Index Sorting Research Articles

Evaluation of single-cell sorting accuracy using antibody-derived tag-based qPCR.

Single-cell sorting (index sorting) is a widely used method to isolate one cell at a time using fluorescence-activated cell sorting (FACS) for downstream applications such as single-cell sequencing or single-cell expansion. Despite widespread use, few assays are available to evaluate the proteomic features of the sorted single cell and further confirm the accuracy of single-cell sorting. With this caveat, we developed a novel assay to confirm the protein expression of sorted single cells by co-staining cells with the same marker using both antibody-derived tags (ADTs) and fluorescent antibodies. After single-cell sorting, we amplified the oligo of the ADT reagent as a surrogate signal for the protein expression using multiplex TaqMan™ qPCR on sorted cells. This assay is not only useful for confirming the identity of a sorted single cell but also an efficient method to profile proteomic features at the single-cell level. Finally, we appliedthis assay to characterize protein expression on whole cell lysate. Because of the sensitivity of the TaqMan™ qPCR, we can detect protein expression from a small number of cells. In summary, the ADT-based qPCR assay developed here can be utilized to confirm single-cell sorting accuracy and characterizing protein expression on both single cells and whole cell lysate.

182 Purification and Characterization of Human Neural Stem and Progenitor Cells

INTRODUCTION: The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. METHODS: We combine high-dimensional flow cytometry with single cell transcriptomics to comprehensively purify and functionally characterize NSPCs from the developing human brain during mid-gestation. We utilize index sorting based on the combinatorial expression of cell-surface markers (immunophenotype) to directly link surface-marker expression to the expressed transcriptomes of single cells, allowing for rigorous validation of sort purity. We further demonstrate the functional properties of each identified cell type in vitro and in vivo. RESULTS: CD24-THY1-/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain.THY1hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24+ THY1-/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a novel transcriptomically distinct THY1hiEGFRhiPDGFRA- bipotent glial progenitor cell, which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. CONCLUSIONS: The results presented here are strong proof-of-concept that distinct cell types from the developing brain can be prospectively isolated based on the expression of multiple surface markers. The combined results from scRNA-seq, in vitro differentiation, and in vivo transplantation offers the most rigorous interrogation of NSPCs’ transcriptomic identities and functional behavior. The modular nature of our antibody panel allows for the easy expansion or simplification of the panel, based on the cell-type of interest, thus providing a valuable tool for future investigation of both molecular and functional NSPC heterogeneity.

Towards a Decision Engine for Therapeutic Optimization of Relapsed Refractory Multiple Myeloma

Introduction Substantial progress in the treatment of Multiple Myeloma (MM) extends survival for many patients (Pts), even after multiple relapses, owing to a broad and rapidly extending therapeutic arsenal, with major advances in the arena of bispecific antibodies and CART therapy. However, this also creates an increasingly complex challenge in selecting optimal therapeutic modalities for each patient, as current guidelines rely on empirical considerations. To address this, our study employs a population-scale single cell genomics database of over 250 patient samples covering all disease stages and cycles of treatment with complete patient medical records and machine learning approaches to analyze molecular signatures of myeloma cells and bone marrow immune microenvironment. Methods This study constitutes a meta-trial of RRMM patients who underwent single-cell RNA sequencing (scRNA-seq) between 6.2017 and 6.2023 in our lab. We gathered comprehensive demographic and myeloma clinical data, with a particular focus on therapeutic history and drug refractoriness, defined based on IMWG criteria (progression on or within 60 days from treatment). Our clinical scRNA-seq pipeline, includes a tailored protocol involving index sorting of CD38+ CD138+ positive cells for myeloma plasma cells (PCs) (Ledergor et al, Nat Med 2018; Cohen et al Nat Med 2020), together with CD45+ sorting and scRNA-seq cells from the tumor microenvironment for to identify tumor -immune molecular signatures associated with drug resistance. Results A total of 243 patient samples were included in the study, comprising RRMM (N=128), NDMM (N=21), SMM (N=9), and MGUS (N=4) patients with clinical annotation. Among RRMM patients, the median age was 72 (range 40-89), and 59% exhibited high-risk FISH cytogenetics (t(4:14), del17p, t(14:16), t(14:20), +1q21), while 17% had double-hit myeloma. Refractoriness to specific drugs was observed, such as bortezomib (49%), carfilzomib (27%), lenalidomide (49%), pomalidomide (33%), daratumumab (49). In total, we sequenced 158,444 myeloma/plasma cells, with an average of 880 cells per patient, along with 800 CD45+ cells. By using plasma cells from 11 healthy donors as a reference, we calculated transcriptional changes for each patient, determining the upregulated and downregulated genes profile. Clustering the patient's tumor profiles revealed 7 main tumor archetypes [TA] defined by the expression of 8 gene modules. Refractoriness score, defined as the average number of drugs to which myeloma is refractory, exhibited variations across different tumor archetypes. Notably, the expression levels of several tumor drivers and crucial targets of novel immunotherapies also showed diversity among the tumor archetypes. For instance, TA#6, primarily comprising newly diagnosed patients, displayed lower expression of GPRC5D. Conversely, in TA#1, which consisted of ultra-refractory patients with high MYC and CD47 expression, both GPRC5D and TNFRSF17 (BCMA) were downregulated. Examination of bone marrow CD45+ immune cells revealed a striking bimodal immune composition, characterized by one subtype with increased T-cell abundance and another enriched with myeloid cells. Each immune cell subtype contributed to the drug responsiveness score and provided additional insights complementary to the tumor cells' profile. Further clinical and transcriptional correlations involving both MM cells and immune cells will be presented at the meeting. Conclusions We constructed a clinically-annotated transcriptional matrix revealing the blueprint of relapsed refractory myeloma. This novel decision engine provides a basis for optimizing treatment selection by considering both tumor and tumor microenvironment characteristics. By incorporating critical information on drug resistance patterns and expression of key targets like GPRC5D, TNFRSF17 (BCMA) and FcRH5, we enable precision medicine approaches for improved management of relapsed refractory myeloma. These findings open avenues for tailored and more effective therapeutic strategies, promising better outcomes for patients with relapsed refractory myeloma.

An adaptive multiobjective evolutionary algorithm for dynamic multiobjective flexible scheduling problem

There are various uncertain disturbances in the actual manufacturing environment, which makes dynamic multiobjective flexible scheduling problem of flexible job shop (MDFJSP) become the research focus in the field of optimal scheduling. In this paper, MDFJSP in the environment of temporary order insertion uncertainty is studied, and a multiobjective dynamic scheduling scheme based on rescheduling index and adaptive nondominated sorting genetic algorithm (NSGA-II) is proposed. First, based on the actual manufacturing environment, the mathematical model of the traditional flexible job shop scheduling problem is improved, and the multiobjective dynamic rescheduling model of flexible work center is established. Then, the existing rescheduling mechanisms are summarized, and a rescheduling hybrid driving mechanism based on the rescheduling index is proposed to enable it to reschedule and drive according to the actual situation. Finally, the shortcomings of the traditional multiobjective scheduling algorithm NSGA-II are analyzed, the adaptive cross mutation strategy and the simplified harmonic normalized distance measure method are proposed to improve it, and an adaptive multiobjective dynamic scheduling algorithm NSGA-II (MDSA-NSGA-II) is formed. To analyze the performance of this algorithm, the performance of this algorithm is compared with five classical flexible job shop multiobjective scheduling algorithms in international general examples, and the effectiveness is verified by real aircraft production examples. The experimental results fully show that MDSA-NSGA-II has good performance in solving MDFJSP.

Immune Phenotypes and Target Antigens of Clonally Expanded Bone Marrow T Cells in Treatment-Naïve Multiple Myeloma.

Multiple myeloma is a hematologic malignancy of monoclonal plasma cells that accumulate in the bone marrow. Despite their clinical and pathophysiologic relevance, the roles of bone marrow-infiltrating T cells in treatment-naïve patients are incompletely understood. We investigated whether clonally expanded T cells (i) were detectable in multiple myeloma bone marrow, (ii) showed characteristic immune phenotypes, and (iii) whether dominant clones recognized antigens selectively presented on multiple myeloma cells. Single-cell index sorting and T-cell receptor (TCR) αβ sequencing of bone marrow T cells from 13 treatment-naïve patients showed dominant clonal expansion within CD8+ cytolytic effector compartments, and only a minority of expanded T-cell clones expressed the classic immune-checkpoint molecules PD-1, CTLA-4, or TIM-3. To identify their molecular targets, TCRs of 68 dominant bone marrow clones from five selected patients were reexpressed and incubated with multiple myeloma and non-multiple myeloma cells from corresponding patients. Only 1 of 68 TCRs recognized antigen presented on multiple myeloma cells. This TCR was HLA-C-restricted, self-peptide-specific and could be activated by multiple myeloma cells of multiple patients. The remaining dominant T-cell clones did not recognize multiple myeloma cells and were, in part, specific for antigens associated with chronic viral infections. In conclusion, we showed that dominant bone marrow T-cell clones in treatment-naïve patients rarely recognize antigens presented on multiple myeloma cells and exhibit low expression of classic immune-checkpoint molecules. Our data provide experimental context for experiences from clinical immune-checkpoint inhibition trials and will inform future T cell-dependent therapeutic strategies.

Immune phenotypes and checkpoint molecule expression of clonally expanded lymph node-infiltrating T cells in classical Hodgkin lymphoma

Lymph node-infiltrating T cells have been of particular interest in classical Hodgkin lymphoma (cHL). High rates of complete therapeutic responses to antibody-mediated immune checkpoint blockade, even in relapsed/refractory patients, suggest the existence of a T cell-dominated, antigen-experienced, functionally inhibited and lymphoma-directed immune microenvironment. We asked whether clonally expanded T cells (1) were detectable in cHL lymph nodes, (2) showed characteristic immune phenotypes, and (3) were inhibited by immune checkpoint molecule expression. We applied high-dimensional FACS index sorting and single cell T cell receptor αβ sequencing to lymph node-infiltrating T cells from 10 treatment-naïve patients. T cells were predominantly CD4+ and showed memory differentiation. Expression of classical immune checkpoint molecules (CTLA-4, PD-1, TIM-3) was generally low (< 12.0% of T cells) and not different between CD4+ and CD8+ T cells. Degrees of clonal T cell expansion varied between patients (range: 1–18 expanded clones per patient) and was almost exclusively restricted to CD8+ T cells. Clonally expanded T cells showed non-naïve phenotypes and low checkpoint molecule expression similar to non-expanded T cells. Our data suggest that the therapeutic effects of immune checkpoint blockade require mechanisms in addition to dis-inhibition of pre-existing lymphoma-directed T cell responses. Future studies on immune checkpoint blockade-associated effects will identify molecular T cell targets, address dynamic aspects of cell compositions over time, and extend their focus beyond lymph node-infiltrating T cells.

P748: SINGLE CELL GENOTYPING OF MATCHED BONE MARROW AND PERIPHERAL BLOOD CELLS IN TREATMENT NAIVE AND AZA-TREATED MDS AND CMML

Background: Somatic mutations in hematopoietic stem cells (HSCs) are a central pathogenic event in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) where they induce proliferative advantages and impaired differentiation with subsequent cytopenias in peripheral blood (PB). Patients with high-risk disease who are ineligible for allogenic stem cell transplantation are treated with hypomethylating agents, including 5-azacytidine (AZA). AZA treatment can improve PB counts and delay progression to Akute Myeloid Leukemia. However, AZA response does not require eradication of mutated HSCs and may be related to improved differentiation capacity of mutated hematopoietic stem and progenitor cells (HSPCs). However, the contribution of mutated HSPCs to steadystate hematopoiesis in MDS and CMML is unclear. Aims: In this project, we aimed to determine the distribution of individual subclones and even wild-type cells within the HSPC-compartment and the contribution of these subclones and wild-type cells to effective hematopoiesis in patients with MDS or CMML. Methods: We used a combination of index sorting and single cell genotyping to characterize the somatic mutations of individual stem- and progenitor cells and matched high-turnover circulating cells in three patients, one treatment naïve and two AZA-treated. Using this approach, myeloid driver mutations previously detected in a traditional approach were tracked in HSC/multipotent progenitors (MPP), MDS stem cells MDS-SC, progenitor cells (common myeloid progenitor (CMP), granulocyte monocyte progenitor (GMP), megakaryocyte erythroid progenitor (MEP)), as well as in monocytes, neutrophils and naïve B cells (nBC). Results: Patient H198302 and Patient H198303 both had CMML and had been treated with AZA for ̴10 years with complete response. Patient H198304 had MDS-EB1 and has never been treated with hypomethylating agents. For each patient, the figure shows the proportions of cells across the haematopoietic hierarchy carrying zero, one, two, three, or four mutations in the specified alleles. Only very small numbers of HSPCs carrying no mutation could be detected. In all three patients, most stem and progenitor cells carried a majority of tracked mutations, with no major differences between stem cells, progenitor cells and MDS-stem cells. In one patient, H198304, MDS-stem cells had a higher proportion of cells with 3 or 4 mutations detected. Overall, in all three patients, a similar frequency of mutations was observed in differentiated monocytes and neutrophils with a substantial proportion of the circulating cells derived from highly mutated progenitors. One notable exception were nBCs in Patient H198304, which were mostly wild-type, suggesting that the small wildtype HSC population is the predominant origin of nBCs in this individual. Analysis of additional lymphoid populations in PB from this patient revealed that naïve T, but not NK cells were also predominantly wild-type, suggesting specific impairment of B- and T-lineage maturation in the mutated cells. Image:Summary/Conclusion: In conclusion, attrition of highly mutated cells during myeloid maturation was not observed in any of the three patients, irrespective of HMA therapy. This suggests that in vivo, highly mutated stem and progenitor cells retain the capacity to differentiate to mature myeloid and in some patients mature lymphoid cells and contribute significantly to circulating blood cells in MDS and CMML, prior to and following AZA treatment.

In utero origin of myelofibrosis presenting in adult monozygotic twins

The latency between acquisition of an initiating somatic driver mutation by a single-cell and clinical presentation with cancer is largely unknown. We describe a remarkable case of monozygotic twins presenting with CALR mutation-positive myeloproliferative neoplasms (MPNs) (aged 37 and 38 years), with a clinical phenotype of primary myelofibrosis. The CALR mutation was absent in T cells and dermal fibroblasts, confirming somatic acquisition. Whole-genome sequencing lineage tracing revealed a common clonal origin of the CALR-mutant MPN clone, which occurred in utero followed by twin-to-twin transplacental transmission and subsequent similar disease latency. Index sorting and single-colony genotyping revealed phenotypic hematopoietic stem cells (HSCs) as the likely MPN-propagating cell. Furthermore, neonatal blood spot analysis confirmed in utero origin of the JAK2V617F mutation in a patient presenting with polycythemia vera (aged 34 years). These findings provide a unique window into the prolonged evolutionary dynamics of MPNs and fitness advantage exerted by MPN-associated driver mutations in HSCs.

Expansion of Quiescent Hematopoietic Stem Cells under Stress and Nonstress Conditions in Mice.

Hematopoietic stem cells (HSCs) are maintained in the quiescent state for protection from stress. How quiescent HSCs expand in vivo under stress and nonstress conditions, however, is poorly understood. Using the fluorescent ubiquitination-based cell cycle indicator (Fucci) mice, we analyzed quiescent and cycling HSCs in the bone marrow after transplantation and during development and aging. The cell cycle of HSCs in Fucci mice were analyzed by flow cytometry. Single-cell colony assays suggested that cycling cells were likely in the process of differentiation. Long-term competitive repopulation and limiting dilution assays revealed that given a higher frequency of functional HSCs in quiescent cells, durable self-renewal potential was greater in quiescent cells than cycling cells. In the bone marrow,functional HSC pool, represented by quiescent HSCs, was rapidly re-established by three weeks after transplantation, significantly expanded by three weeks of age in development, and gradually accumulated with aging. Single-cell RNA-sequencing with flow cytometric index sorting suggested that high levels of CD201 and Sca-1 expression and a low level of mitochondrial activity were associated with quiescent HSCs. A set of candidate quiescent genes in HSCs were also provided. This study implied that controlling quiescence in HSCs is important for their in vivo expansion and maintenance.

Development of HFU-based permeability prediction models using core data for characterisation of a heterogeneous Oligocene sand in the Nam Con Son basin

Core data by both routine and special core analysis are required to understand and predict reservoir petrophysical characteristics. In this research, a total number of 50 core plugs taken from an Oligocene sand (T30) in the Nam Con Son basin, offshore southern Vietnam, were tested in the core laboratory of the Vietnam Petroleum Institute (VPI).&#x0D; The results of routine core analysis (RCA) including porosity and permeability measurements were employed to divide the study reservoir into hydraulic flow units (HFUs) using the global hydraulic elements (GHEs) approach. Based on five classified HFUs, 16 samples were selected for special core analysis, i.e., mercury injection capillary pressure (MICP) and grain size analyses for establishing non-linear porosity-permeability model of each HFU based on Kozeny-Carman equation, which provides an improved prediction of permeability (R2 = 0.846) comparing to that by the empirical poro-perm relationship (R2 = 0.633).&#x0D; In addition, another permeability model, namely the Winland R35 method, was applied and gave very satisfactory results (R2 = 0.919). Finally, by integrating the results from MICP and grain size analyses, a good trendline of pore size distribution index (λ) and grain sorting was successfully obtained to help characterise the study reservoir. High λ came with poor sorting, and vice versa, the low λ corresponded to good sorting of grain size.

Single-Cell and Spatial Analyses Characterize Distinct Subsets of Malignant T Cells in Angioimmunoblastic T Cell Lymphoma

IntroductionAngioimmunoblastic T-cell lymphoma (AITL) is the most frequent of nodal peripheral T-cell lymphomas. AITL results from the transformation of T follicular helper (T FH) cells and is characterized by chemo-resistance and poor survival (5-year OS around 30%). Recent data from prospective clinical trials suggest that disease outcome may be impacted by factors other than genomic features, such as the tumor microenvironment (TME) and overall intra-tumoral heterogeneity. Our understanding of AITL intra-tumoral genetic, transcriptional and functional heterogeneity is limited because most molecular data generated so far have come from bulk analyses. Single-cell RNA sequencing (scRNA-seq) enables fine characterization of cell types and functional cell states. When focused on T or B cells, 5'-end scRNA-seq also yields the TCR or BCR sequences that allow tracking clonally related cells. Here we studied the intra-tumor heterogeneity of AITL tumors using integrative scRNA-seq.MethodsWe analyzed lymph node live cell suspensions from AITL patients (n=10) using droplet-based 10x Genomics 5'-end scRNA-seq. Malignant T cells from 4 AITL samples were also analyzed by FACS index sorting and plate-based 5'-end scRNA-seq to link cell surface phenotype and gene expression profile. We identified malignant T cell clones by intersecting the gene expression and TCR sequencing data, and performed separate focused analyses of TME subsets and malignant T cells. We compared subsets of malignant T cells from all patients using marker gene-based metaclustering to identify AITL T cell states conserved across patients. We explored the genetic heterogeneity of malignant T cells by mapping RHOA G17V mutations and inferring copy number variation (CNV) subclones from scRNA-seq data. In select cases, we performed in situ analysis by immunohistochemistry (IHC) or spatial transcriptomics to characterize the spatial distribution of malignant T cell subsets identified by scRNA-seq.ResultsBased on gene expression, malignant T cells grouped in patient-specific clusters, while non-malignant T, B and myeloid TME cells from all patients clustered by cell type or cell state. Among TME cells, we identified 7 subsets of B cells (including activated B cells, plasma cells, and one patient-specific monoclonal B cell proliferation), 6 subsets of myeloid cells (including macrophages, conventional and plasmacytoid dendritic cells), and 8 subsets of non-malignant T cells (including activated cytotoxic T lymphocytes (CTL) with clonal expansions). Patient-specific malignant T cells were heterogeneous and divided into several gene-expression based clusters. Metaclustering of malignant T cell subsets identified T central memory (T CM)-like and T FH-like states in 10/10 samples. We also identified in 3/10 samples clusters of CTL-like malignant T cells expressing characteristic marker genes (including NKG7, GNLY, GZMK, PRF1). We observed an intra-sample continuum of gene expression states from quiescent T CM-like to proliferating T FH-like states. T FH-like cells were larger in size and expressed higher levels of surface PD1 and ICOS than T CM-like and CTL-like subsets. We detected the RHOA G17V mutation in malignant T cells of 4/4 mutated cases, with no evidence of subclonal heterogeneity for that mutation. We detected clonal and subclonal CNV in most AITL malignant T cells. CTL-like states were enriched in specific CNV subclones, but the T CM-like to T FH-like continuum was observed in all CNV subclones, suggesting that functional plasticity and subclonal genetic evolution may occur independently. In situ staining of markers for T FH-like (PD1, ICOS, CD200) and CTL-like (GZMK, GZMA) cells showed that T FH-like and CTL-like cells occupied distinct tissue niches within the tumor. In spatial transcriptomics analysis, T FH-like cells mapped to follicular dendritic cell (FDC)-rich areas, while T CM-like cells were associated with T-zone reticular cells.ConclusionsOur analyses recapitulate known characteristics of AITL TME, and uncover previously unrecognized heterogeneity among malignant T cells across multiple patients. The distinct gene expression programs, phenotypes, genetics, and locations of T FH-like, T CM-like and CTL-like states suggest that AITL malignant T cells undergo significant functional plasticity and genetic divergence, which could influence response to therapy and overall clinical course. [Display omitted] DisclosuresLemonnier: Institut Roche: Research Funding; Gilead: Other: travel grant. Gaulard: Gilead: Consultancy; Innate Pharma: Research Funding; Sanofi: Research Funding; Alderaan: Research Funding; Takeda: Consultancy, Honoraria. Milpied: Institut Roche: Research Funding; Innate Pharma: Research Funding; Bristol Myers Squibb: Research Funding.

CD14 Expressing Precursors Give Rise to Highly Functional Conventional Dendritic Cells for Use as Dendritic Cell Vaccine.

Simple SummaryDendritic cells are attractive candidates for immunotherapy to prevent disease recurrence in cancer patients. Dendritic cells are a plastic population of antigen presenting cells and a variety of protocols have been described to generate dendritic cells from either monocytes or stem cells. To induce long lasting immunity, dendritic cells need to have a fully mature phenotype and activate naïve T-cells. Here, we describe a good manufacturer protocol to generate very potent conventional DC-like cells, derived from cord blood stem cells via a CD14+CD115+ precursor stage. They express high levels of CD1c and strongly activate both naïve as well as antigen-experienced T-cells. Implementation of this protocol in the clinic could advance the efficiency of dendritic cell based vaccines.Induction of long-lasting immunity by dendritic cells (DCs) makes them attractive candidates for anti-tumor vaccination. Although DC vaccinations are generally considered safe, clinical responses remain inconsistent in clinical trials. This initiated studies to identify subsets of DCs with superior capabilities to induce effective and memory anti-tumor responses. The use of primary DCs has been suggested to overcome the functional limitations of ex vivo monocyte-derived DCs (moDC). The ontogeny of primary DCs has recently been revised by the introduction of DC3, which phenotypically resembles conventional (c)DC2 as well as moDC. Previously, we developed a protocol to generate cDC2s from cord blood (CB)-derived stem cells via a CD115-expressing precursor. Here, we performed index sorting and single-cell RNA-sequencing to define the heterogeneity of in vitro developed DC precursors and identified CD14+CD115+ expressing cells that develop into CD1c++DCs and the remainder cells brought about CD123+DCs, as well as assessed their potency. The maturation status and T-cell activation potential were assessed using flow cytometry. CD123+DCs were specifically prone to take up antigens but only modestly activated T-cells. In contrast, CD1c++ are highly mature and specialized in both naïve as well as antigen-experienced T-cell activation. These findings show in vitro functional diversity between cord blood stem cell-derived CD123+DC and CD1c++DCs and may advance the efficiency of DC-based vaccines.

A plate-based single-cell ATAC-seq workflow for fast and robust profiling of chromatin accessibility.

Profiling chromatin accessibility at the single-cell level provides critical information about cell type composition and cell-to-cell variation within a complex tissue. Emerging techniques for the interrogation of chromatin accessibility in individual cells allow investigation of the fundamental mechanisms that lead to the variability of different cells. This protocol describes a fast and robust method for single-cell chromatin accessibility profiling based on the assay for transposase-accessible chromatin using sequencing (ATAC-seq). The method combines up-front bulk Tn5 tagging of chromatin with flow cytometry to isolate single nuclei or cells. Reagents required to generate sequencing libraries are added to the same well in the plate where cells are sorted. The protocol described here generates data of high complexity and excellent signal-to-noise ratio and can be combined with index sorting for in-depth characterization of cell types. The whole experimental procedure can be finished within 1 or 2 d with a throughput of hundreds to thousands of nuclei, and the data can be processed by the provided computational pipeline. The execution of the protocol only requires basic techniques and equipment in a molecular biology laboratory with flow cytometry support.

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 4days 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 3days from isolation of the cells to the production of a library ready for sequencing.

Distinct Transcriptional Signatures Distinguish the Emergence of Multipotent Progenitors and Hematopoietic Stem Cells from Endothelial Precursors in the Murine Embryo

During embryonic development, blood cells emerge from hemogenic endothelium (HE), producing waves of hematopoietic progenitors prior to the emergence of rare hematopoietic stem cells (HSCs), which have the unique ability to self-renew and generate all cell types of the adult hematopoietic system. HSCs have significant potential for use in cellular therapies and disease modeling. However, efforts to generate HSCs in vitro from pluripotent stem cells (PSCs) have been limited by an incomplete understanding of the unique phenotypic markers and transcriptional programs that distinguish HE with HSC potential. Previous studies have demonstrated that yolk sac-derived erythromyeloid progenitors and HSCs originate from distinct populations of HE. However, it is not known whether the earliest lymphoid-competent progenitors, multipotent progenitors, and HSCs originate from HE with common phenotypic and transcriptional properties. To investigate this, we combined index sorting of single hemogenic precursors with stromal co-culture that enables simultaneous detection of HSC and multilineage hematopoietic potential, to functionally validate surface markers that may distinguish hemogenic precursors with different hematopoietic fates. We previously found that the co-expression of two markers, CD61 and EPCR, identifies a subset of VE-Cadherin+ endothelial cells from the mouse P-Sp/AGM region (para-aortic splanchnopleura/aorta-gonad-mesonephros, where the first HSCs are generated from HE between E9 and E11 in development) enriched phenotypically for arterial endothelial surface markers (e.g. Dll4, CD44) and functionally for hemogenic precursors with HSC potential. However, this population remains heterogeneous, containing clonal hemogenic precursors with the potential for HSC as well as multilineage progenitor-restricted fates. Here, we report that expression of arterial marker CXCR4 further enriched for functional HSC potential in hemogenic precursors in the P-Sp/AGM between E9 and E10, when the first clonal HSC precursors are detected at rare frequency. In contrast, we detected more abundant clonal HE with multilineage hematopoietic potential (producing lymphoid, erythroid, and myeloid progeny in vitro but lacking HSC potential) at the same stage, which are distinguished by comparatively lower CXCR4 expression. To investigate transcriptional differences between HE populations differentially expressing CXCR4, we performed single-cell RNA sequencing of E9 P-Sp-derived VE-Cadherin+CD61+EPCR+ cells. Using an unbiased gene module analysis based on graph autocorrelation in the Monocle 3 platform to identify genes that co-vary over pseudotime, we found that Cxcr4 is uniquely expressed in a subset of cells simultaneously enriched for arterial-specific genes (including Dll4, Efnb2, Hey2, Sox17, Cd44) and genes with established roles in HSC maintenance and self-renewal (including Mecom, Cdkn1c, H19, Txnip, Kmt2a). Conversely, expression of these genes is decreased in cells undergoing the endothelial to hematopoietic transition at this stage based on pseudotemporal ordering, concomitant with increasing expression of hematopoietic-specifying transcription factors Runx1 and Gfi1, and other genes associated with definitive hematopoiesis (egs. Myb, Kit, Hlf, Gata2, Mpl, Lyl1). We also examined the aggregate expression of established HSC-specific signature genes from published data sets across pseudotime, and found that they exhibit similar expression dynamics to that of Cxcr4 and Dll4, reaching peak expression prior to the initiation of Runx1 and Gfi1 expression. Altogether, our studies support a model in which the initial populations of multipotent progenitors and HSCs emerge independently from HE in the P-Sp/AGM. Furthermore, our findings suggest that HE with HSC competence is uniquely defined by co-expression of arterial endothelial genes and genes encoding HSC self-renewal factors, providing insight into the earliest transcriptional programs that must be coordinated to drive HSC fate from endothelial precursors. Future studies will focus on identifying the signal pathways whose integration promotes expression of these HSC-defining transcriptional programs in endothelial cells, with the goal of advancing methods for HSC generation in vitro. Disclosures Bernstein: Lyell Immunopharma: Current equity holder in publicly-traded company, Other: Laboratory Support; Deverra Therapeutics: Current equity holder in publicly-traded company.

1004 – INSIGHT ON NOTCH MECHANISMS TO ORCHESTRATE THE DEVELOPMENT OF HEMATOPOIETIC STEM CELLS IN THE EMBRYO

A conserved Notch function is crucial for the specification and generation of Hematopoietic Stem Cells (HSC) across evolutionary distinct organisms. By using genetically modified organisms, most of the elements that are functionally relevant have been identified mainly in mouse and zebrafish. However, due to the disruptive nature of these studies, it is still uncertain the physiological sequence of events that lead to and result from this specific Notch function. This is especially important in terms of reproducing this activity in vitro. The Notch system provides cells with a binary decision mechanism that takes place among neighboring cells. In vertebrate systems, several Notch ligands and receptors co-exist in the same cell and cells compete for activating the Notch receptor that will condition the fate of that cell. In the AGM, HSCs develop from hemogenic endothelial cells that reside in the ventral side of the dorsal aorta. Cells in the developing aortic endothelium co-express different types of Notch ligands and receptors. Several groups including ours have contributed to define the elements involved in this signal, but still several gaps prevent to have a complete understanding. In addition to the Notch1 receptor, Jagged1 and hes1, we have recently shown how Dll4 has also an important structural function in the hematopoietic cluster composition of the aorta. We provided data on how blocking Dll4 with a specific antibody impinges on the number of cells recruited into the cluster and the HSC activity. Thanks to current reagents and methodologies, we now have taken a step backwards to analyze the dynamics of Notch ligands and receptors at the protein and RNA level by single cell index sorting. We are characterizing the different hemogenic and hematopoietic clusters by the expression and activity of the Notch pathway. Latest results will be presented in the meeting.

3063 – DISTINCT POPULATIONS OF MULTIPOTENT PROGENITORS AND HEMATOPOIETIC STEM CELLS EMERGE FROM HEMOGENIC ENDOTHELIUM IN THE MURINE EMBRYO

During embryonic development, blood cells emerge from hemogenic endothelium (HE), producing waves of hematopoietic progenitors prior to the emergence of rare hematopoietic stem cells (HSC), which have the unique ability to self-renew and generate all cell types of the adult hematopoietic system. Prior studies have demonstrated that yolk sac-derived erythromyeloid progenitors and HSC originate from distinct populations of HE. However, it is not known whether the earliest lymphoid-competent and multipotent progenitors originate from the same population of HE that gives rise to HSC. To investigate this, we combined index sorting of single hemogenic precursors with stromal co-culture that enables simultaneous detection of HSC and multilineage hematopoietic potential, to functionally validate surface markers that may distinguish hemogenic precursors with different hematopoietic fates. We found that expression of arterial marker CXCR4 specifically enriches for functional HSC potential in hemogenic precursors in the para-aortic splanchnopleura (P-Sp), where the first HSC precursors are detected at around E9 in the murine embryo. In contrast, we detected clonal HE with lymphoid and multilineage hematopoietic potential at the same stage that are distinct from HSC precursors and largely lack CXCR4 expression. Single-cell RNA sequencing demonstrated that Cxcr4 is uniquely expressed in P-Sp-derived cells enriched for arterial-specific genes and genes essential for HSC maintenance and self-renewal, whereas expression of these genes is decreased in the majority of Runx1-expressing HE. Altogether, our studies suggest a model in which the earliest lymphoid and multipotent progenitors emerge from HE that is phenotypically and transcriptionally distinct from HSC precursors, and that co-expression of arterial endothelial and self-renewal factors uniquely defines HE with HSC competence.

3037 – COMBINED SINGLE-CELL DNA METHYLOME AND TRANSCRIPTOME ANALYSIS IDENTIFIES MOLECULAR STATES OF EARLY LINEAGE COMMITMENT

5-Methylcytosine is a stable epigenetic modification, whose remodeling at specific CpG residues appears integral to the process of enforcing lineage-restricted gene expression. In our previously generated DNA methylation map of the entire murine hematopoietic system we have observed that DNA methylation programming appears to be both progressive and irreversible during hematopoietic differentiation, suggesting that this modification could be used to unambiguously identify molecular marks of lineage commitment. To identify DNA methylation patterns relevant to lineage commitment, we applied combined single-cell whole-genome bisulfite and transcriptome sequencing on index-sorted Lin- Sca1+ cKit+ hematopoietic stem and multipotent progenitor cells (HSPCs) and on mature lymphoid cells and monocytes. We used our bulk DNA methylome map of the murine hematopoietic system as a template to identify DNA methylation programs of co-regulated CpGs in data obtained from single cells. This allowed us to overcome the critical limitation that single-cell DNA methylomes typically only represent 1-5% of genome-wide CpGs. We were thus able to recapitulate lineage-specific DNA methylation programs identified in bulk-sorted cell populations. In mature cells, DNA methylation programming was mutually exclusive for a specific lineage, supporting the use of single-cell methylomes to establish discreet points at which lineage commitment occurs. Importantly, lineage-specific CpGs were already identified within individual HSPCs, providing a molecular basis for early lineage-priming. This finding was substantiated by the identification of uniform and lineage-specific molecular states within the HSPC compartment, which might still be compatible with a step-wise differentiation process. Lastly, integrative analysis of combined single-cell methylome, transcriptome and index sorting datasets demonstrated that the identified methylome states of lineage commitment are accompanied by changes in lineage-specific gene expression, while the conventional immunophenotypic definition of HSPCs showed only limited correlation with the molecular states. Together, our work provides proof-of-principle that a high-resolution reference DNA methylation map facilitates the analysis of single-cell DNA methylome data and gives novel insights into the epigenetic regulation of hematopoietic commitment decisions.

Cancer-associated fibroblast compositions change with breast cancer progression linking the ratio of S100A4+ and PDPN+ CAFs to clinical outcome.

Tumors are supported by cancer-associated fibroblasts (CAFs). CAFs are heterogeneous and carry out distinct cancer-associated functions. Understanding the full repertoire of CAFs and their dynamic changes as tumors evolve could improve the precision of cancer treatment. Here we comprehensively analyze CAFs using index and transcriptional single-cell sorting at several time points along breast tumor progression in mice, uncovering distinct subpopulations. Notably, the transcriptional programs of these subpopulations change over time and in metastases, transitioning from an immunoregulatory program to wound-healing and antigen-presentation programs, indicating that CAFs and their functions are dynamic. Two main CAF subpopulations are also found in human breast tumors, where their ratio is associated with disease outcome across subtypes and is particularly correlated with BRCA mutations in triple-negative breast cancer. These findings indicate that the repertoire of CAF changes over time in breast cancer progression, with direct clinical implications.

Utilizing borehole electrical images to evaluate sorting of conglomeratic formation: example from Lower Triassic Baikouquan Formation in Well M152, Mahu Depression, Junggar Basin, China

Grain-size sorting of buried formation in openholes only comes from the particle size analysis of drilled cores. However, cores impossibly cover the whole formation, and the sorting of uncored interval is absent. With ultrahigh-resolution, borehole electrical image contains particular geological attributes, such as grain-sizes, sedimentary structures, and facies. In this paper, a sorting evaluation method is proposed based on conductivity spectrum of borehole electrical image from Baikouquan Formation in Well M152, Mahu Depression. The first stage is revising the depth of the cores by calibrating the cores to the borehole electrical images. The second stage is computing the two-dimensional conductivity spectrum of conductivity image. The third stage is calculating the sorting index from conductivity spectrum. The fourth stage is converting the sorting index to the evaluated sorting by a mathematical equation between sorting index and laboratorial grain-size sorting. In the cored interval of the Well M152, the evaluated sorting is compared with the grain-size of gravels; what can be concluded is that the evaluated sorting values from borehole electrical images gradually reduce with the decrease of the gravelly grain-sizes. The proposed method is demonstrated be beneficial to reflect the vertical diameters and sorting of gravelly grains in deeply buried conglomeratic formation continuously.