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Background: Pathomics facilitates automated, reproducible and precise histopathology analysis and morphological phenotyping. Similar to molecular omics, pathomics datasets are high-dimensional, but also face large outlier variability and inherent data missingness, making quick and comprehensible data analysis challenging. To facilitate pathomics data analysis and interpretation as well as support a broad implementation we developed tRigon (Toolbox foR InteGrative (path-)Omics data aNalysis), a Shiny application for fast, comprehensive and reproducible pathomics analysis. Results: tRigon is available via the CRAN repository (https://cran.r-project.org/web/packages/tRigon) with its source code available on GitLab (https://git-ce.rwth-aachen.de/labooratory-ai/trigon). The tRigon package can be installed locally and its application can be executed from the R console via the command 'tRigon::run_tRigon()'. Alternatively, the application is hosted online and can be accessed at https://labooratory.shinyapps.io/tRigon. We show fast computation of small, medium and large datasets in a low- and high-performance hardware setting, indicating broad applicability of tRigon. Conclusions: tRigon allows researchers without coding abilities to perform exploratory feature analyses of pathomics and non-pathomics datasets on their own using a variety of hardware. [ABSTRACT FROM AUTHOR]

Although clinical applications represent the next challenge in single-cell genomics and digital pathology, we still lack computational methods to analyze single-cell or pathomics data to find sample-level trajectories or clusters associated with diseases. This remains challenging as single-cell/pathomics data are multi-scale, i.e., a sample is represented by clusters of cells/structures, and samples cannot be easily compared with each other. Here we propose PatIent Level analysis with Optimal Transport (PILOT). PILOT uses optimal transport to compute the Wasserstein distance between two individual single-cell samples. This allows us to perform unsupervised analysis at the sample level and uncover trajectories or cellular clusters associated with disease progression. We evaluate PILOT and competing approaches in single-cell genomics or pathomics studies involving various human diseases with up to 600 samples/patients and millions of cells or tissue structures. Our results demonstrate that PILOT detects disease-associated samples from large and complex single-cell or pathomics data. Moreover, PILOT provides a statistical approach to find changes in cell populations, gene expression, and tissue structures related to the trajectories or clusters supporting interpretation of predictions. Synopsis: PILOT is a computational framework of analysis of multi-scale single cell or pathomics data measured over distinct patients. It allows the estimation of sample-level clustering and trajectories Statistical methods allow the interpretation of results, i.e., association of clusters/trajectories with cell clusters, genes and tissue structures. PILOT is showcased in scRNA-seq of myocardial infarction and pathomics data of kidney IgA nephropathy. PILOT is a computational framework of analysis of multi-scale single cell or pathomics data measured over distinct patients. [ABSTRACT FROM AUTHOR]

Cell–cell communication is mediated by membrane receptors and their ligands, such as the Eph/ephrin system, orchestrating cell migration during development and in diverse cancer types. Epigenetic mechanisms are key for integrating external "signals", e.g., from neighboring cells, into the transcriptome in health and disease. Previously, we reported ephrinA5 to trigger transcriptional changes of lncRNAs and protein-coding genes in cerebellar granule cells, a cell model for medulloblastoma. LncRNAs represent important adaptors for epigenetic writers through which they regulate gene expression. Here, we investigate a lncRNA-mediated targeting of DNMT1 to specific gene loci by the combined power of in silico modeling of RNA/DNA interactions and wet lab approaches, in the context of the clinically relevant use case of ephrinA5-dependent regulation of cellular motility of cerebellar granule cells. We provide evidence that Snhg15, a cancer-related lncRNA, recruits DNMT1 to the Ncam1 promoter through RNA/DNA triplex structure formation and the interaction with DNMT1. This mediates DNA methylation-dependent silencing of Ncam1, being abolished by ephrinA5 stimulation-triggered reduction of Snhg15 expression. Hence, we here propose a triple helix recognition mechanism, underlying cell motility regulation via lncRNA-targeted DNA methylation in a clinically relevant context. [ABSTRACT FROM AUTHOR]

Summary The increasing availability of single-cell multi-omics data allows to quantitatively characterize gene regulation. We here describe scMEGA (Single-cell Multiomic Enhancer-based Gene Regulatory Network Inference) that enables an end-to-end analysis of multi-omics data for gene regulatory network inference including modalities integration, trajectory analysis, enhancer-to-promoter association, network analysis and visualization. This enables to study the complex gene regulation mechanisms for dynamic biological processes, such as cellular differentiation and disease-driven cellular remodeling. We provide a case study on gene regulatory networks controlling myofibroblast activation in human myocardial infarction. Availability and implementation scMEGA is implemented in R, released under the MIT license and available from https://github.com/CostaLab/scMEGA. Tutorials are available from https://costalab.github.io/scMEGA. Supplementary information Supplementary data are available at Bioinformatics Advances online. [ABSTRACT FROM AUTHOR]

Transcription factors play a determining role in lineage commitment and cell differentiation. Interferon regulatory factor 8 (IRF8) is a lineage determining transcription factor in hematopoiesis and master regulator of dendritic cells (DC), an important immune cell for immunity and tolerance. IRF8 is prominently upregulated in DC development by autoactivation and controls both DC differentiation and function. However, it is unclear how Irf8 autoactivation is controlled and eventually limited. Here, we identified a novel long non-coding RNA transcribed from the +32 kb enhancer downstream of Irf8 transcription start site and expressed specifically in mouse plasmacytoid DC (pDC), referred to as lncIrf8. The lncIrf8 locus interacts with the lrf8 promoter and shows differential epigenetic signatures in pDC versus classical DC type 1 (cDC1). Interestingly, a sequence element of the lncIrf8 promoter, but not lncIrf8 itself, is crucial for mouse pDC and cDC1 differentiation, and this sequence element confers feedback inhibition of Irf8 expression. Taken together, in DC development Irf8 autoactivation is first initiated by flanking enhancers and then second controlled by feedback inhibition through the lncIrf8 promoter element in the +32 kb enhancer. Our work reveals a previously unrecognized negative feedback loop of Irf8 that orchestrates its own expression and thereby controls DC differentiation. [ABSTRACT FROM AUTHOR]

Mast cells (MCs) represent a population of hematopoietic cells with a key role in innate and adaptive immunity and are well known for their detrimental role in allergic responses. Yet, MCs occur in low abundance, which hampers their detailed molecular analysis. Here, we capitalized on the potential of induced pluripotent stem (iPS) cells to give rise to all cells in the body and established a novel and robust protocol for human iPS cell differentiation toward MCs. Relying on a panel of systemic mastocytosis (SM) patient-specific iPS cell lines carrying the KIT D816V mutation, we generated functional MCs that recapitulate SM disease features: increased number of MCs, abnormal maturation kinetics and activated phenotype, CD25 and CD30 surface expression and a transcriptional signature characterized by upregulated expression of innate and inflammatory response genes. Therefore, human iPS cell-derived MCs are a reliable, inexhaustible, and close-to-human tool for disease modeling and pharmacological screening to explore novel MC therapeutics. [ABSTRACT FROM AUTHOR]

Background: Massive amounts of data are produced by combining next-generation sequencing with complex biochemistry techniques to characterize regulatory genomics profiles, such as protein–DNA interaction and chromatin accessibility. Interpretation of such high-throughput data typically requires different computation methods. However, existing tools are usually developed for a specific task, which makes it challenging to analyze the data in an integrative manner. Results: We here describe the Regulatory Genomics Toolbox (RGT), a computational library for the integrative analysis of regulatory genomics data. RGT provides different functionalities to handle genomic signals and regions. Based on that, we developed several tools to perform distinct downstream analyses, including the prediction of transcription factor binding sites using ATAC-seq data, identification of differential peaks from ChIP-seq data, and detection of triple helix mediated RNA and DNA interactions, visualization, and finding an association between distinct regulatory factors. Conclusion: We present here RGT; a framework to facilitate the customization of computational methods to analyze genomic data for specific regulatory genomics problems. RGT is a comprehensive and flexible Python package for analyzing high throughput regulatory genomics data and is available at: https://github.com/CostaLab/reg-gen. The documentation is available at: https://reg-gen.readthedocs.io [ABSTRACT FROM AUTHOR]

Changes in gene expression programs are intimately linked to cell fate decisions. Post-translational modifications of core histones contribute to control gene expression. Methylation of lysine 4 of histone H3 (H3K4) correlates with active promoters and gene transcription. This modification is catalyzed by KMT2 methyltransferases, which require interaction with 4 core subunits, WDR5, RBBP5, ASH2L and DPY30, for catalytic activity. Ash2l is necessary for organismal development and for tissue homeostasis. In mouse embryo fibroblasts (MEFs), Ash2l loss results in gene repression, provoking a senescence phenotype. We now find that upon knockout of Ash2l both H3K4 mono- and tri-methylation (H3K4me1 and me3, respectively) were deregulated. In particular, loss of H3K4me3 at promoters correlated with gene repression, especially at CpG island promoters. Ash2l loss resulted in increased loading of histone H3 and reduced chromatin accessibility at promoters, accompanied by an increase of repressing and a decrease of activating histone marks. Moreover, we observed altered binding of CTCF upon Ash2l loss. Lost and gained binding was noticed at promoter-associated and intergenic sites, respectively. Thus, Ash2l loss and reduction of H3K4me3 correlate with altered chromatin accessibility and transcription factor binding. These findings contribute to a more detailed understanding of mechanistic consequences of H3K4me3 loss and associated repression of gene transcription and thus of the observed cellular consequences. [ABSTRACT FROM AUTHOR]

Background: Single-cell RNA sequencing (scRNA-seq) allows the detection of rare cell types in complex tissues. The detection of markers for rare cell types is useful for further biological analysis of, for example, flow cytometry and imaging data sets for either physical isolation or spatial characterization of these cells. However, only a few computational approaches consider the problem of selecting specific marker genes from scRNA-seq data. Results: Here, we propose sc2marker, which is based on the maximum margin index and a database of proteins with antibodies, to select markers for flow cytometry or imaging. We evaluated the performances of sc2marker and competing methods in ranking known markers in scRNA-seq data of immune and stromal cells. The results showed that sc2marker performed better than the competing methods in accuracy, while having a competitive running time. [ABSTRACT FROM AUTHOR]

• Cultured BM stromal cells are distinct from in vivo mesenchymal stromal cells. • Cultured BM stromal cells are transcriptionally similar to in vivo fibroblasts. • Cultured BM stromal cells retain plasticity ex vivo. • Cultured BM stromal cells have hematopoiesis-support capacity, increased through TGFb inhibition. Within the heterogenous pool of bone marrow stromal cells, mesenchymal stromal cells (MSCs) are of particular interest because of their hematopoiesis-supporting capacities, contribution to disease progression, therapy resistance, and leukemic initiation. Cultured bone marrow-derived stromal cells (cBMSCs) are used for in vitro modeling of hematopoiesis–stroma interactions, validation of disease mechanisms, and screening for therapeutic targets. Here, we place cBMSCs (mouse and human) in a bone marrow tissue context by systematically comparing the transcriptome of plastic-adherent cells on a single-cell level with in vivo counterparts. Cultured BMSCs encompass a rather homogenous cell population, independent of the isolation method used and, although still possessing hematopoiesis-supporting capacity, are distinct from freshly isolated MSCs and more akin to in vivo fibroblast populations. Informed by combined cell trajectories and pathway analyses, we illustrate that TGFb inhibition in vitro can preserve a more "MSC"-like phenotype [ABSTRACT FROM AUTHOR]

Genomic and epigenomic studies revealed dysregulation of long non-coding RNAs in many cancer entities, including liver cancer. We identified an epigenetic mechanism leading to upregulation of the long intergenic non-coding RNA 152 (LINC00152) expression in human hepatocellular carcinoma (HCC). Here, we aimed to characterize a potential competing endogenous RNA (ceRNA) network, in which LINC00152 exerts oncogenic functions by sponging miRNAs, thereby affecting their target gene expression. Database and gene expression data of human HCC were integrated to develop a potential LINC00152-driven ceRNA in silico. RNA immunoprecipitation and luciferase assay were used to identify miRNA binding to LINC00152 in human HCC cells. Functionally active players in the ceRNA network were analyzed using gene editing, siRNA or miRNA mimic transfection, and expression vectors in vitro. RNA expression in human HCC in vivo was validated by RNA in situ hybridization. Let-7c-5p, miR-23a-3p, miR-125a-5p, miR-125b-5p, miR-143a-3p, miR-193-3p, and miR-195-5p were detected as new components of the potential LINC00152 ceRNA network in human HCC. LINC00152 was confirmed to sponge miR143a-3p in human HCC cell lines, thereby limiting its binding to their respective target genes, like KLC2. KLC2 was identified as a central mediator promoting pro-tumorigenic effects of LINC00152 overexpression in HCC cells. Furthermore, co-expression of LINC00152 and KLC2 was observed in human HCC cohorts and high KLC2 expression was associated with shorter patient survival. Functional assays demonstrated that KLC2 promoted cell proliferation, clonogenicity and migration in vitro. The LINC00152-miR-143a-3p-KLC2 axis may represent a therapeutic target in human HCC. [ABSTRACT FROM AUTHOR]

Nephrotic syndrome (NS) is characterized by severe proteinuria as a consequence of kidney glomerular injury due to podocyte damage. In vitro models mimicking in vivo podocyte characteristics are a prerequisite to resolve NS pathogenesis. The detailed characterization of organoid podocytes resulting from a hybrid culture protocol showed a podocyte population that resembles adult podocytes and was superior compared with 2D counterparts, based on single-cell RNA sequencing, super-resolution imaging and electron microscopy. In this study, these next-generation podocytes in kidney organoids enabled personalized idiopathic nephrotic syndrome modeling, as shown by activated slit diaphragm signaling and podocyte injury following protamine sulfate, puromycin aminonucleoside treatment and exposure to NS plasma containing pathogenic permeability factors. Organoids cultured from cells of a patient with heterozygous NPHS2 mutations showed poor NPHS2 expression and aberrant NPHS1 localization, which was reversible after genetic correction. Repaired organoids displayed increased VEGFA pathway activity and transcription factor activity known to be essential for podocyte physiology, as shown by RNA sequencing. This study shows that organoids are the preferred model of choice to study idiopathic and congenital podocytopathies. [ABSTRACT FROM AUTHOR]

Cell type specification during pancreatic development is tightly controlled by a transcriptional and epigenetic network. The precise role of most transcription factors, however, has been only described in mice. To convey such concepts to human pancreatic development, alternative model systems such as pancreatic in vitro differentiation of human pluripotent stem cells can be employed. Here, we analyzed stage-specific RNA-, ChIP-, and ATAC-sequencing data to dissect transcriptional and regulatory mechanisms during pancreatic development. Transcriptome and open chromatin maps of pancreatic differentiation from human pluripotent stem cells provide a stage-specific pattern of known pancreatic transcription factors and indicate ONECUT1 as a crucial fate regulator in pancreas progenitors. Moreover, our data suggest that ONECUT1 is also involved in preparing pancreatic progenitors for later endocrine specification. The dissection of the transcriptional and regulatory circuitry revealed an important role for ONECUT1 within such network and will serve as resource to study human development and disease. Heller et al analyze transcriptomic and epigenetic datasets from human PSCs differentiating into pancreatic progenitors to elucidate the regulatory mechanisms behind pancreatic development. The authors report that the transcription factor ONECUT1 is expressed specifically in pancreatic development and associates with other key TFs (such as FOXA2, GATA6, PDX1) during endocrine specification. [ABSTRACT FROM AUTHOR]

Abstract Chronic inflammation in the liver provokes fibrosis and, on long-term, carcinogenesis. This sequence is prototypically recapitulated in mice with hepatocyte-specific knock-out of the NF-κB essential modulator (NEMO), termed NEMOLPC-KO mice, in which increased hepatocyte apoptosis and compensatory regeneration cause steatosis, inflammation and fibrosis. Natural killer T (NKT) cells carrying the chemokine receptor CXCR6 participate in liver inflammation and injury responses. Here, we investigated the role of CXCR6 in the NEMOLPC-KO mouse model. Unexpectedly, genetic deletion of CXCR6 enhanced hepatocyte death, inflammation and fibrosis in NEMOLPC-KO mice. Although CXCR6 expression is restricted to immune cells in the liver, the adoptive transfer of CXCR6+ cells did not protect NEMOLPC-KO Cxcr6 −/− mice from hepatic injury. Gene array analyses revealed up-regulated stress response and metabolism pathways in hepatocytes from NEMOLPC-KO Cxcr6 −/− mice, functionally corresponding to an increased susceptibility of these hepatocytes to TNFα-induced cell death in vitro. These data revealed a novel CXCR6-dependent mechanism of suppressing inflammatory hepatocytic responses to cellular stress. Highlights • NEMOLPC-KO Cxcr6 −/− mice display enhanced liver damage and fibrosis compared to NEMOLPC-KO mice. • NEMOLPC-KO Cxcr6 −/− mice are hallmarked by a pro-inflammatory and pro-apoptotic liver environment. • Stress response and metabolism pathways are upregulated in hepatocytes from NEMOLPC-KO Cxcr6 −/− mice. • CXCR6 protects hepatocytes from TNFα-induced hepatotoxicity. [ABSTRACT FROM AUTHOR]

E-type cyclins E1 (CcnEI) and E2 (CcnE2) are regulatory subunits of cyclin-dependent kinase 2 (Cdk2) and thought to control the transition of quiescent cells into the cell cycle. Initial findings indicated that CcnEI and CcnE2 have largely overlapping functions for cancer development in several tumor entities including hepatocellular carcinoma (HCC). In the present study, we dissected the differential contributions of CcnEI, CcnE2, and Cdk2 for initiation and progression of HCC in mice and patients. To this end, we tested the HCC susceptibility in mice with constitutive deficiency for CcnEI or CcnE2 as well as in mice lacking Cdk2 in hepatocytes. Genetic inactivation of CcnEI largely prevented development of liver cancer in mice in two established HCC models, while ablation of CcnE2 had no effect on hepatocarcinogenesis. Importantly, CcnEI-driven HCC initiation was dependent on Cdk2. However, isolated primary hepatoma cells typically acquired independence on CcnEI and Cdk2 with increasing progression in vitro, which was associated with a gene signature involving secondary induction of CcnE2 and up-regulation of cell cycle and DNA repair pathways. Importantly, a similar expression profile was also found in HCC patients with elevated CcnE2 expression and poor survival. In general, overall survival in HCC patients was synergistically affected by expression of CcnEI and CcnE2, but not through Cdk2. Our study suggests that HCC initiation specifically depends on CcnEI and Cdk2, while HCC progression requires expression of any E-cyclin, but no Cdk2. [ABSTRACT FROM AUTHOR]

Colonies of induced pluripotent stem cells (iPSCs) reveal aspects of self-organization even under culture conditions that maintain pluripotency. To investigate the dynamics of this process under spatial confinement, we used either polydimethylsiloxane (PDMS) pillars or micro-contact printing of vitronectin. There was a progressive upregulation of OCT4, E-cadherin, and NANOG within 70 μm from the outer rim of iPSC colonies. Single-cell RNA-sequencing and spatial reconstruction of gene expression demonstrated that OCT4 high subsets, residing at the edge of the colony, have pronounced up-regulation of the TGF-β pathway, particularly of NODAL and its inhibitor LEFTY. Interestingly, after 5–7 days, iPSC colonies detached spontaneously from micro-contact printed substrates to form 3D aggregates. This new method allowed generation of embryoid bodies (EBs) of controlled size without enzymatic or mechanical treatment. Within the early 3D aggregates, radial organization and differential gene expression continued in analogy to the changes observed during self-organization of iPSC colonies. Early self-detached aggregates revealed up-regulated germline-specific gene expression patterns as compared to conventional EBs. However, there were no marked differences after further directed differentiation toward hematopoietic, mesenchymal, and neuronal lineages. Our results provide further insight into the gradual self-organization within iPSC colonies and at their transition into EBs. [ABSTRACT FROM AUTHOR]

Simple Summary: The cell cycle regulator Cyclin E1 is a key mediator and biomarker of liver cancer progression in mice and man independent of its canonical interacting partner Cyclin-dependent kinase 2. Over-expression of Cyclin E1 during hepatocarcinogenesis modulates several distinct biological processes such as proliferation, DNA damage response, stemness, invasion and the tumour microenvironment. Interventional depletion of Cyclin E1 in the course of liver cancer progression significantly reduces tumour burden. In contrast, the expression of Cyclin-dependent kinase 2 is dispensable for the progression of liver cancer in mice and lacked diagnostic or prognostic value in patients. Thus, specific inhibition of Cyclin E1 expression represents a promising strategy for the treatment of liver cancer. Cyclin E1 (CCNE1) is a regulatory subunit of Cyclin-dependent kinase 2 (CDK2) and is thought to control the transition of quiescent cells into cell cycle progression. Recently, we identified CCNE1 and CDK2 as key factors for the initiation of hepatocellular carcinoma (HCC). In the present study, we dissected the contributions of CCNE1 and CDK2 for HCC progression in mice and patients. Therefore, we generated genetically modified mice allowing inducible deletion of Ccne1 or Cdk2. After initiation of HCC, using the hepatocarcinogen diethylnitrosamine (DEN), we deleted Ccne1 or Cdk2 and subsequently analysed HCC progression. The relevance of CCNE1 or CDK2 for human HCC progression was investigated by in silico database analysis. Interventional deletion of Ccne1, but not of Cdk2, substantially reduced the HCC burden in mice. Ccne1-deficient HCCs were characterised by attenuated proliferation, impaired DNA damage response and downregulation of markers for stemness and microinvasion. Additionally, the tumour microenvironment of Ccne1-deficient mice showed a reduction in immune mediators, myeloid cells and cancer-associated fibroblasts. In sharp contrast, Cdk2 was dispensable for HCC progression in mice. In agreement with our mouse data, CCNE1 was overexpressed in HCC patients independent of risk factors, and associated with reduced disease-free survival, a common signature for enhanced chromosomal instability, proliferation, dedifferentiation and invasion. However, CDK2 lacked diagnostic or prognostic value in HCC patients. In summary, CCNE1 drives HCC progression in a CDK2-independent manner in mice and man. Therefore, interventional inactivation of CCNE1 represents a promising strategy the treatment of liver cancer. [ABSTRACT FROM AUTHOR]

Key aspects of intestinal T cells, including their antigen specificity and their selection by the microbiota and other intestinal antigens, as well as the contribution of individual T cell clones to regulatory and effector functions, remain unresolved. Here we tracked adoptively transferred T cell populations to specify the interrelation of T cell receptor repertoire and the gut antigenic environment. We show that dominant TCRα clonotypes were shared between interferon-γ- and interleukin-17-producing but not regulatory Foxp3+ T cells. Identical TCRα clonotypes accumulated in the colon of different individuals, whereas antibiotics or defined colonization correlated with the expansion of distinct expanded T cell clonotypes. Our results demonstrate key aspects of intestinal CD4+ T cell activation and suggest that few microbial species exert a dominant effect on the intestinal T cell repertoire during colitis. We speculate that dominant proinflammatory T cell clones might provide a therapeutic target in human inflammatory bowel disease. [Display omitted] • During colitis, the regulatory and effector T cell repertoire are dissimilar • In vitro expansion generates colitogenic T cells of similar clonality • Recipients transferred with identical donor T cells show similar TCRα repertoires • Discrete microbial species exert dominant effects on the intestinal T cell repertoire Microbiota-reactive intestinal T cells have been reported but the breadth of antigens shaping the gut T population is unknown. Muschaweck et al. combined TCR repertoire sequencing and adoptive transfer of in vitro expanded T cells to show that microbiota-driven expansion of CD4+ T cells dominates in the inflamed colon. [ABSTRACT FROM AUTHOR]

Background: The complex composition of different cell types within a tissue can be estimated by deconvolution of bulk gene expression profiles or with various single-cell sequencing approaches. Alternatively, DNA methylation (DNAm) profiles have been used to establish an atlas for multiple human tissues and cell types. DNAm is particularly suitable for deconvolution of cell types because each CG dinucleotide (CpG site) has only two states per DNA strand—methylated or non-methylated—and these epigenetic modifications are very consistent during cellular differentiation. So far, deconvolution of DNAm profiles implies complex signatures of many CpGs that are often measured by genome-wide analysis with Illumina BeadChip microarrays. In this study, we investigated if the characterization of cell types in tissue is also feasible with individual cell type-specific CpG sites, which can be addressed by targeted analysis, such as pyrosequencing. Results: We compiled and curated 579 Illumina 450k BeadChip DNAm profiles of 14 different non-malignant human cell types. A training and validation strategy was applied to identify and test for cell type-specific CpGs. We initially focused on estimating the relative amount of fibroblasts using two CpGs that were either hypermethylated or hypomethylated in fibroblasts. The combination of these two DNAm levels into a "FibroScore" correlated with the state of fibrosis and was associated with overall survival in various types of cancer. Furthermore, we identified hypomethylated CpGs for leukocytes, endothelial cells, epithelial cells, hepatocytes, glia, neurons, fibroblasts, and induced pluripotent stem cells. The accuracy of this eight CpG signature was tested in additional BeadChip datasets of defined cell mixtures and the results were comparable to previously published signatures based on several thousand CpGs. Finally, we established and validated pyrosequencing assays for the relevant CpGs that can be utilized for classification and deconvolution of cell types. Conclusion: This proof of concept study demonstrates that DNAm analysis at individual CpGs reflects the cellular composition of cellular mixtures and different tissues. Targeted analysis of these genomic regions facilitates robust methods for application in basic research and clinical settings. [ABSTRACT FROM AUTHOR]

Tubular injury is the hallmark of acute kidney injury (AKI) with a tremendous impact on patients and health-care systems. During injury, any differentiated proximal tubular cell (PT) may transition into a specific injured phenotype, so-called "scattered tubular cell" (STC)-phenotype. To understand the fate of this specific phenotype, we generated transgenic mice allowing inducible, reversible, and irreversible tagging of these cells in a murine AKI model, the unilateral ischemia-reperfusion injury (IRI). For lineage tracing, we analyzed the kidneys using single-cell profiling during disease development at various time points. Labeled cells, which we defined by established endogenous markers, already appeared 8 h after injury and showed a distinct expression set of genes. We show that STCs re-differentiate back into fully differentiated PTs upon the resolution of the injury. In summary, we show the dynamics of the phenotypic transition of PTs during injury, revealing a reversible transcriptional program as an adaptive response during disease., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

The myeloproliferative disease polycythemia vera (PV) driven by the JAK2 V617F mutation can transform into myelofibrosis (post-PV-MF). It remains an open question how JAK2 V617F in hematopoietic stem cells induces MF. Megakaryocytes are major players in murine PV models but are difficult to study in the human setting. We generated induced pluripotent stem cells (iPSCs) from JAK2 V617F PV patients and differentiated them into megakaryocytes. In differentiation assays, JAK2 V617F iPSCs recapitulated the pathognomonic skewed megakaryocytic and erythroid differentiation. JAK2 V617F iPSCs had a TPO-independent and increased propensity to differentiate into megakaryocytes. RNA sequencing of JAK2 V617F iPSC-derived megakaryocytes reflected a proinflammatory, profibrotic phenotype and decreased ribosome biogenesis. In three-dimensional (3D) coculture, JAK2 V617F megakaryocytes induced a profibrotic phenotype through direct cell contact, which was reversed by the JAK2 inhibitor ruxolitinib. The 3D coculture system opens the perspective for further disease modeling and drug discovery., Competing Interests: Declaration of interests W.W. and V.T. are involved in Cygenia GmbH (www.cygenia.com), which provides services for DNA methylation analysis to other scientists. S.K. reports funding from Novartis, Bristol-Myers Squibb, and Janssen/Geron; advisory board honoraria from Pfizer, Incyte, Ariad, Novartis, AOP Pharma, BMS, Celgene, Geron, Janssen, CTI, Roche, Baxalta, GSK, Sierra Oncology, and Sanofi; a patent for Bromodomain and Extra-Terminal (BET) inhibitor at RWTH Aachen University; honoraria from Novartis, Bristol Myers Squibb, Celgene, Geron, Janssen, Pfizer, Incyte, Ariad, Shire, Roche, and AOP Pharma; and other financial support (e.g., travel support) from Alexion, Novartis, Bristol Myers Squibb, Incyte, Ariad, AOP Pharma, Baxalta, CTI, Pfizer, Sanofi, Celgene, Shire, Janssen, Geron, Abbvie, Imago Biosciences, Sierra Oncology, GSK, and Karthos. T.H.B. served as a consultant for Janssen, Merck, Novartis, and Pfizer; received research funding form Novartis and Pfizer; and received honoraria from Janssen, Merck, Novartis, and Pfizer., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The role of hematopoietic Hedgehog signaling in myeloproliferative neoplasms (MPNs) remains incompletely understood despite data suggesting that Hedgehog (Hh) pathway inhibitors have therapeutic activity in patients. We aim to systematically interrogate the role of canonical vs. non-canonical Hh signaling in MPNs. We show that Gli1 protein levels in patient peripheral blood mononuclear cells (PBMCs) mark fibrotic progression and that, in murine MPN models, absence of hematopoietic Gli1, but not Gli2 or Smo, significantly reduces MPN phenotype and fibrosis, indicating that GLI1 in the MPN clone can be activated in a non-canonical fashion. Additionally, we establish that hematopoietic Gli1 has a significant effect on stromal cells, mediated through a druggable MIF-CD74 axis. These data highlight the complex interplay between alterations in the MPN clone and activation of stromal cells and indicate that Gli1 represents a promising therapeutic target in MPNs, particularly that Hh signaling is dispensable for normal hematopoiesis., Competing Interests: Declaration of interests N.B. is employed by Prelude Therapeutics. S.K. received research grants from Geron, Janssen, AOP Pharma, and Novartis; received consulting fees from Pfizer, Incyte, Ariad, Novartis, AOP Pharma, Bristol Myers Squibb, Celgene, Geron, Janssen, CTI BioPharma, Roche, Bayer, PharmaEssentia, Sierra Oncology, Imago Biosciences, and GSK; payment or honoraria from Novartis, BMS/Celgene, and Pfizer; received travel/accommodation support from Alexion, Novartis, Bristol Myers Squibb, Incyte, AOP Pharma, CTI BioPharma, Pfizer, Celgene, Janssen, Geron, Roche, AbbVie, Sierra Oncology, and Karthos; and participated on advisory boards for Pfizer, Incyte, Ariad, Novartis, AOP Pharma, BMS, Celgene, Geron, Janssen, CTI BioPharma, Roche, Bayer, Sierra Oncology, PharmaEssentia, Imago Biosciences, and GSK. R.L.L. is on the supervisory board of QIAGEN and is a scientific advisor to Imago, Mission Bio, Zentalis, Ajax, Auron, Prelude, C4 Therapeutics, and Isoplexis; receives research support from Ajax, Zentalis, and Abbvie; consulted for Incyte, Janssen, and Astra Zeneca; and received honoraria from Astra Zeneca for invited lectures. R.K. has grants from Travere Therapeutics, Galapagos, Chugai, and Novo Nordisk and is a consultant for Bayer, Pfizer, Novo Nordisk, and Gruenenthal. I.G.C. has a grant from Illumina. R.K. and R.K.S. are founders and shareholders of Sequantrix GmbH. N.F. is employed by Sequantrix GmbH., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Progressive respiratory failure is the primary cause of death in the coronavirus disease 2019 (COVID-19) pandemic. It is the final outcome of the acute respiratory distress syndrome (ARDS), characterized by an initial exacerbated inflammatory response, metabolic derangement and ultimate tissue scarring. A positive balance of cellular energy may result crucial for the recovery of clinical COVID-19. Hence, we asked if two key pathways involved in cellular energy generation, AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) signaling and fatty acid oxidation (FAO) could be beneficial. We tested the drugs metformin (AMPK activator) and baicalin (CPT1A activator) in different experimental models mimicking COVID-19 associated inflammation in lung and kidney. We also studied two different cohorts of COVID-19 patients that had been previously treated with metformin. These drugs ameliorated lung damage in an ARDS animal model, while activation of AMPK/ACC signaling increased mitochondrial function and decreased TGF-β-induced fibrosis, apoptosis and inflammation markers in lung epithelial cells. Similar results were observed with two indole derivatives, IND6 and IND8 with AMPK activating capacity. Consistently, a reduced time of hospitalization and need of intensive care was observed in COVID-19 patients previously exposed to metformin. Baicalin also mitigated the activation of pro-inflammatory bone marrow-derived macrophages (BMDMs) and reduced kidney fibrosis in two animal models of kidney injury, another key target of COVID-19. In human epithelial lung and kidney cells, both drugs improved mitochondrial function and prevented TGF-β-induced renal epithelial cell dedifferentiation. Our results support that favoring cellular energy production through enhanced FAO may prove useful in the prevention of COVID-19-induced lung and renal damage., Competing Interests: Declaration of competing interest The authors have no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Cell-type specific DNA methylation (DNAm) can be employed to determine the numbers of leukocyte subsets in blood. In contrast to conventional methods for leukocyte counts, which are based on cellular morphology or surface marker protein expression, the cellular deconvolution based on DNAm levels is applicable for frozen or dried blood. Here, we further enhanced targeted DNAm assays for leukocyte counts in clinical application., Methods: DNAm profiles of 40 different studies were compiled to identify CG dinucleotides (CpGs) with cell-type specific DNAm using a computational framework, CimpleG. DNAm levels at these CpGs were then measured with digital droplet PCR in venous blood from 160 healthy donors and 150 patients with various hematological disorders. Deconvolution was further validated with venous blood (n = 75) and capillary blood (n = 31) that was dried on Whatman paper or on Mitra microsampling devices., Results: In venous blood, automated cell counting or flow cytometry correlated well with epigenetic estimates of relative leukocyte counts for granulocytes (r = 0.95), lymphocytes (r = 0.97), monocytes (r = 0.82), CD4 T cells (r = 0.84), CD8 T cells (r = 0.94), B cells (r = 0.96), and NK cells (r = 0.72). Similar correlations and precisions were achieved for dried blood samples. Spike-in with a reference plasmid enabled accurate epigenetic estimation of absolute leukocyte counts from dried blood samples, correlating with conventional venous (r = 0.86) and capillary (r = 0.80) blood measurements., Conclusions: The advanced selection of cell-type specific CpGs and utilization of digital droplet PCR analysis provided accurate epigenetic blood counts. Analysis of dried blood facilitates self-sampling with a finger prick, thereby enabling easier accessibility to testing., (© Association for Diagnostics & Laboratory Medicine 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

It is currently not well known how necroptosis and necroptosis responses manifest in vivo. Here, we uncovered a molecular switch facilitating reprogramming between two alternative modes of necroptosis signaling in hepatocytes, fundamentally affecting immune responses and hepatocarcinogenesis. Concomitant necrosome and NF-κB activation in hepatocytes, which physiologically express low concentrations of receptor-interacting kinase 3 (RIPK3), did not lead to immediate cell death but forced them into a prolonged "sublethal" state with leaky membranes, functioning as secretory cells that released specific chemokines including CCL20 and MCP-1. This triggered hepatic cell proliferation as well as activation of procarcinogenic monocyte-derived macrophage cell clusters, contributing to hepatocarcinogenesis. In contrast, necrosome activation in hepatocytes with inactive NF-κB-signaling caused an accelerated execution of necroptosis, limiting alarmin release, and thereby preventing inflammation and hepatocarcinogenesis. Consistently, intratumoral NF-κB-necroptosis signatures were associated with poor prognosis in human hepatocarcinogenesis. Therefore, pharmacological reprogramming between these distinct forms of necroptosis may represent a promising strategy against hepatocellular carcinoma., Competing Interests: Declaration of interests A.L.S. and J.M.M. contribute to a project developing necroptosis inhibitors in collaboration with Anaxis Pharma., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The management of patients with chronic myeloid leukemia (CML) has been revolutionized by the introduction of tyrosine kinase inhibitors (TKIs), which induce deep molecular responses so that treatment can eventually be discontinued, leading to treatment-free remission (TFR) in a subset of patients. Unfortunately, leukemic stem cells (LSCs) often persist and a fraction of these can again expand in about half of patients that attempt TKI discontinuation. In this study, we show that presence of myelofibrosis (MF) at the time of diagnosis is a factor associating with TFR failure. Fibrotic transformation is governed by the action of several cytokines, and interestingly, some of them have also been described to support LSC persistence. At the cellular level, these could be produced by both malignant cells and by components of the bone marrow (BM) niche, including megakaryocytes (MKs) and mesenchymal stromal cells (MSCs). In our cohort of 57 patients, around 40% presented with MF at diagnosis and the number of blasts in the peripheral blood and BM was significantly elevated in patients with higher grade of MF. Employing a CML transgenic mouse model, we could observe higher levels of alpha-smooth muscle actin (α-SMA) in the BM when compared to control mice. Short-term treatment with the TKI nilotinib, efficiently reduced spleen weight and BCR::ABL1 mRNA levels, while α-SMA expression was only partially reduced. Interestingly, the number of MKs was increased in the spleen of CML mice and elevated in both BM and spleen upon nilotinib treatment. Analysis of human CML-vs healthy donor (HD)-derived MSCs showed an altered expression of gene signatures reflecting fibrosis as well as hematopoietic support, thus suggesting MSCs as a potential player in these two processes. Finally, in our cohort, 12 patients qualified for TKI discontinuation, and here we observed that all patients who failed TFR had BM fibrosis at diagnosis, whereas this was only the case in 25% of patients with achieved TFR, further supporting the link between fibrosis and LSC persistence., Competing Interests: TB served as a consultant or speaker at satellite symposia for AstraZeneca, Janssen, Merck, Novartis and Pfizer and received research funding form Novartis and Pfizer. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jacobi, Vieri, Bütow, Namasu, Flüter, Costa, Maié, Lindemann-Docter, Chatain, Beier, Huber, Wagner, Crysandt, Brümmendorf and Schemionek.)

Transcription factors play a determining role in lineage commitment and cell differentiation. Interferon regulatory factor 8 (IRF8) is a lineage determining transcription factor in hematopoiesis and master regulator of dendritic cells (DC), an important immune cell for immunity and tolerance. IRF8 is prominently upregulated in DC development by autoactivation and controls both DC differentiation and function. However, it is unclear how Irf8 autoactivation is controlled and eventually limited. Here, we identified a novel long non-coding RNA transcribed from the +32 kb enhancer downstream of Irf8 transcription start site and expressed specifically in mouse plasmacytoid DC (pDC), referred to as lncIrf8 . The lncIrf8 locus interacts with the lrf8 promoter and shows differential epigenetic signatures in pDC versus classical DC type 1 (cDC1). Interestingly, a sequence element of the lncIrf8 promoter, but not lncIrf8 itself, is crucial for mouse pDC and cDC1 differentiation, and this sequence element confers feedback inhibition of Irf8 expression. Taken together, in DC development Irf8 autoactivation is first initiated by flanking enhancers and then second controlled by feedback inhibition through the lncIrf8 promoter element in the +32 kb enhancer. Our work reveals a previously unrecognized negative feedback loop of Irf8 that orchestrates its own expression and thereby controls DC differentiation., Competing Interests: HX, ZL, CK, KG, TL, Md, KS, IC, MZ No competing interests declared, (© 2023, Xu et al.)

Pathology diagnostics relies on the assessment of morphology by trained experts, which remains subjective and qualitative. Here we developed a framework for large-scale histomorphometry (FLASH) performing deep learning-based semantic segmentation and subsequent large-scale extraction of interpretable, quantitative, morphometric features in non-tumour kidney histology. We use two internal and three external, multi-centre cohorts to analyse over 1000 kidney biopsies and nephrectomies. By associating morphometric features with clinical parameters, we confirm previous concepts and reveal unexpected relations. We show that the extracted features are independent predictors of long-term clinical outcomes in IgA-nephropathy. We introduce single-structure morphometric analysis by applying techniques from single-cell transcriptomics, identifying distinct glomerular populations and morphometric phenotypes along a trajectory of disease progression. Our study provides a concept for Next-generation Morphometry (NGM), enabling comprehensive quantitative pathology data mining, i.e., pathomics., (© 2023. The Author(s).)

DNA:DNA:RNA triplexes that are formed through Hoogsteen base-pairing of the RNA in the major groove of the DNA duplex have been observed in vitro, but the extent to which these interactions occur in cells and how they impact cellular functions remains elusive. Using a combination of bioinformatic techniques, RNA/DNA pulldown and biophysical studies, we set out to identify functionally important DNA:DNA:RNA triplex-forming long non-coding RNAs (lncRNA) in human endothelial cells. The lncRNA HIF1α-AS1 was retrieved as a top hit. Endogenous HIF1α-AS1 reduces the expression of numerous genes, including EPH Receptor A2 and Adrenomedullin through DNA:DNA:RNA triplex formation by acting as an adapter for the repressive human silencing hub complex (HUSH). Moreover, the oxygen-sensitive HIF1α-AS1 is down-regulated in pulmonary hypertension and loss-of-function approaches not only result in gene de-repression but also enhance angiogenic capacity. As exemplified here with HIF1α-AS1, DNA:DNA:RNA triplex formation is a functionally important mechanism of trans-acting gene expression control., (© 2022. The Author(s).)

Gene expression is controlled in part by post-translational modifications of core histones. Methylation of lysine 4 of histone H3 (H3K4), associated with open chromatin and gene transcription, is catalyzed by type 2 lysine methyltransferase complexes that require WDR5, RBBP5, ASH2L and DPY30 as core subunits. Ash2l is essential during embryogenesis and for maintaining adult tissues. To expand on the mechanistic understanding of Ash2l, we generated mouse embryo fibroblasts (MEFs) with conditional Ash2l alleles. Upon loss of Ash2l, methylation of H3K4 and gene expression were downregulated, which correlated with inhibition of proliferation and cell cycle progression. Moreover, we observed induction of senescence concomitant with a set of downregulated signature genes but independent of SASP. Many of the signature genes are FoxM1 responsive. Indeed, exogenous FOXM1 was sufficient to delay senescence. Thus, although the loss of Ash2l in MEFs has broad and complex consequences, a distinct set of downregulated genes promotes senescence., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Myocardial infarction is a leading cause of death worldwide 1 . Although advances have been made in acute treatment, an incomplete understanding of remodelling processes has limited the effectiveness of therapies to reduce late-stage mortality 2 . Here we generate an integrative high-resolution map of human cardiac remodelling after myocardial infarction using single-cell gene expression, chromatin accessibility and spatial transcriptomic profiling of multiple physiological zones at distinct time points in myocardium from patients with myocardial infarction and controls. Multi-modal data integration enabled us to evaluate cardiac cell-type compositions at increased resolution, yielding insights into changes of the cardiac transcriptome and epigenome through the identification of distinct tissue structures of injury, repair and remodelling. We identified and validated disease-specific cardiac cell states of major cell types and analysed them in their spatial context, evaluating their dependency on other cell types. Our data elucidate the molecular principles of human myocardial tissue organization, recapitulating a gradual cardiomyocyte and myeloid continuum following ischaemic injury. In sum, our study provides an integrative molecular map of human myocardial infarction, represents an essential reference for the field and paves the way for advanced mechanistic and therapeutic studies of cardiac disease., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Motivation: The advent of multi-modal single-cell sequencing techniques have shed new light on molecular mechanisms by simultaneously inspecting transcriptomes, epigenomes and proteomes of the same cell. However, to date, the existing computational approaches for integration of multimodal single-cell data are either computationally expensive, require the delineation of parameters or can only be applied to particular modalities., Results: Here we present a single-cell multi-modal integration method, named Multi-mOdal Joint IntegraTion of cOmpOnents (MOJITOO). MOJITOO uses canonical correlation analysis for a fast and parameter free detection of a shared representation of cells from multimodal single-cell data. Moreover, estimated canonical components can be used for interpretation, i.e. association of modality-specific molecular features with the latent space. We evaluate MOJITOO using bi- and tri-modal single-cell datasets and show that MOJITOO outperforms existing methods regarding computational requirements, preservation of original latent spaces and clustering., Availability and Implementation: The software, code and data for benchmarking are available at https://github.com/CostaLab/MOJITOO and https://doi.org/10.5281/zenodo.6348128., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2022. Published by Oxford University Press.)

The growing availability of single-cell data, especially transcriptomics, has sparked an increased interest in the inference of cell-cell communication. Many computational tools were developed for this purpose. Each of them consists of a resource of intercellular interactions prior knowledge and a method to predict potential cell-cell communication events. Yet the impact of the choice of resource and method on the resulting predictions is largely unknown. To shed light on this, we systematically compare 16 cell-cell communication inference resources and 7 methods, plus the consensus between the methods' predictions. Among the resources, we find few unique interactions, a varying degree of overlap, and an uneven coverage of specific pathways and tissue-enriched proteins. We then examine all possible combinations of methods and resources and show that both strongly influence the predicted intercellular interactions. Finally, we assess the agreement of cell-cell communication methods with spatial colocalisation, cytokine activities, and receptor protein abundance and find that predictions are generally coherent with those data modalities. To facilitate the use of the methods and resources described in this work, we provide LIANA, a LIgand-receptor ANalysis frAmework as an open-source interface to all the resources and methods., (© 2022. The Author(s).)

How genetic haploinsufficiency contributes to the clonal dominance of hematopoietic stem cells (HSCs) in del(5q) myelodysplastic syndrome (MDS) remains unresolved. Using a genetic barcoding strategy, we performed a systematic comparison on genes implicated in the pathogenesis of del(5q) MDS in direct competition with each other and wild-type (WT) cells with single-clone resolution. Csnk1a1 haploinsufficient HSCs expanded (oligo)clonally and outcompeted all other tested genes and combinations. Csnk1a1-/+ multipotent progenitors showed a proproliferative gene signature and HSCs showed a downregulation of inflammatory signaling/immune response. In validation experiments, Csnk1a1-/+ HSCs outperformed their WT counterparts under a chronic inflammation stimulus, also known to be caused by neighboring genes on chromosome 5. We therefore propose a crucial role for Csnk1a1 haploinsufficiency in the selective advantage of 5q-HSCs, implemented by creation of a unique competitive advantage through increased HSC self-renewal and proliferation capacity, as well as increased fitness under inflammatory stress., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Kidney failure is frequently observed during and after COVID-19, but it remains elusive whether this is a direct effect of the virus. Here, we report that SARS-CoV-2 directly infects kidney cells and is associated with increased tubule-interstitial kidney fibrosis in patient autopsy samples. To study direct effects of the virus on the kidney independent of systemic effects of COVID-19, we infected human-induced pluripotent stem-cell-derived kidney organoids with SARS-CoV-2. Single-cell RNA sequencing indicated injury and dedifferentiation of infected cells with activation of profibrotic signaling pathways. Importantly, SARS-CoV-2 infection also led to increased collagen 1 protein expression in organoids. A SARS-CoV-2 protease inhibitor was able to ameliorate the infection of kidney cells by SARS-CoV-2. Our results suggest that SARS-CoV-2 can directly infect kidney cells and induce cell injury with subsequent fibrosis. These data could explain both acute kidney injury in COVID-19 patients and the development of chronic kidney disease in long COVID., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Single-cell technologies have rapidly developed in recent years and have already had a significant impact on the research of myeloproliferative neoplasms. The increasing number of publicly available data sets allows characterization of the bone marrow niche in patients and mouse models at unprecedented resolution. Single-cell RNA sequencing has successfully been used to identify and characterize disease-driving cell populations and to identify the alarmin S100A8/A9 as an important mediator of myelofibrosis and potent therapeutic target. It is now possible to execute a streamlined set of experiments to specifically identify and validate actionable target genes functionally with the advance of reliable in vivo models and the possibility of conducting single-cell analyses with a minimal amount of patient material. The advent of large-scale analyses of both hematopoietic and non-hematopoietic bone marrow cells will allow comprehensive network analyses guiding an increasingly detailed mapping of the MPN interactome., (Copyright © 2021 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Calreticulin (CALR) mutations are driver mutations in myeloproliferative neoplasms (MPNs), leading to activation of the thrombopoietin receptor and causing abnormal megakaryopoiesis. Here, we generated patient-derived CALRins5- or CALRdel52-positive induced pluripotent stem cells (iPSCs) to establish an MPN disease model for molecular and mechanistic studies. We demonstrated myeloperoxidase deficiency in granulocytic cells derived from homozygous CALR mutant iPSCs, rescued by repairing the mutation using CRISPR/Cas9. iPSC-derived megakaryocytes showed characteristics of primary megakaryocytes such as formation of demarcation membrane system and cytoplasmic pro-platelet protrusions. Importantly, CALR mutations led to enhanced megakaryopoiesis and accelerated megakaryocytic development in a thrombopoietin-independent manner. Mechanistically, our study identified differentially regulated pathways in mutated versus unmutated megakaryocytes, such as hypoxia signaling, which represents a potential target for therapeutic intervention. Altogether, we demonstrate key aspects of mutated CALR-driven pathogenesis dependent on its zygosity, and found novel therapeutic targets, making our model a valuable tool for clinical drug screening in MPNs., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

A major drawback of single-cell ATAC-seq (scATAC-seq) is its sparsity, i.e., open chromatin regions with no reads due to loss of DNA material during the scATAC-seq protocol. Here, we propose scOpen, a computational method based on regularized non-negative matrix factorization for imputing and quantifying the open chromatin status of regulatory regions from sparse scATAC-seq experiments. We show that scOpen improves crucial downstream analysis steps of scATAC-seq data as clustering, visualization, cis-regulatory DNA interactions, and delineation of regulatory features. We demonstrate the power of scOpen to dissect regulatory changes in the development of fibrosis in the kidney. This identifies a role of Runx1 and target genes by promoting fibroblast to myofibroblast differentiation driving kidney fibrosis., (© 2021. The Author(s).)

Genes involved in distinct diabetes types suggest shared disease mechanisms. Here we show that One Cut Homeobox 1 (ONECUT1) mutations cause monogenic recessive syndromic diabetes in two unrelated patients, characterized by intrauterine growth retardation, pancreas hypoplasia and gallbladder agenesis/hypoplasia, and early-onset diabetes in heterozygous relatives. Heterozygous carriers of rare coding variants of ONECUT1 define a distinctive subgroup of diabetic patients with early-onset, nonautoimmune diabetes, who respond well to diabetes treatment. In addition, common regulatory ONECUT1 variants are associated with multifactorial type 2 diabetes. Directed differentiation of human pluripotent stem cells revealed that loss of ONECUT1 impairs pancreatic progenitor formation and a subsequent endocrine program. Loss of ONECUT1 altered transcription factor binding and enhancer activity and NKX2.2/NKX6.1 expression in pancreatic progenitor cells. Collectively, we demonstrate that ONECUT1 controls a transcriptional and epigenetic machinery regulating endocrine development, involved in a spectrum of diabetes, encompassing monogenic (recessive and dominant) as well as multifactorial inheritance. Our findings highlight the broad contribution of ONECUT1 in diabetes pathogenesis, marking an important step toward precision diabetes medicine., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)