 7.5 (E7.5), a cell fraction also endowed with endothelial potential. In Eng-knockout embryos, hematopoietic colony activity and numbers of CD71(+)Ter119(+) erythroid progenitors were severely reduced. This coincided with severely reduced expression of embryonic globin and key bone morphogenic protein (BMP) target genes, including the hematopoietic regulators Scl, Gata1, Gata2, and Msx-1. To interrogate molecular pathways active in the earliest hematopoietic progenitors, we applied transcriptional profiling to sorted cells from E7.5 embryos. Eng(+)Flk-1(+) progenitors coexpressed TGFß and BMP receptors and target genes. Furthermore, Eng(+)Flk-1(+) cells presented high levels of phospho-SMAD1/5, indicating active TGFß and/or BMP signaling. Remarkably, under hematopoietic serum-free culture conditions, hematopoietic outgrowth of Eng-expressing cells was dependent on the TGFß superfamily ligands BMP4, BMP2, or TGF-ß1. These data demonstrate that the E(+)F(+) fraction at E7.5 represents mesodermal cells competent to respond to TGFß1, BMP4, or BMP2, shaping their hematopoietic development, and that Eng acts as a critical regulator in this process by modulating TGF/BMP signaling.

Clonal analysis reveals a common progenitor for endothelial, myeloid, and lymphoid precursors in umbilical cord blood.

RATIONALE: 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. METHODS AND RESULTS: using a single-cell sorting approach and in vitro multilineage differentiation assays, here we show that individual CD34(+)CD45(+)CD133(+)CD38(+) cells from cord blood uniquely have the ability to differentiate into T- and B-lymphoid, myeloid, and endothelial cells. The latter were characterized by the expression of VE-cadherin, KDR, von Willebrand factor, endothelial nitric oxide synthase, the lack of CD45, CD133, and c-fms (colony stimulating factor-1 receptor). Unexpectedly when transplanted into hindlimb ischemic NOD-scid IL2Rγ(null) mice, freshly isolated CD34(+)CD45(+)CD133(+)CD38(+) cells maintained their hematopoietic identity and were rarely found to integrate into host blood vessels. Nevertheless, they significantly improved perfusion, most likely through a paracrine mechanism. On the other hand, CD34(+)CD45(+)CD133(+)CD38(+) cells differentiated in vitro into endothelial cells were able to form vessels in vivo in both Matrigel plug and hindlimb ischemia transplantation assays. CONCLUSIONS: these findings indicate that the CD34(+)CD45(+)CD133(+)CD38(+) cell fraction contains a common progenitor for the hematopoietic and vascular lineages and may represent a valuable cell source for therapeutic applications.

ARHGAP21 modulates FAK activity and impairs glioblastoma cell migration.

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. In the present work, we demonstrate that the RhoGTPase Activating Protein 21 (ARHGAP21) is expressed in the nuclear and perinuclear regions of several cell lines. In T98G and U138MG, glioblastoma derived cell lines, ARHGAP21 interacts with the C-terminal region of Focal Adhesion Kinase (FAK). ARHGAP21 depletion by shRNAi in T98G cells alters cellular morphology and increases: FAK phosphorylation states and activation of downstream signaling; the activity state of Cdc42; the production of metalloproteinase 2 (MMP-2) and cell migration rates. These modifications were found to be mainly due to the loss of ARHGAP21 action on FAK and, consequently, the activation of downstream effectors. These results suggest not only that ARHGAP21 might act as a tumor suppressor gene, but also indicate that ARHGAP21 might be a master regulator of migration having a crucial role in controlling the progression of different tumor types.