Mapping the cellular biogeography of human bone marrow niches using single-cell transcriptomics and proteomic imaging.

Non-hematopoietic cells are essential contributors to hematopoiesis. However, heterogeneity and spatial organization of these cells in human bone marrow remain largely uncharacterized. We used single-cell RNA sequencing (scRNA-seq) to profile 29,325 non-hematopoietic cells and discovered nine transcriptionally distinct subtypes. We simultaneously profiled 53,417 hematopoietic cells and predicted their interactions with non-hematopoietic subsets. We employed co-detection by indexing (CODEX) to spatially profile over 1.2 million cells. We integrated scRNA-seq and CODEX data to link predicted cellular signaling with spatial proximity. Our analysis revealed a hyperoxygenated arterio-endosteal neighborhood for early myelopoiesis, and an adipocytic localization for early hematopoietic stem and progenitor cells (HSPCs). We used our CODEX atlas to annotate new images and uncovered mesenchymal stromal cell (MSC) expansion and spatial neighborhoods co-enriched for leukemic blasts and MSCs in acute myeloid leukemia (AML) patient samples. This spatially resolved, multiomic atlas of human bone marrow provides a reference for investigation of cellular interactions that drive hematopoiesis.

Mapping the Cellular Biogeography of Human Bone Marrow Niches Using Single-Cell Transcriptomics and Proteomic Imaging.

The bone marrow is the organ responsible for blood production. Diverse non-hematopoietic cells contribute essentially to hematopoiesis. However, these cells and their spatial organization remain largely uncharacterized as they have been technically challenging to study in humans. Here, we used fresh femoral head samples and performed single-cell RNA sequencing (scRNA-Seq) to profile 29,325 enriched non-hematopoietic bone marrow cells and discover nine transcriptionally distinct subtypes. We next employed CO-detection by inDEXing (CODEX) multiplexed imaging of 18 individuals, including both healthy and acute myeloid leukemia (AML) samples, to spatially profile over one million single cells with a novel 53-antibody panel. We discovered a relatively hyperoxygenated arterio-endosteal niche for early myelopoiesis, and an adipocytic, but not endosteal or perivascular, niche for early hematopoietic stem and progenitor cells. We used our atlas to predict cell type labels in new bone marrow images and used these predictions to uncover mesenchymal stromal cell (MSC) expansion and leukemic blast/MSC-enriched spatial neighborhoods in AML patient samples. Our work represents the first comprehensive, spatially-resolved multiomic atlas of human bone marrow and will serve as a reference for future investigation of cellular interactions that drive hematopoiesis.

Identification and targeting of treatment resistant progenitor populations in T-cell Acute Lymphoblastic Leukemia.

Refractoriness to initial chemotherapy and relapse after remission are the main obstacles to cure in T-cell Acute Lymphoblastic Leukemia (T-ALL). Biomarker guided risk stratification and targeted therapy have the potential to improve outcomes in high-risk T-ALL; however, cellular and genetic factors contributing to treatment resistance remain unknown. Previous bulk genomic studies in T-ALL have implicated tumor heterogeneity as an unexplored mechanism for treatment failure. To link tumor subpopulations with clinical outcome, we created an atlas of healthy pediatric hematopoiesis and applied single-cell multiomic (CITE-seq/snATAC-seq) analysis to a cohort of 40 cases of T-ALL treated on the Children's Oncology Group AALL0434 clinical trial. The cohort was carefully selected to capture the immunophenotypic diversity of T-ALL, with early T-cell precursor (ETP) and Near/Non-ETP subtypes represented, as well as enriched with both relapsed and treatment refractory cases. Integrated analyses of T-ALL blasts and normal T-cell precursors identified a bone-marrow progenitor-like (BMP-like) leukemia sub-population associated with treatment failure and poor overall survival. The single-cell-derived molecular signature of BMP-like blasts predicted poor outcome across multiple subtypes of T-ALL within two independent patient cohorts using bulk RNA-sequencing data from over 1300 patients. We defined the mutational landscape of BMP-like T-ALL, finding that NOTCH1 mutations additively drive T-ALL blasts away from the BMP-like state. We transcriptionally matched BMP-like blasts to early thymic seeding progenitors that have low NR3C1 expression and high stem cell gene expression, corresponding to a corticosteroid and conventional cytotoxic resistant phenotype we observed in ex vivo drug screening. To identify novel targets for BMP-like blasts, we performed in silico and in vitro drug screening against the BMP-like signature and prioritized BMP-like overexpressed cell-surface (CD44, ITGA4, LGALS1) and intracellular proteins (BCL-2, MCL-1, BTK, NF-κB) as candidates for precision targeted therapy. We established patient derived xenograft models of BMP-high and BMP-low leukemias, which revealed vulnerability of BMP-like blasts to apoptosis-inducing agents, TEC-kinase inhibitors, and proteasome inhibitors. Our study establishes the first multi-omic signatures for rapid risk-stratification and targeted treatment of high-risk T-ALL.

Decade-long leukaemia remissions with persistence of CD4+ CAR T cells.

The adoptive transfer of T lymphocytes reprogrammed to target tumour cells has demonstrated potential for treatment of various cancers1-7. However, little is known about the long-term potential and clonal stability of the infused cells. Here we studied long-lasting CD19-redirected chimeric antigen receptor (CAR) T cells in two patients with chronic lymphocytic leukaemia1-4 who achieved a complete remission in 2010. CAR T cells remained detectable more than ten years after infusion, with sustained remission in both patients. Notably, a highly activated CD4+ population emerged in both patients, dominating the CAR T cell population at the later time points. This transition was reflected in the stabilization of the clonal make-up of CAR T cells with a repertoire dominated by a small number of clones. Single-cell profiling demonstrated that these long-persisting CD4+ CAR T cells exhibited cytotoxic characteristics along with ongoing functional activation and proliferation. In addition, longitudinal profiling revealed a population of gamma delta CAR T cells that prominently expanded in one patient concomitant with CD8+ CAR T cells during the initial response phase. Our identification and characterization of these unexpected CAR T cell populations provide novel insight into the CAR T cell characteristics associated with anti-cancer response and long-term remission in leukaemia.

Single-cell multiomics reveals increased plasticity, resistant populations, and stem-cell-like blasts in KMT2A-rearranged leukemia.

KMT2A-rearranged (KMT2A-r) infant acute lymphoblastic leukemia (ALL) is a devastating malignancy with a dismal outcome, and younger age at diagnosis is associated with increased risk of relapse. To discover age-specific differences and critical drivers that mediate poor outcome in KMT2A-r ALL, we subjected KMT2A-r leukemias and normal hematopoietic cells from patients of different ages to single-cell multiomics analyses. We uncovered the following critical new insights: leukemia cells from patients <6 months have significantly increased lineage plasticity. Steroid response pathways are downregulated in the most immature blasts from younger patients. We identify a hematopoietic stem and progenitor-like (HSPC-like) population in the blood of younger patients that contains leukemic blasts and form an immunosuppressive signaling circuit with cytotoxic lymphocytes. These observations offer a compelling explanation for the ability of leukemias in young patients to evade chemotherapy and immune-mediated control. Our analysis also revealed preexisting lymphomyeloid primed progenitors and myeloid blasts at initial diagnosis of B-ALL. Tracking of leukemic clones in 2 patients whose leukemia underwent a lineage switch documented the evolution of such clones into frank acute myeloid leukemia (AML). These findings provide critical insights into KMT2A-r ALL and have clinical implications for molecularly targeted and immunotherapy approaches. Beyond infant ALL, our study demonstrates the power of single-cell multiomics to detect tumor intrinsic and extrinsic factors affecting rare but critical subpopulations within a malignant population that ultimately determines patient outcome.

Integrative Bulk and Single-Cell Profiling of Premanufacture T-cell Populations Reveals Factors Mediating Long-Term Persistence of CAR T-cell Therapy.

The adoptive transfer of chimeric antigen receptor (CAR) T cells represents a breakthrough in clinical oncology, yet both between- and within-patient differences in autologously derived T cells are a major contributor to therapy failure. To interrogate the molecular determinants of clinical CAR T-cell persistence, we extensively characterized the premanufacture T cells of 71 patients with B-cell malignancies on trial to receive anti-CD19 CAR T-cell therapy. We performed RNA-sequencing analysis on sorted T-cell subsets from all 71 patients, followed by paired Cellular Indexing of Transcriptomes and Epitopes (CITE) sequencing and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) on T cells from six of these patients. We found that chronic IFN signaling regulated by IRF7 was associated with poor CAR T-cell persistence across T-cell subsets, and that the TCF7 regulon not only associates with the favorable naïve T-cell state, but is maintained in effector T cells among patients with long-term CAR T-cell persistence. These findings provide key insights into the underlying molecular determinants of clinical CAR T-cell function. SIGNIFICANCE: To improve clinical outcomes for CAR T-cell therapy, there is a need to understand the molecular determinants of CAR T-cell persistence. These data represent the largest clinically annotated molecular atlas in CAR T-cell therapy to date, and significantly advance our understanding of the mechanisms underlying therapeutic efficacy.This article is highlighted in the In This Issue feature, p. 2113.

Diverse molecular compositions of dissolved organic matter derived from different composts using ESI FT-ICR MS.

Dissolved organic matter (DOM) derived from various composts can promote significant changes of soil properties. However, little is known about the DOM compositions and their similarities and differences at the molecular level. In this study, the molecular compositions of DOM derived from kitchen waste compost (KWC), green waste compost (GWC), manure waste compost (MWC), and sewage sludge compost (SSC) were characterized by electrospray ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS). The molecular formulas were classified into four subcategories: CHO, CHON, CHOS, and CHONS. The KWC, MWC, and SSC DOM represented the highest fraction (35.8%-47.4%) of CHON subcategory, while the GWC DOM represented the highest fraction (68.4%) of CHO subcategory. The GWC DOM was recognized as the nitrogen- and sulfur-deficient compounds that were less saturated, more aromatic, and more oxidized compared with other samples. Further analysis of the oxygen, nitrogen-containing (N-containing), and sulfur-containing (S-containing) functional groups in the four subcategories revealed higher organic molecular complexity. Comparison of the similarities and differences of the four samples revealed 22.8% ubiquitous formulas and 17.4%, 11.1%, 10.7%, and 6.3% unique formulas of GWC, KWC, SSC, and MWC DOM, respectively, suggesting a large proportion of ubiquitous DOM as well as unique, source-specific molecular signatures. The findings presented herein provide new insight into the molecular characterization of DOM derived from various composts and demonstrated the potential role of these different compounds for agricultural utilization.

Diverse noncoding mutations contribute to deregulation of cis-regulatory landscape in pediatric cancers.

Interpreting the function of noncoding mutations in cancer genomes remains a major challenge. Here, we developed a computational framework to identify putative causal noncoding mutations of all classes by joint analysis of mutation and gene expression data. We identified thousands of SNVs/small indels and structural variants as putative causal mutations in five major pediatric cancers. We experimentally validated the oncogenic role of CHD4 overexpression via enhancer hijacking in B-ALL. We observed a general exclusivity of coding and noncoding mutations affecting the same genes and pathways. We showed that integrated mutation profiles can help define novel patient subtypes with different clinical outcomes. Our study introduces a general strategy to systematically identify and characterize the full spectrum of noncoding mutations in cancers.

Transcriptional regulatory network controlling the ontogeny of hematopoietic stem cells.

Hematopoietic stem cell (HSC) ontogeny is accompanied by dynamic changes in gene regulatory networks. We performed RNA-seq and histone mark ChIP-seq to define the transcriptomes and epigenomes of cells representing key developmental stages of HSC ontogeny in mice. The five populations analyzed were embryonic day 10.5 (E10.5) endothelium and hemogenic endothelium from the major arteries, an enriched population of prehematopoietic stem cells (pre-HSCs), fetal liver HSCs, and adult bone marrow HSCs. Using epigenetic signatures, we identified enhancers for each developmental stage. Only 12% of enhancers are primed, and 78% are active, suggesting the vast majority of enhancers are established de novo without prior priming in earlier stages. We constructed developmental stage-specific transcriptional regulatory networks by linking enhancers and predicted bound transcription factors to their target promoters using a novel computational algorithm, target inference via physical connection (TIPC). TIPC predicted known transcriptional regulators for the endothelial-to-hematopoietic transition, validating our overall approach, and identified putative novel transcription factors, including the broadly expressed transcription factors SP3 and MAZ. Finally, we validated a role for SP3 and MAZ in the formation of hemogenic endothelium. Our data and computational analyses provide a useful resource for uncovering regulators of HSC formation.

Developmental trajectory of prehematopoietic stem cell formation from endothelium.

Hematopoietic stem and progenitor cells (HSPCs) in the bone marrow are derived from a small population of hemogenic endothelial (HE) cells located in the major arteries of the mammalian embryo. HE cells undergo an endothelial to hematopoietic cell transition, giving rise to HSPCs that accumulate in intra-arterial clusters (IAC) before colonizing the fetal liver. To examine the cell and molecular transitions between endothelial (E), HE, and IAC cells, and the heterogeneity of HSPCs within IACs, we profiled ∼40 000 cells from the caudal arteries (dorsal aorta, umbilical, vitelline) of 9.5 days post coitus (dpc) to 11.5 dpc mouse embryos by single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin sequencing. We identified a continuous developmental trajectory from E to HE to IAC cells, with identifiable intermediate stages. The intermediate stage most proximal to HE, which we term pre-HE, is characterized by increased accessibility of chromatin enriched for SOX, FOX, GATA, and SMAD motifs. A developmental bottleneck separates pre-HE from HE, with RUNX1 dosage regulating the efficiency of the pre-HE to HE transition. A distal candidate Runx1 enhancer exhibits high chromatin accessibility specifically in pre-HE cells at the bottleneck, but loses accessibility thereafter. Distinct developmental trajectories within IAC cells result in 2 populations of CD45+ HSPCs; an initial wave of lymphomyeloid-biased progenitors, followed by precursors of hematopoietic stem cells (pre-HSCs). This multiomics single-cell atlas significantly expands our understanding of pre-HSC ontogeny.

scATAC-pro: a comprehensive workbench for single-cell chromatin accessibility sequencing data.

Single-cell chromatin accessibility sequencing has become a powerful technology for understanding epigenetic heterogeneity of complex tissues. However, there is a lack of open-source software for comprehensive processing, analysis, and visualization of such data generated using all existing experimental protocols. Here, we present scATAC-pro for quality assessment, analysis, and visualization of single-cell chromatin accessibility sequencing data. scATAC-pro computes a range of quality control metrics for several key steps of experimental protocols, with a flexible choice of methods. It generates summary reports for both quality assessment and downstream analysis. scATAC-pro is available at https://github.com/tanlabcode/scATAC-pro.

SciBet as a portable and fast single cell type identifier.

Fast, robust and technology-independent computational methods are needed for supervised cell type annotation of single-cell RNA sequencing data. We present SciBet, a supervised cell type identifier that accurately predicts cell identity for newly sequenced cells with order-of-magnitude speed advantage. We enable web client deployment of SciBet for rapid local computation without uploading local data to the server. Facing the exponential growth in the size of single cell RNA datasets, this user-friendly and cross-platform tool can be widely useful for single cell type identification.

Spatial Genome Re-organization between Fetal and Adult Hematopoietic Stem Cells.

Fetal hematopoietic stem cells (HSCs) undergo a developmental switch to become adult HSCs with distinct functional properties. To better understand the molecular mechanisms underlying the developmental switch, we have conducted deep sequencing of the 3D genome, epigenome, and transcriptome of fetal and adult HSCs in mouse. We find that chromosomal compartments and topologically associating domains (TADs) are largely conserved between fetal and adult HSCs. However, there is a global trend of increased compartmentalization and TAD boundary strength in adult HSCs. In contrast, intra-TAD chromatin interactions are much more dynamic and widespread, involving over a thousand gene promoters and distal enhancers. These developmental-stage-specific enhancer-promoter interactions are mediated by different sets of transcription factors, such as TCF3 and MAFB in fetal HSCs, versus NR4A1 and GATA3 in adult HSCs. Loss-of-function studies of TCF3 confirm the role of TCF3 in mediating condition-specific enhancer-promoter interactions and gene regulation in fetal HSCs.

Analytical dataset on the molecular compositional changes of dissolved organic matter during hyperthermophilic composting.

The aim of this research work was to determine the molecular compositional changes of dissolved organic matter (DOM) taken from different phases of the hyperthermophilic composting (HTC) process. The DOM samples were extracted by the standard protocol of C18 extraction methodology, and then analyzed by electrospray ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS). The profiles of negative ion mass spectrum and DOM molecular formulas of four compost samples were reported. Data related to the molecular compositional changes of DOM during HTC were also presented. Further interpretation and discussion on these datasets can be found in the related article entitled "Molecular insights into the transformation of dissolved organic matter during hyperthermophilic composting using ESI FT-ICR MS" [1].

Molecular insights into the transformation of dissolved organic matter during hyperthermophilic composting using ESI FT-ICR MS.

The aim of this work was to study the molecular compositional changes of dissolved organic matter (DOM) during hyperthermophilic composting (HTC) using electrospray ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry. Our results reveal that DOM in hyperthermophilic compost mainly consisted of lignins/carboxylic-rich alicyclic molecules (72%) with relatively lower H/C (1.24), and the higher double bound equivalent (5.98) and aromaticity index (0.22) when compared with the DOM in composting materials, suggesting that HTC led to an increase in carboxyl-rich, unsaturated, and aromatic compounds. Profiles of the DOM's transformation indicated that low O/C (O/C < 0.3) and high H/C (H/C < 1.5) compounds were preferentially decomposed in the hyperthermophilic phase of HTC. Abundant produced intermediates, such as lignin phenols and amino sugars, were further transformed to refractory humic substances. This investigation extends the current understanding of the molecular mechanisms on humification of HTC, and reveals further applications for hyperthermophilic compost.

A dual-signal output ratiometric electrochemiluminescent sensor for NADH detection.

Ratiometric electrochemiluminescence (ECL) has attracted great attention in the field of electrochemical analysis. In this study, a dual-signal-output ratiometric ECL sensor was developed for the detection of nicotinamide adenine dinucleotide (NADH). Nitrogen-doped graphene quantum dots (NGQDs) exhibit double ECL signal output capability, without the requirement of additional coreactants. NADH can amplify the anodic ECL response of NGQDs, while it can diminish the cathodic ECL response of NGQDs. Based on the principle between relative enhancing ECL intensity ratio and NADH concentrations, the constructed ratiometric ECL sensor was applied to NADH assays, with a wide concentration range of 10-400 µM and a low limit of detection (LOD) of 2.5 µM (S/N = 3). Furthermore, the proposed method was applied for the determination of spiked NADH, which was proved to be feasible in the biological sample matrix. The proposed strategy of modulating multiple-ECL signals of the single NGQD emitter not only provides a new ECL system for the accurate detection of NADH but also broadens the design pathway for ratiometric sensing fabrication.

Ternary Z-scheme heterojunction of Bi2WO6 with reduced graphene oxide (rGO) and meso-tetra (4-carboxyphenyl) porphyrin (TCPP) for enhanced visible-light photocatalysis.

A highly efficient and new ternary TCPP/rGO/Bi2WO6 Z-scheme heterojunction was designed and fabricated via a facile hydrothermal approach and a liquid ultrasonic route in sequence. The crystal structures, morphologies, microstructures, chemical compositions, elemental states, optical and photo-electrochemical properties of the heterojunction were characterized. This Z-scheme TCPP/rGO/Bi2WO6 photocatalyst has significantly enhanced photocatalytic activity for the tetracycline (TC) degradation under the irradiation of visible light (λ > 420 nm) within 60 min, as compared to pure Bi2WO6, rGO/Bi2WO6 and TCPP/Bi2WO6 composites. The effects of the photocatalyst dosages, pollutant concentrations, coexisting ions and illumination conditions on the photodegradation were investigated. According to the trapping experiments and electron spin resonance analyses, the hole (h+) and superoxide radical (O2-) mainly contribute to the TC decomposition in the TCPP/rGO/Bi2WO6 photocatalytic system. The photodegradation process in the TCPP/rGO/Bi2WO6 ternary composites can be well described by the proposed Z-scheme mechanism. The results indicate that more efficient charge separation, better light absorption, and larger surface area from the developed photocatalyst collectively contribute to the excellent photocatalytic performances. Besides, the photocatalyst has great stability and recyclability with a removal efficiency of 79.27% even after five times of repeated treatment. This work reports a new strategy for the preparation of Z-scheme heterojunction photocatalyst with high photocatalytic activity and provides an alternative for the effective removal of antibiotic wastewater through photocatalysis.

RUNX1 and the endothelial origin of blood.

The transcription factor RUNX1 is required in the embryo for formation of the adult hematopoietic system. Here, we describe the seminal findings that led to the discovery of RUNX1 and of its critical role in blood cell formation in the embryo from hemogenic endothelium (HE). We also present RNA-sequencing data demonstrating that HE cells in different anatomic sites, which produce hematopoietic progenitors with dissimilar differentiation potentials, are molecularly distinct. Hemogenic and non-HE cells in the yolk sac are more closely related to each other than either is to hemogenic or non-HE cells in the major arteries. Therefore, a major driver of the different lineage potentials of the committed erythro-myeloid progenitors that emerge in the yolk sac versus hematopoietic stem cells that originate in the major arteries is likely to be the distinct molecular properties of the HE cells from which they are derived. We used bioinformatics analyses to predict signaling pathways active in arterial HE, which include the functionally validated pathways Notch, Wnt, and Hedgehog. We also used a novel bioinformatics approach to assemble transcriptional regulatory networks and predict transcription factors that may be specifically involved in hematopoietic cell formation from arterial HE, which is the origin of the adult hematopoietic system.