Resilient anatomy and local plasticity of naive and stress haematopoiesis.

The bone marrow adjusts blood cell production to meet physiological demands in response to insults. The spatial organization of normal and stress responses are unknown owing to the lack of methods to visualize most steps of blood production. Here we develop strategies to image multipotent haematopoiesis, erythropoiesis and lymphopoiesis in mice. We combine these with imaging of myelopoiesis1 to define the anatomy of normal and stress haematopoiesis. In the steady state, across the skeleton, single stem cells and multipotent progenitors distribute through the marrow enriched near megakaryocytes. Lineage-committed progenitors are recruited to blood vessels, where they contribute to lineage-specific microanatomical structures composed of progenitors and immature cells, which function as the production sites for each major blood lineage. This overall anatomy is resilient to insults, as it was maintained after haemorrhage, systemic bacterial infection and granulocyte colony-stimulating factor (G-CSF) treatment, and during ageing. Production sites enable haematopoietic plasticity as they differentially and selectively modulate their numbers and output in response to insults. We found that stress responses are variable across the skeleton: the tibia and the sternum respond in opposite ways to G-CSF, and the skull does not increase erythropoiesis after haemorrhage. Our studies enable in situ analyses of haematopoiesis, define the anatomy of normal and stress responses, identify discrete microanatomical production sites that confer plasticity to haematopoiesis, and uncover unprecedented heterogeneity of stress responses across the skeleton.

An immunophenotype-coupled transcriptomic atlas of human hematopoietic progenitors.

Analysis of the human hematopoietic progenitor compartment is being transformed by single-cell multimodal approaches. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) enables coupled surface protein and transcriptome profiling, thereby revealing genomic programs underlying progenitor states. To perform CITE-seq systematically on primary human bone marrow cells, we used titrations with 266 CITE-seq antibodies (antibody-derived tags) and machine learning to optimize a panel of 132 antibodies. Multimodal analysis resolved >80 stem, progenitor, immune, stromal and transitional cells defined by distinctive surface markers and transcriptomes. This dataset enables flow cytometry solutions for in silico-predicted cell states and identifies dozens of cell surface markers consistently detected across donors spanning race and sex. Finally, aligning annotations from this atlas, we nominate normal marrow equivalents for acute myeloid leukemia stem cell populations that differ in clinical response. This atlas serves as an advanced digital resource for hematopoietic progenitor analyses in human health and disease.

Slow cycling and durable Flt3+ progenitors contribute to hematopoiesis under native conditions.

The dynamics of the hematopoietic flux responsible for blood cell production in native conditions remains a matter of debate. Using CITE-seq analyses, we uncovered a distinct progenitor population that displays a cell cycle gene signature similar to the one found in quiescent hematopoietic stem cells. We further determined that the CD62L marker can be used to phenotypically enrich this population in the Flt3+ multipotent progenitor (MPP4) compartment. Functional in vitro and in vivo analyses validated the heterogeneity of the MPP4 compartment and established the quiescent/slow-cycling properties of the CD62L- MPP4 cells. Furthermore, studies under native conditions revealed a novel hierarchical organization of the MPP compartments in which quiescent/slow-cycling MPP4 cells sustain a prolonged hematopoietic activity at steady-state while giving rise to other lineage-biased MPP populations. Altogether, our data characterize a durable and productive quiescent/slow-cycling hematopoietic intermediary within the MPP4 compartment and highlight early paths of progenitor differentiation during unperturbed hematopoiesis.

Decision level integration of unimodal and multimodal single cell data with scTriangulate.

Decisively delineating cell identities from uni- and multimodal single-cell datasets is complicated by diverse modalities, clustering methods, and reference atlases. We describe scTriangulate, a computational framework to mix-and-match multiple clustering results, modalities, associated algorithms, and resolutions to achieve an optimal solution. Rather than ensemble approaches which select the "consensus", scTriangulate picks the most stable solution through coalitional iteration. When evaluated on diverse multimodal technologies, scTriangulate outperforms alternative approaches to identify high-confidence cell-populations and modality-specific subtypes. Unlike existing integration strategies that rely on modality-specific joint embedding or geometric graphs, scTriangulate makes no assumption about the distributions of raw underlying values. As a result, this approach can solve unprecedented integration challenges, including the ability to automate reference cell-atlas construction, resolve clonal architecture within molecularly defined cell-populations and subdivide clusters to discover splicing-defined disease subtypes. scTriangulate is a flexible strategy for unified integration of single-cell or multimodal clustering solutions, from nearly unlimited sources.

Social distancing during COVID-19: threat and efficacy among university students in seven nations.

COVID-19 has been spreading fast worldwide, and until effective and safe vaccines have been widely adopted, preventive measures such as social distancing are crucial to keep the pandemic under control. The study's research questions asked which psychosocial factors predict social distancing behavior and whether there are country-level differences in social distancing? Using the Extended Parallel Process Model (EPPM) as a theoretical lens, we examined the predictive effects of threat and efficacy and demographic variables on adherence to the COVID-19 preventive behavior of social distancing using a survey among an international sample of university students. Using path modeling and analysis of covariance, we confirmed the predictive effects of the EPPM on social distancing behavior. Our final model showed that perceived susceptibility to COVID-19 was both directly and indirectly (through response efficacy) associated with social distancing behavior; that perceived severity of COVID-19 yielded a significant indirect effect on social distancing behavior through both self-efficacy and response efficacy; that perceived susceptibility is indirectly and positively associated with social distancing behavior through response efficacy; and that self-efficacy and response efficacy were directly associated with social distancing behavior. Additionally, there were country-level differences in social distancing. Possible explanations for and implications of these findings are discussed.

Isolation of primary immune cells from fibrotic skin, esophageal, and gut tissue.

Understanding the interplay between immune and structural cells is important for studying fibrosis and inflammation; however, primary immune cell isolation from organs that are typically enriched in stromal cells, like the lung, esophagus, or gut, proves to be an ongoing challenge. In fibrotic conditions, this challenge becomes even greater as infiltrating cells become trapped in the robust extracellular matrix (ECM). This protocol details a method to isolate cells at high yield from stroma-rich organs that can be used for further analyses via flow cytometry, stimulation, or culturing. Validation of this method is confirmed by flow cytometry data assessing immune cell populations of interest. This protocol can be completed in approximately 5-6 h.

A primer on single-cell genomics in myeloid biology.

PURPOSE OF REVIEW: Understanding the fast-moving field of single-cell technologies, as applied to myeloid biology, requires an appreciation of basic molecular, informatics, and biological concepts. Here, we highlight both key and recent articles to illustrate basic concepts for those new to molecular single-cell analyses in myeloid hematology. RECENT FINDINGS: Recent studies apply single-cell omics to discover novel cell populations, construct relationships between cell populations, reconfigure the organization of hematopoiesis, and study hematopoietic lineage tree and fate choices. Accompanying development of technologies, new informatic tools have emerged, providing exciting new insights. SUMMARY: Hematopoietic stem and progenitor cells are regulated by complex intrinsic and extrinsic factors to produce blood cell types. In this review, we discuss recent advances in single-cell omics to profile these cells, methods to infer cell type identify, and trajectories from molecular omics data to ultimately derive new insights into hematopoietic stem and progenitor cell biology. We further discuss future applications of these technologies to understand hematopoietic cell interactions, function, and development. The goal is to offer a comprehensive overview of current single-cell technologies and their impact on our understanding of myeloid cell development for those new to single-cell analyses.

Mouse models of neutropenia reveal progenitor-stage-specific defects.

Advances in genetics and sequencing have identified a plethora of disease-associated and disease-causing genetic alterations. To determine causality between genetics and disease, accurate models for molecular dissection are required; however, the rapid expansion of transcriptional populations identified through single-cell analyses presents a major challenge for accurate comparisons between mutant and wild-type cells. Here we generate mouse models of human severe congenital neutropenia (SCN) using patient-derived mutations in the GFI1 transcription factor. To determine the effects of SCN mutations, we generated single-cell references for granulopoietic genomic states with linked epitopes1, aligned mutant cells to their wild-type equivalents and identified differentially expressed genes and epigenetic loci. We find that GFI1-target genes are altered sequentially, as cells go through successive states of differentiation. These insights facilitated the genetic rescue of granulocytic specification but not post-commitment defects in innate immune effector function, and underscore the importance of evaluating the effects of mutations and therapy within each relevant cell state.

Hepatocellular cancer-derived alpha fetoprotein uptake reduces CD1 molecules on monocyte-derived dendritic cells.

Alpha fetoprotein (AFP) is produced by over 50% of hepatocellular carcinomas (HCC). Uptake of tumor-derived AFP (tAFP) can impair activity of human dendritic cells (DC). The expression pattern of the lipid antigen presenting genes from the CD1 family is reduced in AFP-treated monocyte-derived DC. Surface CD1 family proteins, particularly CD1d, were reduced in AFP-exposed DC (by both normal cord blood-derived AFP (nAFP) and tAFP). NKT cells recognize lipid antigens presented by CD1d molecules. They play an important role in connecting the innate and adaptive immune systems, and in anti-tumor immunity. We hypothesized that AFP might impair the ability of DC to stimulate natural killer T (NKT) cells. No significant impact of AFP was observed on NKT cell stimulation. By examining secreted cytokines, we observed non-significant AFP-induced changes in several secreted proteins. These data indicate that AFP downregulates CD1 molecules on DC, but the impact on NKT cell activations is minimal.