Lin, M;Hartl, K;Heuberger, J;Beccaceci, G;Berger, H;Li, H;Liu, L;Müllerke, S;Conrad, T;Heymann, F;Woehler, A;Tacke, F;Rajewsky, N;Sigal, M;
PMID: 37230989 | DOI: 10.1038/s41467-023-38780-3
The cellular organization of gastrointestinal crypts is orchestrated by different cells of the stromal niche but available in vitro models fail to fully recapitulate the interplay between epithelium and stroma. Here, we establish a colon assembloid system comprising the epithelium and diverse stromal cell subtypes. These assembloids recapitulate the development of mature crypts resembling in vivo cellular diversity and organization, including maintenance of a stem/progenitor cell compartment in the base and their maturation into secretory/absorptive cell types. This process is supported by self-organizing stromal cells around the crypts that resemble in vivo organization, with cell types that support stem cell turnover adjacent to the stem cell compartment. Assembloids that lack BMP receptors either in epithelial or stromal cells fail to undergo proper crypt formation. Our data highlight the crucial role of bidirectional signaling between epithelium and stroma, with BMP as a central determinant of compartmentalization along the crypt axis.
Zhang, CH;Gao, Y;Hung, HH;Zhuo, Z;Grodzinsky, AJ;Lassar, AB;
PMID: 36435829 | DOI: 10.1038/s41467-022-35010-0
While prior work has established that articular cartilage arises from Prg4-expressing perichondrial cells, it is not clear how this process is specifically restricted to the perichondrium of synovial joints. We document that the transcription factor Creb5 is necessary to initiate the expression of signaling molecules that both direct the formation of synovial joints and guide perichondrial tissue to form articular cartilage instead of bone. Creb5 promotes the generation of articular chondrocytes from perichondrial precursors in part by inducing expression of signaling molecules that block a Wnt5a autoregulatory loop in the perichondrium. Postnatal deletion of Creb5 in the articular cartilage leads to loss of both flat superficial zone articular chondrocytes coupled with a loss of both Prg4 and Wif1 expression in the articular cartilage; and a non-cell autonomous up-regulation of Ctgf. Our findings indicate that Creb5 promotes joint formation and the subsequent development of articular chondrocytes by driving the expression of signaling molecules that both specify the joint interzone and simultaneously inhibit a Wnt5a positive-feedback loop in the perichondrium.
Tang, WC;Tsao, SW;Jones, GE;Liu, X;Tsai, MH;Delecluse, HJ;Dai, W;You, C;Zhang, J;Huang, SCM;Leung, MM;Liu, T;Ching, YP;Chen, H;Lo, KW;Li, X;Tsang, CM;
PMID: 36420735 | DOI: 10.1002/path.6036
Invadopodia are actin-rich membrane protrusions that digest the matrix barrier during cancer metastasis. Since the discovery of invadopodia, they were visualized as localized and dot-like structures in different types of cancer cells on top of a 2D matrix. In this investigation of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC), a highly invasive cancer frequently accompanied by neck lymph node and distal organ metastases, we revealed a new form of invadopodium with mobilizing features. Integration of live-cell imaging and molecular assays revealed the interaction of macrophage-released TNFα and EBV-encoded latent membrane protein 1 (LMP1) in co-activating the EGFR/Src/ERK/cortactin and Cdc42/N-WASP signaling axes for mobilizing the invadopodia with lateral movements. This phenomenon endows the invadopodia with massive degradative power, visualized as a shift of focal dot-like digestion patterns on a 2D gelatin to a dendrite-like digestion pattern. Notably, single stimulation of either LMP1 or TNFα could only enhance the number of ordinary dot-like invadopodia, suggesting that the EBV infection sensitizes the NPC cells to form mobilizing invadopodia when encountering a TNFα-rich tumor microenvironment. This study unveils the interplay of EBV and stromal components in driving the invasive potential of NPC via unleashing the propulsion of invadopodia in overcoming matrix hurdles. This article is protected by
Pathology - Research and Practice
Schwab, C;Domke, L;Rose, F;Hausser, I;Schirmacher, P;Longerich, T;
| DOI: 10.1016/j.prp.2022.154000
Pulmonary capillary microthrombosis has been proposed as a major pathogenetic factor driving severe COVID-19. Autopsy studies reported endothelialitis but it is under debate if it is caused by SARS-CoV-2 infection of endothelial cells. In this study, RNA in situ hybridization was used to detect viral RNA and to identify the infected cell types in lung tissue of 40 patients with fatal COVID-19. SARS-CoV-2 Spike protein-coding RNA showed a steadily decreasing signal abundance over a period of three weeks. Besides the original virus strain the variants of concern Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529) could also be detected by the assay. Viral RNA was mainly detected in alveolar macrophages and pulmonary epithelial cells, while only single virus-positive endothelial cells were observed even in cases with high viral load suggesting that viral infection of endothelial cells is not a key factor for the development of pulmonary capillary microthrombosis.
A cellular and spatial map of the choroid plexus across brain ventricles and ages
Dani, N;Herbst, RH;McCabe, C;Green, GS;Kaiser, K;Head, JP;Cui, J;Shipley, FB;Jang, A;Dionne, D;Nguyen, L;Rodman, C;Riesenfeld, SJ;Prochazka, J;Prochazkova, M;Sedlacek, R;Zhang, F;Bryja, V;Rozenblatt-Rosen, O;Habib, N;Regev, A;Lehtinen, MK;
PMID: 33932339 | DOI: 10.1016/j.cell.2021.04.003
The choroid plexus (ChP) in each brain ventricle produces cerebrospinal fluid (CSF) and forms the blood-CSF barrier. Here, we construct a single-cell and spatial atlas of each ChP in the developing, adult, and aged mouse brain. We delineate diverse cell types, subtypes, cell states, and expression programs in epithelial and mesenchymal cells across ages and ventricles. In the developing ChP, we predict a common progenitor pool for epithelial and neuronal cells, validated by lineage tracing. Epithelial and fibroblast cells show regionalized expression by ventricle, starting at embryonic stages and persisting with age, with a dramatic transcriptional shift with maturation, and a smaller shift in each aged cell type. With aging, epithelial cells upregulate host-defense programs, and resident macrophages upregulate interleukin-1β (IL-1β) signaling genes. Our atlas reveals cellular diversity, architecture and signaling across ventricles during development, maturation, and aging of the ChP-brain barrier.
Bockmayr, M;Harnisch, K;Pohl, L;Schweizer, L;Mohme, T;Körner, M;Alawi, M;Suwala, A;Dorostkar, M;Monoranu, C;Hasselblatt, M;Wefers, A;Capper, D;Hench, J;Frank, S;Richardson, T;Tran, I;Liu, E;Snuderl, M;Engertsberger, L;Benesch, M;von Deimling, A;Obrecht, D;Mynarek, M;Rutkowski, S;Glatzel, M;Neumann, J;Schüller, U;
| DOI: 10.1093/neuonc/noac079.143
Myxopapillary ependymoma (MPE) is a heterogeneous disease regarding histopathology and outcome. The underlying molecular biology is poorly understood, and markers that reliably predict the patients’ clinical course are unknown. We assembled a cohort of 185 tumors classified as MPE based on DNA methylation from pediatric, adolescent, and adult patients. Methylation patterns, copy number profiles, and MGMT promoter methylation were analyzed for all tumors, 106 tumors were evaluated histomorphologically, and RNA sequencing was performed for 37 cases. Based on methylation profiling, we defined two subtypes MPE-A and MPEB, and explored associations with epidemiological, clinical, pathological, and molecular characteristics of these tumors. Tumors in the methylation class MPE were histologically diagnosed as WHO grade I (59%), WHO grade II (37%), or WHO grade III tumors (4%). 75/77 analyzed tumors expressed HOXB13, which is a diagnostic feature not detected in other spinal ependymal tumors. Based on DNA methylation, our series split into two subtypes. MPE-A occurred in younger patients (median age 27 vs. 45 years, p=7.3e-05). They were enriched with WHO grade I tumors and associated with papillary morphology and MGMT promoter hypermethylation (all p<0.001). MPE-B included most tumors initially diagnosed as WHO grade II and cases with tanycytic morphology. Copy number alterations were more common in MPE-A. RNA sequencing revealed an enrichment for extracellular matrix and immune system-related signatures in MPE-A. 15/30 MPE-A could not be totally resected compared to 1/58 MPE-B (p=6.3e-08), and progression-free survival was significantly better for MPE-B (p=3.4e-06, 10-year relapse rate 33% vs. 85%). We unraveled the morphological and clinical heterogeneity of MPE by identifying two molecularly distinct subtypes. These subtypes significantly differed in progression-free survival and will likely need different protocols for surveillance and treatment.
Broeckel, RM;Feldmann, F;McNally, KL;Chiramel, AI;Sturdevant, GL;Leung, JM;Hanley, PW;Lovaglio, J;Rosenke, R;Scott, DP;Saturday, G;Bouamr, F;Rasmussen, AL;Robertson, SJ;Best, SM;
PMID: 34855915 | DOI: 10.1371/journal.ppat.1009678
Kyasanur Forest disease virus (KFDV) and the closely related Alkhurma hemorrhagic disease virus (AHFV) are emerging flaviviruses that cause severe viral hemorrhagic fevers in humans. Increasing geographical expansion and case numbers, particularly of KFDV in southwest India, class these viruses as a public health threat. Viral pathogenesis is not well understood and additional vaccines and antivirals are needed to effectively counter the impact of these viruses. However, current animal models of KFDV pathogenesis do not accurately reproduce viral tissue tropism or clinical outcomes observed in humans. Here, we show that pigtailed macaques (Macaca nemestrina) infected with KFDV or AHFV develop viremia that peaks 2 to 4 days following inoculation. Over the course of infection, animals developed lymphocytopenia, thrombocytopenia, and elevated liver enzymes. Infected animals exhibited hallmark signs of human disease characterized by a flushed appearance, piloerection, dehydration, loss of appetite, weakness, and hemorrhagic signs including epistaxis. Virus was commonly present in the gastrointestinal tract, consistent with human disease caused by KFDV and AHFV where gastrointestinal symptoms (hemorrhage, vomiting, diarrhea) are common. Importantly, RNAseq of whole blood revealed that KFDV downregulated gene expression of key clotting factors that was not observed during AHFV infection, consistent with increased severity of KFDV disease observed in this model. This work characterizes a nonhuman primate model for KFDV and AHFV that closely resembles human disease for further utilization in understanding host immunity and development of antiviral countermeasures.
Am J Respir Crit Care Med. 2018 Dec 15.
Reyfman PA, Walter JM, Joshi N, Anekalla KR, McQuattie-Pimentel AC, Chiu S, Fernandez R, Akbarpour M, Chen CI, Ren Z, Verma R, Abdala-Valencia H, Nam K, Chi M, Han S, Gonzalez-Gonzalez FJ, Soberanes S, Watanabe S, Williams KJN, Flozak AS, Nicholson TT, Morgan VK, Winter DR, Hinchcliff M, Hrusch CL, Guzy RD, Bonham CA, Sperling AI, Bag R, Hamanaka RB, Mutlu GM, Yeldandi AV, Marshall SA, Shilatifard A, Amaral LAN, Perlman H, Sznajder JI, Argento AC, Gillespie CT, Dematte J, Jain M, Singer BD, Ridge KM, Lam AP, Bharat A, Bhorade SM, Gottardi CJ, Budinger GRS, Misharin AV.
PMID: 30554520 | DOI: 10.1164/rccm.201712-2410OC
Abstract RATIONALE: The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell populations during pulmonary fibrosis that are important for disease pathogenesis. OBJECTIVES: To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells or other cell types in lung tissue from subjects with pulmonary fibrosis compared with controls. METHODS: We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data in using in situ RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. MEASUREMENTS AND MAIN RESULTS: We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to non-overlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data. CONCLUSIONS: We generated a single cell atlas of pulmonary fibrosis. Using this atlas we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.
Kinchen J, Chen HH, Parikh K, Antanaviciute A, Jagielowicz M, Fawkner-Corbett D, Ashley N, Cubitt L, Mellado-Gomez E, Attar M, Sharma E, Wills Q, Bowden R, Richter FC, Ahern D, Puri KD, Henault J, Gervais F, Koohy H, Simmons A.
PMID: - | DOI: 10.1016/j.cell.2018.08.067
Intestinal mesenchymal cells play essential roles in epithelial homeostasis, matrix remodeling, immunity, and inflammation. But the extent of heterogeneity within the colonic mesenchyme in these processes remains unknown. Using unbiased single-cell profiling of over 16,500 colonic mesenchymal cells, we reveal four subsets of fibroblasts expressing divergent transcriptional regulators and functional pathways, in addition to pericytes and myofibroblasts. We identified a niche population located in proximity to epithelial crypts expressing SOX6, F3 (CD142), and WNT genes essential for colonic epithelial stem cellfunction. In colitis, we observed dysregulation of this niche and emergence of an activated mesenchymal population. This subset expressed TNF superfamily member 14 (TNFSF14), fibroblastic reticular cell-associated genes, IL-33, and Lysyl oxidases. Further, it induced factors that impaired epithelial proliferation and maturation and contributed to oxidative stress and disease severity in vivo. Our work defines how the colonic mesenchyme remodels to fuel inflammation and barrier dysfunction in IBD.
He, S;Bhatt, R;Brown, C;Brown, EA;Buhr, DL;Chantranuvatana, K;Danaher, P;Dunaway, D;Garrison, RG;Geiss, G;Gregory, MT;Hoang, ML;Khafizov, R;Killingbeck, EE;Kim, D;Kim, TK;Kim, Y;Klock, A;Korukonda, M;Kutchma, A;Lewis, ZR;Liang, Y;Nelson, JS;Ong, GT;Perillo, EP;Phan, JC;Phan-Everson, T;Piazza, E;Rane, T;Reitz, Z;Rhodes, M;Rosenbloom, A;Ross, D;Sato, H;Wardhani, AW;Williams-Wietzikoski, CA;Wu, L;Beechem, JM;
PMID: 36203011 | DOI: 10.1038/s41587-022-01483-z
Resolving the spatial distribution of RNA and protein in tissues at subcellular resolution is a challenge in the field of spatial biology. We describe spatial molecular imaging, a system that measures RNAs and proteins in intact biological samples at subcellular resolution by performing multiple cycles of nucleic acid hybridization of fluorescent molecular barcodes. We demonstrate that spatial molecular imaging has high sensitivity (one or two copies per cell) and very low error rate (0.0092 false calls per cell) and background (~0.04 counts per cell). The imaging system generates three-dimensional, super-resolution localization of analytes at ~2 million cells per sample. Cell segmentation is morphology based using antibodies, compatible with formalin-fixed, paraffin-embedded samples. We measured multiomic data (980 RNAs and 108 proteins) at subcellular resolution in formalin-fixed, paraffin-embedded tissues (nonsmall cell lung and breast cancer) and identified >18 distinct cell types, ten unique tumor microenvironments and 100 pairwise ligand-receptor interactions. Data on >800,000 single cells and ~260 million transcripts can be accessed at http://nanostring.com/CosMx-dataset .
Lovatt, D;Tamburino, A;Krasowska-Zoladek, A;Sanoja, R;Li, L;Peterson, V;Wang, X;Uslaner, J;
PMID: 36261573 | DOI: 10.1038/s42003-022-03970-0
Patients with peripheral nerve injury, viral infection or metabolic disorder often suffer neuropathic pain due to inadequate pharmacological options for relief. Developing novel therapies has been challenged by incomplete mechanistic understanding of the cellular microenvironment in sensory nerve that trigger the emergence and persistence of pain. In this study, we report a high resolution transcriptomics map of the cellular heterogeneity of naïve and injured rat sensory nerve covering more than 110,000 individual cells. Annotation reveals distinguishing molecular features of multiple major cell types totaling 45 different subtypes in naïve nerve and an additional 23 subtypes emerging after injury. Ligand-receptor analysis revealed a myriad of potential targets for pharmacological intervention. This work forms a comprehensive resource and unprecedented window into the cellular milieu underlying neuropathic pain and demonstrates that nerve injury is a dynamic process orchestrated by multiple cell types in both the endoneurial and epineurial nerve compartments.
Ademi, H;Djari, C;Mayère, C;Neirijnck, Y;Sararols, P;Rands, CM;Stévant, I;Conne, B;Nef, S;
PMID: 35705036 | DOI: 10.1016/j.celrep.2022.110935
Leydig cells (LCs) are the major androgen-producing cells in the testis. They arise from steroidogenic progenitors (SPs), whose origins, maintenance, and differentiation dynamics remain largely unknown. Single-cell transcriptomics reveal that the mouse steroidogenic lineage is specified as early as embryonic day 12.5 (E12.5) and has a dual mesonephric and coelomic origin. SPs specifically express the Wnt5a gene and evolve rapidly. At E12.5 and E13.5, they give rise first to an intermediate population of pre-LCs, and finally to fetal LCs. At E16.5, SPs possess the characteristics of the dormant progenitors at the origin of adult LCs and are also transcriptionally closely related to peritubular myoid cells (PMCs). In agreement with our in silico analysis, in vivo lineage tracing indicates that Wnt5a-expressing cells are bona fide progenitors of PMCs as well as fetal and adult LCs, contributing to most of the LCs present in the fetal and adult testis.
