Protocol for Surgery or Cast of the EpicoNdyle in Children's Elbows (SCIENCE).

Aims: The management of fractures of the medial epicondyle is one of the greatest controversies in paediatric fracture care, with uncertainty concerning the need for surgery. The British Society of Children's Orthopaedic Surgery prioritized this as their most important research question in paediatric trauma. This is the protocol for a randomized controlled, multicentre, prospective superiority trial of operative fixation versus nonoperative treatment for displaced medial epicondyle fractures: the Surgery or Cast of the EpicoNdyle in Children's Elbows (SCIENCE) trial. Methods: Children aged seven to 15 years old inclusive, who have sustained a displaced fracture of the medial epicondyle, are eligible to take part. Baseline function using the Patient-Reported Outcomes Measurement Information System (PROMIS) upper limb score, pain measured using the Wong Baker FACES pain scale, and quality of life (QoL) assessed with the EuroQol five-dimension questionnaire for younger patients (EQ-5D-Y) will be collected. Each patient will be randomly allocated (1:1, stratified using a minimization algorithm by centre and initial elbow dislocation status (i.e. dislocated or not-dislocated at presentation to the emergency department)) to either a regimen of the operative fixation or non-surgical treatment. Outcomes: At six weeks, and three, six, and 12 months, data on function, pain, sports/music participation, QoL, immobilization, and analgesia will be collected. These will also be repeated annually until the child reaches the age of 16 years. Four weeks after injury, the main outcomes plus data on complications, resource use, and school absence will be collected. The primary outcome is the PROMIS upper limb score at 12 months post-randomization. All data will be obtained through electronic questionnaires completed by the participants and/or parents/guardians. The NHS number of participants will be stored to enable future data linkage to sources of routinely collected data (i.e. Hospital Episode Statistics).

Expression profiling of cerebrospinal fluid identifies dysregulated antiviral mechanisms in multiple sclerosis.

Despite the overwhelming evidence that multiple sclerosis is an autoimmune disease, relatively little is known about the precise nature of the immune dysregulation underlying the development of the disease. Reasoning that the CSF from patients might be enriched for cells relevant in pathogenesis, we have completed a high-resolution single-cell analysis of 96 732 CSF cells collected from 33 patients with multiple sclerosis (n = 48 675) and 48 patients with other neurological diseases (n = 48 057). Completing comprehensive cell type annotation, we identified a rare population of CD8+ T cells, characterized by the upregulation of inhibitory receptors, increased in patients with multiple sclerosis. Applying a Multi-Omics Factor Analysis to these single-cell data further revealed that activity in pathways responsible for controlling inflammatory and type 1 interferon responses are altered in multiple sclerosis in both T cells and myeloid cells. We also undertook a systematic search for expression quantitative trait loci in the CSF cells. Of particular interest were two expression quantitative trait loci in CD8+ T cells that were fine mapped to multiple sclerosis susceptibility variants in the viral control genes ZC3HAV1 (rs10271373) and IFITM2 (rs1059091). Further analysis suggests that these associations likely reflect genetic effects on RNA splicing and cell-type specific gene expression respectively. Collectively, our study suggests that alterations in viral control mechanisms might be important in the development of multiple sclerosis.

Single-cell multi-omics analysis of COVID-19 patients with pre-existing autoimmune diseases shows aberrant immune responses to infection.

In COVID-19, hyperinflammatory and dysregulated immune responses contribute to severity. Patients with pre-existing autoimmune conditions can therefore be at increased risk of severe COVID-19 and/or associated sequelae, yet SARS-CoV-2 infection in this group has been little studied. Here, we performed single-cell analysis of peripheral blood mononuclear cells from patients with three major autoimmune diseases (rheumatoid arthritis, psoriasis, or multiple sclerosis) during SARS-CoV-2 infection. We observed compositional differences between the autoimmune disease groups coupled with altered patterns of gene expression, transcription factor activity, and cell-cell communication that substantially shape the immune response under SARS-CoV-2 infection. While enrichment of HLA-DRlow CD14+ monocytes was observed in all three autoimmune disease groups, type-I interferon signaling as well as inflammatory T cell and monocyte responses varied widely between the three groups of patients. Our results reveal disturbed immune responses to SARS-CoV-2 in patients with pre-existing autoimmunity, highlighting important considerations for disease treatment and follow-up.

Preleukemic single-cell landscapes reveal mutation-specific mechanisms and gene programs predictive of AML patient outcomes.

Acute myeloid leukemia (AML) and myeloid neoplasms develop through acquisition of somatic mutations that confer mutation-specific fitness advantages to hematopoietic stem and progenitor cells. However, our understanding of mutational effects remains limited to the resolution attainable within immunophenotypically and clinically accessible bulk cell populations. To decipher heterogeneous cellular fitness to preleukemic mutational perturbations, we performed single-cell RNA sequencing of eight different mouse models with driver mutations of myeloid malignancies, generating 269,048 single-cell profiles. Our analysis infers mutation-driven perturbations in cell abundance, cellular lineage fate, cellular metabolism, and gene expression at the continuous resolution, pinpointing cell populations with transcriptional alterations associated with differentiation bias. We further develop an 11-gene scoring system (Stem11) on the basis of preleukemic transcriptional signatures that predicts AML patient outcomes. Our results demonstrate that a single-cell-resolution deep characterization of preleukemic biology has the potential to enhance our understanding of AML heterogeneity and inform more effective risk stratification strategies.

Genotoxic aldehyde stress prematurely ages hematopoietic stem cells in a p53-driven manner.

Aged hematopoietic stem cells (HSCs) display diminished self-renewal and a myeloid differentiation bias. However, the drivers and mechanisms that underpin this fundamental switch are not understood. HSCs produce genotoxic formaldehyde that requires protection by the detoxification enzymes ALDH2 and ADH5 and the Fanconi anemia (FA) DNA repair pathway. We find that the HSCs in young Aldh2-/-Fancd2-/- mice harbor a transcriptomic signature equivalent to aged wild-type HSCs, along with increased epigenetic age, telomere attrition, and myeloid-biased differentiation quantified by single HSC transplantation. In addition, the p53 response is vigorously activated in Aldh2-/-Fancd2-/- HSCs, while p53 deletion rescued this aged HSC phenotype. To further define the origins of the myeloid differentiation bias, we use a GFP genetic reporter to find a striking enrichment of Vwf+ myeloid and megakaryocyte-lineage-biased HSCs. These results indicate that metabolism-derived formaldehyde-DNA damage stimulates the p53 response in HSCs to drive accelerated aging.

Multi-omics analysis defines highly refractory RAS burdened immature subgroup of infant acute lymphoblastic leukemia.

KMT2A-rearranged infant acute lymphoblastic leukemia (ALL) represents the most refractory type of childhood leukemia. To uncover the molecular heterogeneity of this disease, we perform RNA sequencing, methylation array analysis, whole exome and targeted deep sequencing on 84 infants with KMT2A-rearranged leukemia. Our multi-omics clustering followed by single-sample and single-cell inference of hematopoietic differentiation establishes five robust integrative clusters (ICs) with different master transcription factors, fusion partners and corresponding stages of B-lymphopoietic and early hemato-endothelial development: IRX-type differentiated (IC1), IRX-type undifferentiated (IC2), HOXA-type MLLT1 (IC3), HOXA-type MLLT3 (IC4), and HOXA-type AFF1 (IC5). Importantly, our deep mutational analysis reveals that the number of RAS pathway mutations predicts prognosis and that the most refractory subgroup of IC2 possesses 100% frequency and the heaviest burden of RAS pathway mutations. Our findings highlight the previously under-appreciated intra- and inter-patient heterogeneity of KMT2A-rearranged infant ALL and provide a rationale for the future development of genomics-guided risk stratification and individualized therapy.

Endothelial-specific Gata3 expression is required for hematopoietic stem cell generation.

To generate sufficient numbers of transplantable hematopoietic stem cells (HSCs) in vitro, a detailed understanding of how this process takes place in vivo is essential. The endothelial-to-hematopoietic transition (EHT), which culminates in the production of the first HSCs, is a highly complex process during which key regulators are switched on and off at precise moments, and that is embedded into a myriad of microenvironmental signals from surrounding cells and tissues. We have previously demonstrated an HSC-supportive function for GATA3 within the sympathetic nervous system and the sub-aortic mesenchyme, but show here that it also plays a cell-intrinsic role during the EHT. It is expressed in hemogenic endothelial cells and early HSC precursors, where its expression correlates with a more quiescent state. Importantly, endothelial-specific deletion of Gata3 shows that it is functionally required for these cells to mature into HSCs, placing GATA3 at the core of the EHT regulatory network.

p57Kip2 regulates embryonic blood stem cells by controlling sympathoadrenal progenitor expansion.

Hematopoietic stem cells (HSCs) are of major clinical importance, and finding methods for their in vitro generation is a prime research focus. We show here that the cell cycle inhibitor p57Kip2/Cdkn1c limits the number of emerging HSCs by restricting the size of the sympathetic nervous system (SNS) and the amount of HSC-supportive catecholamines secreted by these cells. This regulation occurs at the SNS progenitor level and is in contrast to the cell-intrinsic function of p57Kip2 in maintaining adult HSCs, highlighting profound differences in cell cycle requirements of adult HSCs compared with their embryonic counterparts. Furthermore, this effect is specific to the aorta-gonad-mesonephros (AGM) region and shows that the AGM is the main contributor to early fetal liver colonization, as early fetal liver HSC numbers are equally affected. Using a range of antagonists in vivo, we show a requirement for intact ß2-adrenergic signaling for SNS-dependent HSC expansion. To gain further molecular insights, we have generated a single-cell RNA-sequencing data set of all Ngfr+ sympathoadrenal cells around the dorsal aorta to dissect their differentiation pathway. Importantly, this not only defined the relevant p57Kip2-expressing SNS progenitor stage but also revealed that some neural crest cells, upon arrival at the aorta, are able to take an alternative differentiation pathway, giving rise to a subset of ventrally restricted mesenchymal cells that express important HSC-supportive factors. Neural crest cells thus appear to contribute to the AGM HSC niche via 2 different mechanisms: SNS-mediated catecholamine secretion and HSC-supportive mesenchymal cell production.

STAT1 is essential for HSC function and maintains MHCIIhi stem cells that resist myeloablation and neoplastic expansion.

Adult hematopoietic stem cells (HSCs) are predominantly quiescent and can be activated in response to acute stress such as infection or cytotoxic insults. STAT1 is a pivotal downstream mediator of interferon (IFN) signaling and is required for IFN-induced HSC proliferation, but little is known about the role of STAT1 in regulating homeostatic hematopoietic stem/progenitor cells (HSPCs). Here, we show that loss of STAT1 altered the steady state HSPC landscape, impaired HSC function in transplantation assays, delayed blood cell regeneration following myeloablation, and disrupted molecular programs that protect HSCs, including control of quiescence. Our results also reveal STAT1-dependent functional HSC heterogeneity. A previously unrecognized subset of homeostatic HSCs with elevated major histocompatibility complex class II (MHCII) expression (MHCIIhi) displayed molecular features of reduced cycling and apoptosis and was refractory to 5-fluorouracil-induced myeloablation. Conversely, MHCIIlo HSCs displayed increased megakaryocytic potential and were preferentially expanded in CALR mutant mice with thrombocytosis. Similar to mice, high MHCII expression is a feature of human HSCs residing in a deeper quiescent state. Our results therefore position STAT1 at the interface of stem cell heterogeneity and the interplay between stem cells and the adaptive immune system, areas of broad interest in the wider stem cell field.

Single-cell transcriptomics reveals the identity and regulators of human mast cell progenitors.

Mast cell accumulation is a hallmark of a number of diseases, including allergic asthma and systemic mastocytosis. Immunoglobulin E-mediated crosslinking of the FcεRI receptors causes mast cell activation and contributes to disease pathogenesis. The mast cell lineage is one of the least studied among the hematopoietic cell lineages, and controversies remain about whether FcεRI expression appears during the mast cell progenitor stage or during terminal mast cell maturation. Here, we used single-cell transcriptomics analysis to reveal a temporal association between the appearance of FcεRI and the mast cell gene signature in CD34+ hematopoietic progenitors in adult peripheral blood. In agreement with these data, the FcεRI+ hematopoietic progenitors formed morphologically, phenotypically, and functionally mature mast cells in long-term culture assays. Single-cell transcriptomics analysis further revealed the expression patterns of prospective cytokine receptors regulating development of mast cell progenitors. Culture assays showed that interleukin-3 (IL-3) and IL-5 promoted disparate effects on progenitor cell proliferation and survival, respectively, whereas IL-33 caused robust FcεRI downregulation. Taken together, we showed that FcεRI expression appears at the progenitor stage of mast cell differentiation in peripheral blood. We also showed that external stimuli regulate FcεRI expression of mast cell progenitors, providing a possible explanation for the variable FcεRI expression levels during mast cell development.

Coagulation factor V is a T-cell inhibitor expressed by leukocytes in COVID-19.

Clotting Factor V (FV) is primarily synthesized in the liver and when cleaved by thrombin forms pro-coagulant Factor Va (FVa). Using whole blood RNAseq and scRNAseq of peripheral blood mononuclear cells, we find that FV mRNA is expressed in leukocytes, and identify neutrophils, monocytes, and T regulatory cells as sources of increased FV in hospitalized patients with COVID-19. Proteomic analysis confirms increased FV in circulating neutrophils in severe COVID-19, and immunofluorescence microscopy identifies FV in lung-infiltrating leukocytes in COVID-19 lung disease. Increased leukocyte FV expression in severe disease correlates with T-cell lymphopenia. Both plasma-derived and a cleavage resistant recombinant FV, but not thrombin cleaved FVa, suppress T-cell proliferation in vitro. Anticoagulants that reduce FV conversion to FVa, including heparin, may have the unintended consequence of suppressing the adaptive immune system.

Unique molecular and functional features of extramedullary hematopoietic stem and progenitor cell reservoirs in humans.

Rare hematopoietic stem and progenitor cell (HSPC) pools outside the bone marrow (BM) contribute to blood production in stress and disease but remain ill-defined. Although nonmobilized peripheral blood (PB) is routinely sampled for clinical management, the diagnosis and monitoring potential of PB HSPCs remain untapped, as no healthy PB HSPC baseline has been reported. Here we comprehensively delineate human extramedullary HSPC compartments comparing spleen, PB, and mobilized PB to BM using single-cell RNA-sequencing and/or functional assays. We uncovered HSPC features shared by extramedullary tissues and others unique to PB. First, in contrast to actively dividing BM HSPCs, we found no evidence of substantial ongoing hematopoiesis in extramedullary tissues at steady state but report increased splenic HSPC proliferative output during stress erythropoiesis. Second, extramedullary hematopoietic stem cells/multipotent progenitors (HSCs/MPPs) from spleen, PB, and mobilized PB share a common transcriptional signature and increased abundance of lineage-primed subsets compared with BM. Third, healthy PB HSPCs display a unique bias toward erythroid-megakaryocytic differentiation. At the HSC/MPP level, this is functionally imparted by a subset of phenotypic CD71+ HSCs/MPPs, exclusively producing erythrocytes and megakaryocytes, highly abundant in PB but rare in other adult tissues. Finally, the unique erythroid-megakaryocytic-skewing of PB is perturbed with age in essential thrombocythemia and ß-thalassemia. Collectively, we identify extramedullary lineage-primed HSPC reservoirs that are nonproliferative in situ and report involvement of splenic HSPCs during demand-adapted hematopoiesis. Our data also establish aberrant composition and function of circulating HSPCs as potential clinical indicators of BM dysfunction.

Simultaneous Analysis of Single-Cell Transcriptomes and Cell Surface Protein Expression of Human Hematopoietic Stem Cells and Progenitors Using the 10x Genomics Platform.

The CITE-seq workflow combines conventional single-cell transcriptomic analysis with simultaneous analysis of cell surface protein expression using oligonucleotide-conjugated antibodies. This addition of immunophenotyping to mRNA data allows for a more detailed characterization of single-cell heterogeneity and can help to identify markers for the prospective isolation of transcriptionally defined novel cell subsets. Here, we describe the workflow for the preparation of human cord blood mononuclear cells and CD34+-enriched hematopoietic progenitors for the simultaneous characterization of protein and RNA using the commercially available TotalSeq™ antibodies from BioLegend and the droplet-based single-cell RNA-seq commercial platform from 10x Genomics.

Implementation of the StandingTall programme to prevent falls in older people: a process evaluation protocol.

INTRODUCTION: One in three people aged 65 years and over fall each year. The health, economic and personal impact of falls will grow substantially in the coming years due to population ageing. Developing and implementing cost-effective strategies to prevent falls and mobility problems among older people is therefore an urgent public health challenge. StandingTall is a low-cost, unsupervised, home-based balance exercise programme delivered through a computer or tablet. StandingTall has a simple user-interface that incorporates physical and behavioural elements designed to promote compliance. A large randomised controlled trial in 503 community-dwelling older people has shown that StandingTall is safe, has high adherence rates and is effective in improving balance and reducing falls. The current project targets a major need for older people and will address the final steps needed to scale this innovative technology for widespread use by older people across Australia and internationally. METHODS AND ANALYSIS: This project will endeavour to recruit 300 participants across three sites in Australia and 100 participants in the UK. The aim of the study is to evaluate the implementation of StandingTall into the community and health service settings in Australia and the UK. The nested process evaluation will use both quantitative and qualitative methods to explore uptake and acceptability of the StandingTall programme and associated resources. The primary outcome is participant adherence to the StandingTall programme over 6 months. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the South East Sydney Local Health District Human Research Ethics Committee (HREC reference 18/288) in Australia and the North West- Greater Manchester South Research Ethics Committee (IRAS ID: 268954) in the UK. Dissemination will be via publications, conferences, newsletter articles, social media, talks to clinicians and consumers and meetings with health departments/managers. TRIAL REGISTRATION NUMBER: ACTRN12619001329156.

Single-Cell Analysis of Hematopoietic Stem Cells.

The study of hematopoiesis has been revolutionized in recent years by the application of single-cell RNA sequencing technologies. The technique coupled with rapidly developing bioinformatic analysis has provided great insight into the cell type compositions of many populations previously defined by their cell surface phenotype. Moreover, transcriptomic information enables the identification of individual molecules and pathways which define novel cell populations and their transitions including cell lineage decisions. Combining single-cell transcriptional profiling with molecular perturbations allows functional analysis of individual factors in gene regulatory networks and better understanding of the earliest stages of malignant transformation. In this chapter we describe a comprehensive protocol for scRNA-Seq analysis of the mouse bone marrow, using both plate-based (low throughput) and droplet-based (high throughput) methods. The protocol includes instructions for sample preparation, an antibody panel for flow cytometric purification of hematopoietic progenitors with index sorting for plate-based analysis or in bulk for droplet-based methods. The plate-based protocol described in this chapter is a combination of the Smart-Seq2 and mcSCRB-Seq protocols, optimized in our laboratory. It utilizes off-the-shelf reagents for cDNA preparation, is amenable to automation using a liquid handler, and takes 4 days from preparation of the cells for sorting to producing a sequencing-ready library. The droplet-based method (using for instance the 10× Genomics platform) relies on the manufacturer's user guide and commercial reagents, and takes 3 days from isolation of the cells to the production of a library ready for sequencing.

Single-cell multi-omics analysis of the immune response in COVID-19.

Analysis of human blood immune cells provides insights into the coordinated response to viral infections such as severe acute respiratory syndrome coronavirus 2, which causes coronavirus disease 2019 (COVID-19). We performed single-cell transcriptome, surface proteome and T and B lymphocyte antigen receptor analyses of over 780,000 peripheral blood mononuclear cells from a cross-sectional cohort of 130 patients with varying severities of COVID-19. We identified expansion of nonclassical monocytes expressing complement transcripts (CD16+C1QA/B/C+) that sequester platelets and were predicted to replenish the alveolar macrophage pool in COVID-19. Early, uncommitted CD34+ hematopoietic stem/progenitor cells were primed toward megakaryopoiesis, accompanied by expanded megakaryocyte-committed progenitors and increased platelet activation. Clonally expanded CD8+ T cells and an increased ratio of CD8+ effector T cells to effector memory T cells characterized severe disease, while circulating follicular helper T cells accompanied mild disease. We observed a relative loss of IgA2 in symptomatic disease despite an overall expansion of plasmablasts and plasma cells. Our study highlights the coordinated immune response that contributes to COVID-19 pathogenesis and reveals discrete cellular components that can be targeted for therapy.

Single-cell molecular profiling provides a high-resolution map of basophil and mast cell development.

BACKGROUND: Basophils and mast cells contribute to the development of allergic reactions. Whereas these mature effector cells are extensively studied, the differentiation trajectories from hematopoietic progenitors to basophils and mast cells are largely uncharted at the single-cell level. METHODS: We performed multicolor flow cytometry, high-coverage single-cell RNA sequencing analyses, and cell fate assays to chart basophil and mast cell differentiation at single-cell resolution in mouse. RESULTS: Analysis of flow cytometry data reconstructed a detailed map of basophil and mast cell differentiation, including a bifurcation of progenitors into two specific trajectories. Molecular profiling and pseudotime ordering of the single cells revealed gene expression changes during differentiation. Cell fate assays showed that multicolor flow cytometry and transcriptional profiling successfully predict the bipotent phenotype of a previously uncharacterized population of peritoneal basophil-mast cell progenitors. CONCLUSIONS: A combination of molecular and functional profiling of bone marrow and peritoneal cells provided a detailed road map of basophil and mast cell development. An interactive web resource was created to enable the wider research community to explore the expression dynamics for any gene of interest.

Manipulating niche composition limits damage to haematopoietic stem cells during Plasmodium infection.

Severe infections are a major stress on haematopoiesis, where the consequences for haematopoietic stem cells (HSCs) have only recently started to emerge. HSC function critically depends on the integrity of complex bone marrow (BM) niches; however, what role the BM microenvironment plays in mediating the effects of infection on HSCs remains an open question. Here, using a murine model of malaria and combining single-cell RNA sequencing, mathematical modelling, transplantation assays and intravital microscopy, we show that haematopoiesis is reprogrammed upon infection, whereby the HSC compartment turns over substantially faster than at steady-state and HSC function is drastically affected. Interferon is found to affect both haematopoietic and mesenchymal BM cells and we specifically identify a dramatic loss of osteoblasts and alterations in endothelial cell function. Osteo-active parathyroid hormone treatment abolishes infection-triggered HSC proliferation and-coupled with reactive oxygen species quenching-enables partial rescuing of HSC function.