Single-cell dissections of Rosette-to-Lumen stage embryoids - and the Glucose-6-phosphate isomerase-induced arthritis model

Single-cell omics (SCO), particularly single-cell RNA-sequencing (scRNA-seq), are widely used to describe dynamic biological and pathophysiological processes. This thesis explores the utility of SCO in characterizing dynamic processes in the in vitro generation of embryo-like structures and a pathophysiological condition in tissue usually sparse in cells, the joint. More specifically, scRNA-seq was used to analyze the transcriptional signatures of cells obtained from a new stem-cell-based embryo model, the Rosette-to-Lumen stage embryoids (RtL-embryoids) and an established glucose-6-phosphate isomerase (G6PI) regulatory T cells (Tregs) depletion model for the induction of acute and chronic arthritis. In 3 dimensional (3D) cell culture environments, blastocyst-derived stem cell lines are self-organized into embryolike structures. Using a 3D cell co-culture system, embryo-like structures can be generated based on transcription factor-mediated reprogramming of embryonic stem cells. In these cultures, embryonic stem cells self-organize into compartmentalized, elongated structures that mimic the inner regions of the early postimplantation embryos. The Smart-Seq 2 scRNA-seq protocol was used to identify transcriptomic profiles similar to epiblasts, primitive-/visceral endoderm, and extraembryonic ectoderms of early murine embryos around E4.5 to E5.5. SCOs revealed how stem-cell-based embryos progressed from rosette formation to lumenogenesis, followed by epiblast-like cells development from naïve- to primed pluripotency. In addition, lineage specification of primordial germ cells and distal/anterior visceral endoderm-like cells were observed respectively in epiblast- or visceral endoderm-like compartments. Applying SCO to RtL-embryoids led to new findings in early embryogenesis. Murine models of induced arthritis are valuable tools for studying the pathogenic process of arthritic inflammation (ArInf). For example, G6PI-induced arthritis is a spontaneously remitting experimental arthritis model in which the depletion of Tregs can further modulate to induce non-remitting, chronic, and destructive arthritis. The Seq-Well scRNA-seq platform was applied to describe mesenchymal and immune cells in ArInf. Sampling over time allowed us to define dynamic processes of transcription in individual cells during ArInf. Here, an increased gradient of Notch signaling was identified between sublining fibroblasts (SLFs) and lining layer fibroblasts (LLFs). Furthermore, in ArInf, a counteractive Prg4 gradient was observed in fibroblasts of the synovium, which was tightly restricted to the lining layer of fibroblasts in the healthy synovium. This counteractive Prg4 gradient was connected to the loss of synovial barrier integrity formed by a distinct population of Cx3cr1+ tissue-resident macrophages, which get lost in ArInf. Arthritis remission and expansion of this specialized macrophage subcluster resulted in a reduced expression of Prg4 in the SLFs. ScRNA-seq provided a cellular framework for understanding the pathomechanisms of ArInf, which is a prerequisite for developing new therapies for this disease. In conclusion, scRNA-seq provided considerable insight into the development of RtL-embryoids but is equally suited to identifying rare cell populations in complex tissue under pathophysiological conditions

Decoding mechanism of action and sensitivity to drug candidates from integrated transcriptome and chromatin state.

Omics-based technologies are driving major advances in precision medicine, but efforts are still required to consolidate their use in drug discovery. In this work, we exemplify the use of multi-omics to support the development of 3-chloropiperidines, a new class of candidate anticancer agents. Combined analyses of transcriptome and chromatin accessibility elucidated the mechanisms underlying sensitivity to test agents. Furthermore, we implemented a new versatile strategy for the integration of RNA- and ATAC-seq (Assay for Transposase-Accessible Chromatin) data, able to accelerate and extend the standalone analyses of distinct omic layers. This platform guided the construction of a perturbation-informed basal signature predicting cancer cell lines' sensitivity and to further direct compound development against specific tumor types. Overall, this approach offers a scalable pipeline to support the early phases of drug discovery, understanding of mechanisms, and potentially inform the positioning of therapeutics in the clinic

Table_3_Identification of drug candidates targeting monocyte reprogramming in people living with HIV.xlsx

IntroductionPeople living with HIV (PLHIV) are characterized by functional reprogramming of innate immune cells even after long-term antiretroviral therapy (ART). In order to assess technical feasibility of omics technologies for application to larger cohorts, we compared multiple omics data layers.MethodsBulk and single-cell transcriptomics, flow cytometry, proteomics, chromatin landscape analysis by ATAC-seq as well as ex vivo drug stimulation were performed in a small number of blood samples derived from PLHIV and healthy controls from the 200-HIV cohort study.ResultsSingle-cell RNA-seq analysis revealed that most immune cells in peripheral blood of PLHIV are altered in their transcriptomes and that a specific functional monocyte state previously described in acute HIV infection is still existing in PLHIV while other monocyte cell states are only occurring acute infection. Further, a reverse transcriptome approach on a rather small number of PLHIV was sufficient to identify drug candidates for reversing the transcriptional phenotype of monocytes in PLHIV.DiscussionThese scientific findings and technological advancements for clinical application of single-cell transcriptomics form the basis for the larger 2000-HIV multicenter cohort study on PLHIV, for which a combination of bulk and single-cell transcriptomics will be included as the leading technology to determine disease endotypes in PLHIV and to predict disease trajectories and outcomes.</p

Table_2_Identification of drug candidates targeting monocyte reprogramming in people living with HIV.xlsx

IntroductionPeople living with HIV (PLHIV) are characterized by functional reprogramming of innate immune cells even after long-term antiretroviral therapy (ART). In order to assess technical feasibility of omics technologies for application to larger cohorts, we compared multiple omics data layers.MethodsBulk and single-cell transcriptomics, flow cytometry, proteomics, chromatin landscape analysis by ATAC-seq as well as ex vivo drug stimulation were performed in a small number of blood samples derived from PLHIV and healthy controls from the 200-HIV cohort study.ResultsSingle-cell RNA-seq analysis revealed that most immune cells in peripheral blood of PLHIV are altered in their transcriptomes and that a specific functional monocyte state previously described in acute HIV infection is still existing in PLHIV while other monocyte cell states are only occurring acute infection. Further, a reverse transcriptome approach on a rather small number of PLHIV was sufficient to identify drug candidates for reversing the transcriptional phenotype of monocytes in PLHIV.DiscussionThese scientific findings and technological advancements for clinical application of single-cell transcriptomics form the basis for the larger 2000-HIV multicenter cohort study on PLHIV, for which a combination of bulk and single-cell transcriptomics will be included as the leading technology to determine disease endotypes in PLHIV and to predict disease trajectories and outcomes.</p

Table_1_Identification of drug candidates targeting monocyte reprogramming in people living with HIV.xlsx

IntroductionPeople living with HIV (PLHIV) are characterized by functional reprogramming of innate immune cells even after long-term antiretroviral therapy (ART). In order to assess technical feasibility of omics technologies for application to larger cohorts, we compared multiple omics data layers.MethodsBulk and single-cell transcriptomics, flow cytometry, proteomics, chromatin landscape analysis by ATAC-seq as well as ex vivo drug stimulation were performed in a small number of blood samples derived from PLHIV and healthy controls from the 200-HIV cohort study.ResultsSingle-cell RNA-seq analysis revealed that most immune cells in peripheral blood of PLHIV are altered in their transcriptomes and that a specific functional monocyte state previously described in acute HIV infection is still existing in PLHIV while other monocyte cell states are only occurring acute infection. Further, a reverse transcriptome approach on a rather small number of PLHIV was sufficient to identify drug candidates for reversing the transcriptional phenotype of monocytes in PLHIV.DiscussionThese scientific findings and technological advancements for clinical application of single-cell transcriptomics form the basis for the larger 2000-HIV multicenter cohort study on PLHIV, for which a combination of bulk and single-cell transcriptomics will be included as the leading technology to determine disease endotypes in PLHIV and to predict disease trajectories and outcomes.</p

DataSheet_1_Identification of drug candidates targeting monocyte reprogramming in people living with HIV.pdf

IntroductionPeople living with HIV (PLHIV) are characterized by functional reprogramming of innate immune cells even after long-term antiretroviral therapy (ART). In order to assess technical feasibility of omics technologies for application to larger cohorts, we compared multiple omics data layers.MethodsBulk and single-cell transcriptomics, flow cytometry, proteomics, chromatin landscape analysis by ATAC-seq as well as ex vivo drug stimulation were performed in a small number of blood samples derived from PLHIV and healthy controls from the 200-HIV cohort study.ResultsSingle-cell RNA-seq analysis revealed that most immune cells in peripheral blood of PLHIV are altered in their transcriptomes and that a specific functional monocyte state previously described in acute HIV infection is still existing in PLHIV while other monocyte cell states are only occurring acute infection. Further, a reverse transcriptome approach on a rather small number of PLHIV was sufficient to identify drug candidates for reversing the transcriptional phenotype of monocytes in PLHIV.DiscussionThese scientific findings and technological advancements for clinical application of single-cell transcriptomics form the basis for the larger 2000-HIV multicenter cohort study on PLHIV, for which a combination of bulk and single-cell transcriptomics will be included as the leading technology to determine disease endotypes in PLHIV and to predict disease trajectories and outcomes.</p

Table_4_Identification of drug candidates targeting monocyte reprogramming in people living with HIV.xlsx

IntroductionPeople living with HIV (PLHIV) are characterized by functional reprogramming of innate immune cells even after long-term antiretroviral therapy (ART). In order to assess technical feasibility of omics technologies for application to larger cohorts, we compared multiple omics data layers.MethodsBulk and single-cell transcriptomics, flow cytometry, proteomics, chromatin landscape analysis by ATAC-seq as well as ex vivo drug stimulation were performed in a small number of blood samples derived from PLHIV and healthy controls from the 200-HIV cohort study.ResultsSingle-cell RNA-seq analysis revealed that most immune cells in peripheral blood of PLHIV are altered in their transcriptomes and that a specific functional monocyte state previously described in acute HIV infection is still existing in PLHIV while other monocyte cell states are only occurring acute infection. Further, a reverse transcriptome approach on a rather small number of PLHIV was sufficient to identify drug candidates for reversing the transcriptional phenotype of monocytes in PLHIV.DiscussionThese scientific findings and technological advancements for clinical application of single-cell transcriptomics form the basis for the larger 2000-HIV multicenter cohort study on PLHIV, for which a combination of bulk and single-cell transcriptomics will be included as the leading technology to determine disease endotypes in PLHIV and to predict disease trajectories and outcomes.</p

Systemic alterations in neutrophils and their precursors in early-stage chronic obstructive pulmonary disease

Summary: Systemic inflammation is established as part of late-stage severe lung disease, but molecular, functional, and phenotypic changes in peripheral immune cells in early disease stages remain ill defined. Chronic obstructive pulmonary disease (COPD) is a major respiratory disease characterized by small-airway inflammation, emphysema, and severe breathing difficulties. Using single-cell analyses we demonstrate that blood neutrophils are already increased in early-stage COPD, and changes in molecular and functional neutrophil states correlate with lung function decline. Assessing neutrophils and their bone marrow precursors in a murine cigarette smoke exposure model identified similar molecular changes in blood neutrophils and precursor populations that also occur in the blood and lung. Our study shows that systemic molecular alterations in neutrophils and their precursors are part of early-stage COPD, a finding to be further explored for potential therapeutic targets and biomarkers for early diagnosis and patient stratification

Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment.

Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRhiCD11chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DRlo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19

Disease severity-specific neutrophil signatures in blood transcriptomes stratify COVID-19 patients

Background!#!The SARS-CoV-2 pandemic is currently leading to increasing numbers of COVID-19 patients all over the world. Clinical presentations range from asymptomatic, mild respiratory tract infection, to severe cases with acute respiratory distress syndrome, respiratory failure, and death. Reports on a dysregulated immune system in the severe cases call for a better characterization and understanding of the changes in the immune system.!##!Methods!#!In order to dissect COVID-19-driven immune host responses, we performed RNA-seq of whole blood cell transcriptomes and granulocyte preparations from mild and severe COVID-19 patients and analyzed the data using a combination of conventional and data-driven co-expression analysis. Additionally, publicly available data was used to show the distinction from COVID-19 to other diseases. Reverse drug target prediction was used to identify known or novel drug candidates based on finding from data-driven findings.!##!Results!#!Here, we profiled whole blood transcriptomes of 39 COVID-19 patients and 10 control donors enabling a data-driven stratification based on molecular phenotype. Neutrophil activation-associated signatures were prominently enriched in severe patient groups, which was corroborated in whole blood transcriptomes from an independent second cohort of 30 as well as in granulocyte samples from a third cohort of 16 COVID-19 patients (44 samples). Comparison of COVID-19 blood transcriptomes with those of a collection of over 3100 samples derived from 12 different viral infections, inflammatory diseases, and independent control samples revealed highly specific transcriptome signatures for COVID-19. Further, stratified transcriptomes predicted patient subgroup-specific drug candidates targeting the dysregulated systemic immune response of the host.!##!Conclusions!#!Our study provides novel insights in the distinct molecular subgroups or phenotypes that are not simply explained by clinical parameters. We show that whole blood transcriptomes are extremely informative for COVID-19 since they capture granulocytes which are major drivers of disease severity