Epithelial organoids derived from intestinal tissue, called ‘enteroids’, recapitulate many aspects of the organ in vitro, and can be used for biological discovery, personalized medicine, and drug development. Here, we interrogated the cell signaling environment within the developing human intestine to identify niche cues that may be important for epithelial development and homeostasis. We identify an EGF family member, EPIREGULIN (EREG), which is robustly expressed in the developing human crypt. Enteroids generated from the developing human intestine grown in standard culture conditions, which contain EGF, are dominated by stem and progenitor cells, feature little differentiation and no spatial organization. Our results demonstrate that EREG can replace EGF in vitro, and EREG leads to spatially resolved enteroids that feature budded and proliferative crypt domains and a differentiated villus-like central lumen. Multiomic (transcriptome plus epigenome) profiling of native crypts, EGF-grown and EREG-grown enteroids show that EGF-enteroids have an altered chromatin landscape that is dependent on EGF concentration, downregulate the master intestinal transcription factor CDX2, and ectopically express stomach genes, a phenomenon that is reversible. This is in contrast to EREG-grown enteroids, which remain intestine-like in culture. Thus, EREG creates a homeostatic intestinal niche in vitro, enabling interrogation of stem cell function, cellular differentiation, and disease modeling.
Charlie J. Childs, Emily M. Holloway, Caden W. Sweet, Yu-Hwai Tsai, Angeline Wu, Abigail Vallie, Madeline K. Eiken, Meghan M. Capeling, Rachel K. Zwick, Brisa Palikuqi, Coralie Trentesaux, Joshua H. Wu, Oscar Pellon-Cardenas, Charles J. Zhang, Ian A. Glass, Claudia Loebel, Qianhui Yu, J. Gray Camp, Jonathan Z. Sexton, Ophir D. Klein, Michael P. Verzi, Jason R. Spence
Volumetric muscle loss (VML) is an acute trauma that results in persistent inflammation, supplantation of muscle tissue with fibrotic scarring, and decreased muscle function. The cell types, nature of cellular communication, and tissue locations that drive the aberrant VML response have remained elusive. Herein, we used spatial transcriptomics on a mouse model of VML and observed VML engenders a unique spatial pro-fibrotic pattern driven by crosstalk between fibrotic and inflammatory macrophages and mesenchymal derived cells. The dysregulated response impinged on muscle stem cell mediated repair, and targeting this circuit resulted in increased regeneration and reductions in inflammation and fibrosis. Collectively, these results enhance our understanding of the cellular crosstalk that drives aberrant regeneration and provides further insight into possible avenues for fibrotic therapy exploration.
Jacqueline A. Larouche, Emily C. Wallace, Bonnie D. Spence, Eric Buras, Carlos A. Aguilar
B cells within secondary lymphoid tissues (SLTs) encompass a diversity of activation states and multiple maturation processes that reflect antigen recognition and transition through the germinal center (GC) reaction, in which mature B cells differentiate into memory and antibody-secreting cells (ASCs). Here, utilizing single-cell RNA-seq, we identify a range of distinct activation and maturation states of tonsillar-derived B cells. In particular, we identify a previously uncharacterized CCL4/CCL3 chemokine-expressing B-cell population with an expression pattern consistent with BCR/CD40 activation. Furthermore, we present a computational method leveraging regulatory network inference and pseudotemporal modeling to identify upstream transcription factor modulation along a GC to ASC axis of transcriptional maturation. Our dataset provides valuable insight into diverse B-cell functional profiles and will be a useful resource for further studies into the B-cell immune compartment.
Diego A. Espinoza, Carole Le Coz, Emylette Cruz Cabrera, Neil Romberg, Amit Bar-Or, Rui Li
To better characterize the heterogeneity of multiple myeloma (MM), we profiled plasma cells (PCs) and their B cell lymphopoiesis in the BM samples from patients with monoclonal gammopathy of undetermined significance, smoldering MM, and active MM by mass cytometry (CyTOF) analysis. Characterization of intra- and interneoplastic heterogeneity of malignant plasmablasts and PCs revealed overexpression of the MM SET domain (MMSET), Notch-1, and CD47. Variations in upregulation of B cell signaling regulators (IFN regulatory factor 4 [IRF-4], CXCR4, B cell lymphoma 6 [Bcl-6], c-Myc, myeloid differentiation primary response protein 88 [MYD88], and spliced X box-binding protein 1 [sXBP-1]) and aberrant markers (CD319, CD269, CD200, CD117, CD56, and CD28) were associated with different clinical outcomes in clonal PC subsets. In addition, prognosis was related to heterogeneity in subclonal expression of stemness markers, including neuroepithelial stem cell protein (Nestin), SRY-box transcription factor 2 (Sox2), Krüppel-like factor 4 (KLF-4), and Nanog. Furthermore, we have defined significantly elevated levels of MMSET, MYD88, c-Myc, CD243, Notch-1, and CD47 from hematopoietic stem cells to PCs in myeloma B cell lymphopoiesis, noted even in premalignant conditions, with variably modulated expression of B cell development regulators, including IRF-4, Bcl-2, Bcl-6, and sXBP-1; aberrant PC markers (such as CD52, CD44, CD200, CD81, CD269, CD117, and CXCR4); and stemness-controlling regulators, including Nanog, KLF-4, octamer-binding transcription factor 3/4 (Oct3/4), Sox2, and retinoic acid receptor α2 (RARα2). This study provides the rationale for precise molecular profiling of patients with MM by CyTOF technology to define disease heterogeneity and prognosis.
Jana Jakubikova, Danka Cholujova, Gabor Beke, Teru Hideshima, Lubos Klucar, Merav Leiba, Krzysztof Jamroziak, Paul G. Richardson, Efstathios Kastritis, David M. Dorfman, Kenneth C. Anderson
A role of CD4+ T cells during the progression from nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH) has been suggested, but which polarization state of these cells characterizes this progression and the development of fibrosis remain unclear. In addition, a gut-liver axis has been suggested to play a role in NASH, but the role of CD4+ T cells in this axis has just begun to be investigated. Combining single-cell RNA sequencing and multiple-parameter flow cytometry, we provide the first cell atlas to our knowledge focused on liver-infiltrating CD4+ T cells in patients with NAFLD and NASH, showing that NASH is characterized by a population of multicytokine-producing CD4+ T cells. Among these cells, only those with a Th17 polarization state were enriched in patients with advanced fibrosis. In parallel, we observed that Bacteroides appeared to be enriched in the intestine of NASH patients and to correlate with the frequency of multicytokine-producing CD4+ T cells. In short, we deliver a CD4+ T cell atlas of NAFLD and NASH, providing the rationale to target CD4+ T cells with a Th17 polarization state to block fibrosis development. Finally, our data offer an early indication to test whether multicytokine-producing CD4+ T cells are part of the gut-liver axis characterizing NASH.
Anna Woestemeier, Pasquale Scognamiglio, Yu Zhao, Jonas Wagner, Franziska Muscate, Christian Casar, Francesco Siracusa, Filippo Cortesi, Theodora Agalioti, Simone Müller, Adrian Sagebiel, Leonie Konczalla, Ramez Wahib, Karl-Frederick Karstens, Anastasios D. Giannou, Anna Duprée, Stefan Wolter, Milagros N. Wong, Anne K. Mühlig, Agata A. Bielecka, Vikas Bansal, Tianran Zhang, Oliver Mann, Victor G. Puelles, Tobias B. Huber, Ansgar W. Lohse, Jakob R. Izbicki, Noah W. Palm, Stefan Bonn, Samuel Huber, Nicola Gagliani
In pulmonary arterial hypertension (PAH), inflammation promotes a fibroproliferative pulmonary vasculopathy. Reductionist studies emphasizing single biochemical reactions suggest a shift toward glycolytic metabolism in PAH; however, key questions remain regarding the metabolic profile of specific cell types within PAH vascular lesions in vivo. We used RNA-seq to profile the transcriptome of pulmonary artery endothelial cells (PAECs) freshly isolated from an inflammatory vascular injury model of PAH ex vivo, and these data were integrated with information from human gene ontology pathways. Network medicine was then used to map all amino acid and glucose pathways to the consolidated human interactome, which includes data on 233,957 physical protein-protein interactions. Glucose and proline pathways were significantly close to the human PAH disease module, suggesting that these pathways are functionally relevant to PAH pathobiology. To test this observation in vivo, we used multi-isotope imaging mass spectrometry (MIMS) to map and quantify utilization of glucose and proline in the PAH pulmonary vasculature at subcellular resolution. Our findings suggest suggest that elevated glucose and proline avidity underlies increased biomass in PAECs and the media of fibrosed PAH pulmonary arterioles. Overall, these data show that anabolic utilization of glucose and proline are fundamental to the vascular pathology of PAH.
Bradley M. Wertheim, Rui-Sheng Wang, Christelle Guillermier, Christiane V.R. Hütter, William M. Oldham, Jörg Menche, Matthew L. Steinhauser, Bradley A. Maron
Patients with the renal phosphate–wasting disease X-linked hypophosphatemia (XLH) and Hyp mice, the murine homolog of XLH, are characterized by loss-of-function mutations in phosphate-regulating endopeptidase homolog X-linked (PHEX), leading to excessive secretion of the bone-derived phosphotropic hormone FGF23. The mineralization defect in patients with XLH and Hyp mice is caused by a combination of hypophosphatemia and local accumulation of mineralization-inhibiting molecules in bone. However, the mechanism by which PHEX deficiency regulates bone cell metabolism remains elusive. Here, we used spatial metabolomics by employing matrix-assisted laser desorption/ionization (MALDI) Fourier-transform ion cyclotron resonance mass spectrometry imaging (MSI) of undecalcified bone cryosections to characterize in situ metabolic changes in bones of Hyp mice in a holistic, unbiased manner. We found complex changes in Hyp bone metabolism, including perturbations in pentose phosphate, purine, pyrimidine, and phospholipid metabolism. Importantly, our study identified an upregulation of several biochemical pathways involved in intra- and extracellular production of the mineralization inhibitor pyrophosphate in the bone matrix of Hyp mice. Our data emphasize the utility of MSI–based spatial metabolomics in bone research and provide holistic in situ insights as to how Phex deficiency–induced changes in biochemical pathways in bone cells are linked to impaired bone mineralization.
Achim Buck, Verena M. Prade, Thomas Kunzke, Reinhold G. Erben, Axel Walch
Mass cytometry, or cytometry by TOF (CyTOF), provides a robust means of determining protein-level measurements of more than 40 markers simultaneously. While the functional states of immune cells occur along continuous phenotypic transitions, cytometric studies surveying cell phenotypes often rely on static metrics, such as discrete cell-type abundances, based on canonical markers and/or restrictive gating strategies. To overcome this limitation, we applied single-cell trajectory inference and nonnegative matrix factorization methods to CyTOF data to trace the dynamics of T cell states. In the setting of cancer immunotherapy, we showed that patient-specific summaries of continuous phenotypic shifts in T cells could be inferred from peripheral blood–derived CyTOF mass cytometry data. We further illustrated that transfer learning enabled these T cell continuous metrics to be used to estimate patient-specific cell states in new sample cohorts from a reference patient data set. Our work establishes the utility of continuous metrics for CyTOF analysis as tools for translational discovery.
Dimitrios N. Sidiropoulos, Genevieve L. Stein-O’Brien, Ludmila Danilova, Nicole E. Gross, Soren Charmsaz, Stephanie Xavier, James Leatherman, Hao Wang, Mark Yarchoan, Elizabeth M. Jaffee, Elana J. Fertig, Won Jin Ho
Bronchiolitis obliterans syndrome (BOS) is the main reason for poor outcomes after lung transplantation (LTx). We and others have recently identified B cells as major contributors to BOS after LTx. The extent of B cell heterogeneity and the relative contributions of B cell subpopulations to BOS, however, remain unclear. Here, we provide a comprehensive analysis of cell population changes and their gene expression patterns during chronic rejection after orthotopic LTx in mice. Of 11 major cell types, Mzb1-expressing plasma cells (PCs) were the most prominently increased population in BOS lungs. These findings were validated in 2 different cohorts of human BOS after LTx. A Bhlhe41, Cxcr3, and Itgb1 triple-positive B cell subset, also expressing classical markers of the innate-like B-1 B cell population, served as the progenitor pool for Mzb1+ PCs. This subset accounted for the increase in IgG2c production within BOS lung grafts. A genetic lack of Igs decreased BOS severity after LTx. In summary, we provide a detailed analysis of cell population changes during BOS. IgG+ PCs and their progenitors — an innate B cell subpopulation — are the major source of local Ab production and a significant contributor to BOS after LTx.
Natalia F. Smirnova, Kent Riemondy, Marta Bueno, Susan Collins, Pavan Suresh, Xingan Wang, Kapil N. Patel, Carlyne Cool, Melanie Königshoff, Nirmal S. Sharma, Oliver Eickelberg
Cancer cells release large quantities of cell-free DNA (cfDNA) into the surrounding tissue and circulation. As cfDNA is a common source of biomarkers for liquid biopsy and has been implicated as a functional mediator for intercellular communication, fundamental characterization of cfDNA topology has widespread biological and clinical ramifications. Whether the topology of cfDNA is such that it exists predominantly in membrane-bound extracellular vesicles (EVs) or in non-vesicular DNA-protein complexes remains poorly understood. Here, we employed a DNA-targeted approach to comprehensively assess total cfDNA topology in cancer. Using preclinical models and patient samples, we demonstrate that nuclear cfDNA is predominantly associated with nucleosomal particles and not EVs, while a substantial subset of mitochondrial cfDNA is membrane-protected and disproportionately derived from non-tumour cells. In addition, discrimination between membrane-protected and accessible mitochondrial cfDNA added diagnostic and prognostic value in a cohort of head and neck cancer patients. Our results support a revised model for cfDNA topology in cancer. Due to its abundance, nuclear cfDNA within nucleosomal particles is the most compelling liquid biopsy substrate, while EV-bound and accessible mitochondrial cfDNA represent distinct reservoirs of potential cancer biomarkers whose structural conformations may also influence their extracellular stability and propensity for uptake by recipient cells.
Ethan Z. Malkin, Steven De Michino, Meghan Lambie, Rita G. Gill, Zhen Zhao, Ariana Rostami, Andrea Arruda, Mark D. Minden, Scott V. Bratman
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