RESUMEN
For many adult human organs, tissue regeneration during chronic disease remains a controversial subject. Regenerative processes are easily observed in animal models, and their underlying mechanisms are becoming well characterized1-4, but technical challenges and ethical aspects are limiting the validation of these results in humans. We decided to address this difficulty with respect to the liver. This organ displays the remarkable ability to regenerate after acute injury, although liver regeneration in the context of recurring injury remains to be fully demonstrated. Here we performed single-nucleus RNA sequencing (snRNA-seq) on 47 liver biopsies from patients with different stages of metabolic dysfunction-associated steatotic liver disease to establish a cellular map of the liver during disease progression. We then combined these single-cell-level data with advanced 3D imaging to reveal profound changes in the liver architecture. Hepatocytes lose their zonation and considerable reorganization of the biliary tree takes place. More importantly, our study uncovers transdifferentiation events that occur between hepatocytes and cholangiocytes without the presence of adult stem cells or developmental progenitor activation. Detailed analyses and functional validations using cholangiocyte organoids confirm the importance of the PI3K-AKT-mTOR pathway in this process, thereby connecting this acquisition of plasticity to insulin signalling. Together, our data indicate that chronic injury creates an environment that induces cellular plasticity in human organs, and understanding the underlying mechanisms of this process could open new therapeutic avenues in the management of chronic diseases.
Asunto(s)
Transdiferenciación Celular , Hepatocitos , Hepatopatías , Hígado , Humanos , Sistema Biliar/citología , Sistema Biliar/metabolismo , Sistema Biliar/patología , Biopsia , Plasticidad de la Célula , Enfermedad Crónica , Progresión de la Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/citología , Células Epiteliales/patología , Hepatocitos/metabolismo , Hepatocitos/citología , Hepatocitos/patología , Insulina/metabolismo , Hígado/patología , Hígado/metabolismo , Hígado/citología , Hepatopatías/patología , Hepatopatías/metabolismo , Regeneración Hepática , Organoides/metabolismo , Organoides/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , RNA-Seq , Transducción de Señal , Análisis de la Célula Individual , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The function of a cell is defined by its intrinsic characteristics and its niche: the tissue microenvironment in which it dwells. Here we combine single-cell and spatial transcriptomics data to discover cellular niches within eight regions of the human heart. We map cells to microanatomical locations and integrate knowledge-based and unsupervised structural annotations. We also profile the cells of the human cardiac conduction system1. The results revealed their distinctive repertoire of ion channels, G-protein-coupled receptors (GPCRs) and regulatory networks, and implicated FOXP2 in the pacemaker phenotype. We show that the sinoatrial node is compartmentalized, with a core of pacemaker cells, fibroblasts and glial cells supporting glutamatergic signalling. Using a custom CellPhoneDB.org module, we identify trans-synaptic pacemaker cell interactions with glia. We introduce a druggable target prediction tool, drug2cell, which leverages single-cell profiles and drug-target interactions to provide mechanistic insights into the chronotropic effects of drugs, including GLP-1 analogues. In the epicardium, we show enrichment of both IgG+ and IgA+ plasma cells forming immune niches that may contribute to infection defence. Overall, we provide new clarity to cardiac electro-anatomy and immunology, and our suite of computational approaches can be applied to other tissues and organs.
Asunto(s)
Microambiente Celular , Corazón , Multiómica , Miocardio , Humanos , Comunicación Celular , Fibroblastos/citología , Ácido Glutámico/metabolismo , Corazón/anatomía & histología , Corazón/inervación , Canales Iónicos/metabolismo , Miocardio/citología , Miocardio/inmunología , Miocardio/metabolismo , Miocitos Cardíacos/citología , Neuroglía/citología , Pericardio/citología , Pericardio/inmunología , Células Plasmáticas/inmunología , Receptores Acoplados a Proteínas G/metabolismo , Nodo Sinoatrial/anatomía & histología , Nodo Sinoatrial/citología , Nodo Sinoatrial/fisiología , Sistema de Conducción Cardíaco/anatomía & histología , Sistema de Conducción Cardíaco/citología , Sistema de Conducción Cardíaco/metabolismoRESUMEN
The lymphocyte genome is prone to many threats, including programmed mutation during differentiation1, antigen-driven proliferation and residency in diverse microenvironments. Here, after developing protocols for expansion of single-cell lymphocyte cultures, we sequenced whole genomes from 717 normal naive and memory B and T cells and haematopoietic stem cells. All lymphocyte subsets carried more point mutations and structural variants than haematopoietic stem cells, with higher burdens in memory cells than in naive cells, and with T cells accumulating mutations at a higher rate throughout life. Off-target effects of immunological diversification accounted for approximately half of the additional differentiation-associated mutations in lymphocytes. Memory B cells acquired, on average, 18 off-target mutations genome-wide for every on-target IGHV mutation during the germinal centre reaction. Structural variation was 16-fold higher in lymphocytes than in stem cells, with around 15% of deletions being attributable to off-target recombinase-activating gene activity. DNA damage from ultraviolet light exposure and other sporadic mutational processes generated hundreds to thousands of mutations in some memory cells. The mutation burden and signatures of normal B cells were broadly similar to those seen in many B-cell cancers, suggesting that malignant transformation of lymphocytes arises from the same mutational processes that are active across normal ontogeny. The mutational landscape of normal lymphocytes chronicles the off-target effects of programmed genome engineering during immunological diversification and the consequences of differentiation, proliferation and residency in diverse microenvironments.
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Linfocitos , Mutación , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Diferenciación Celular , Proliferación Celular , Microambiente Celular , Daño del ADN/genética , Daño del ADN/efectos de la radiación , Centro Germinal/citología , Centro Germinal/inmunología , Humanos , Memoria Inmunológica/genética , Linfocitos/citología , Linfocitos/inmunología , Linfocitos/metabolismo , Linfocitos/patología , Neoplasias/genética , Neoplasias/patologíaRESUMEN
Age-related change in human haematopoiesis causes reduced regenerative capacity1, cytopenias2, immune dysfunction3 and increased risk of blood cancer4-6, but the reason for such abrupt functional decline after 70 years of age remains unclear. Here we sequenced 3,579 genomes from single cell-derived colonies of haematopoietic cells across 10 human subjects from 0 to 81 years of age. Haematopoietic stem cells or multipotent progenitors (HSC/MPPs) accumulated a mean of 17 mutations per year after birth and lost 30 base pairs per year of telomere length. Haematopoiesis in adults less than 65 years of age was massively polyclonal, with high clonal diversity and a stable population of 20,000-200,000 HSC/MPPs contributing evenly to blood production. By contrast, haematopoiesis in individuals aged over 75 showed profoundly decreased clonal diversity. In each of the older subjects, 30-60% of haematopoiesis was accounted for by 12-18 independent clones, each contributing 1-34% of blood production. Most clones had begun their expansion before the subject was 40 years old, but only 22% had known driver mutations. Genome-wide selection analysis estimated that between 1 in 34 and 1 in 12 non-synonymous mutations were drivers, accruing at constant rates throughout life, affecting more genes than identified in blood cancers. Loss of the Y chromosome conferred selective benefits in males. Simulations of haematopoiesis, with constant stem cell population size and constant acquisition of driver mutations conferring moderate fitness benefits, entirely explained the abrupt change in clonal structure in the elderly. Rapidly decreasing clonal diversity is a universal feature of haematopoiesis in aged humans, underpinned by pervasive positive selection acting on many more genes than currently identified.
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Envejecimiento , Hematopoyesis Clonal , Células Clonales , Longevidad , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/genética , Niño , Preescolar , Hematopoyesis Clonal/genética , Células Clonales/citología , Femenino , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patología , Células Madre Hematopoyéticas/citología , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Células Madre Multipotentes/citología , Adulto JovenRESUMEN
The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.
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Envejecimiento , Sistema Nervioso Entérico/citología , Feto/citología , Salud , Intestinos/citología , Intestinos/crecimiento & desarrollo , Ganglios Linfáticos/citología , Ganglios Linfáticos/crecimiento & desarrollo , Adulto , Animales , Niño , Enfermedad de Crohn/patología , Conjuntos de Datos como Asunto , Sistema Nervioso Entérico/anatomía & histología , Sistema Nervioso Entérico/embriología , Sistema Nervioso Entérico/crecimiento & desarrollo , Células Epiteliales/citología , Femenino , Feto/anatomía & histología , Feto/embriología , Humanos , Intestinos/embriología , Intestinos/inervación , Ganglios Linfáticos/embriología , Ganglios Linfáticos/patología , Ratones , Ratones Endogámicos C57BL , Organogénesis , Receptores de IgG/metabolismo , Transducción de Señal , Análisis Espacio-Temporal , Factores de TiempoRESUMEN
All normal somatic cells are thought to acquire mutations, but understanding of the rates, patterns, causes and consequences of somatic mutations in normal cells is limited. The uterine endometrium adopts multiple physiological states over a lifetime and is lined by a gland-forming epithelium1,2. Here, using whole-genome sequencing, we show that normal human endometrial glands are clonal cell populations with total mutation burdens that increase at about 29 base substitutions per year and that are many-fold lower than those of endometrial cancers. Normal endometrial glands frequently carry 'driver' mutations in cancer genes, the burden of which increases with age and decreases with parity. Cell clones with drivers often originate during the first decades of life and subsequently progressively colonize the epithelial lining of the endometrium. Our results show that mutational landscapes differ markedly between normal tissues-perhaps shaped by differences in their structure and physiology-and indicate that the procession of neoplastic change that leads to endometrial cancer is initiated early in life.
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Análisis Mutacional de ADN , Endometrio/citología , Endometrio/metabolismo , Epitelio/metabolismo , Salud , Mutación , Adulto , Edad de Inicio , Anciano , Anciano de 80 o más Años , Envejecimiento/genética , Carcinogénesis/genética , Células Clonales/citología , Neoplasias Endometriales/genética , Endometrio/patología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Epitelio/patología , Femenino , Humanos , Persona de Mediana Edad , Paridad/genética , Factores de Tiempo , Adulto JovenRESUMEN
Cardiovascular disease is the leading cause of death worldwide. Advanced insights into disease mechanisms and therapeutic strategies require a deeper understanding of the molecular processes involved in the healthy heart. Knowledge of the full repertoire of cardiac cells and their gene expression profiles is a fundamental first step in this endeavour. Here, using state-of-the-art analyses of large-scale single-cell and single-nucleus transcriptomes, we characterize six anatomical adult heart regions. Our results highlight the cellular heterogeneity of cardiomyocytes, pericytes and fibroblasts, and reveal distinct atrial and ventricular subsets of cells with diverse developmental origins and specialized properties. We define the complexity of the cardiac vasculature and its changes along the arterio-venous axis. In the immune compartment, we identify cardiac-resident macrophages with inflammatory and protective transcriptional signatures. Furthermore, analyses of cell-to-cell interactions highlight different networks of macrophages, fibroblasts and cardiomyocytes between atria and ventricles that are distinct from those of skeletal muscle. Our human cardiac cell atlas improves our understanding of the human heart and provides a valuable reference for future studies.
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Miocardio/citología , Análisis de la Célula Individual , Transcriptoma , Adipocitos/clasificación , Adipocitos/metabolismo , Adulto , Enzima Convertidora de Angiotensina 2/análisis , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Células Epiteliales/clasificación , Células Epiteliales/metabolismo , Epitelio , Femenino , Fibroblastos/clasificación , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Atrios Cardíacos/anatomía & histología , Atrios Cardíacos/citología , Atrios Cardíacos/inervación , Ventrículos Cardíacos/anatomía & histología , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/inervación , Homeostasis/inmunología , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Miocitos Cardíacos/clasificación , Miocitos Cardíacos/metabolismo , Neuronas/clasificación , Neuronas/metabolismo , Pericitos/clasificación , Pericitos/metabolismo , Receptores de Coronavirus/análisis , Receptores de Coronavirus/genética , Receptores de Coronavirus/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Células del Estroma/clasificación , Células del Estroma/metabolismoRESUMEN
Classic Hodgkin lymphoma (cHL) has a rich immune infiltrate, which is an intrinsic component of the neoplastic process. Malignant Hodgkin Reed-Sternberg cells (HRSCs) create an immunosuppressive microenvironment by the expression of regulatory molecules, preventing T-cell activation. It has also been demonstrated that mononuclear phagocytes (MNPs) in the vicinity of HRSCs express similar regulatory mechanisms in parallel, and their presence in tissue is associated with inferior patient outcomes. MNPs in cHL have hitherto been identified by a small number of canonical markers and are usually described as tumor-associated macrophages. The organization of MNP networks and interactions with HRSCs remains unexplored at high resolution. Here, we defined the global immune-cell composition of cHL and nonlymphoma lymph nodes, integrating data across single-cell RNA sequencing, spatial transcriptomics, and multiplexed immunofluorescence. We observed that MNPs comprise multiple subsets of monocytes, macrophages, and dendritic cells (DCs). Classical monocytes, macrophages and conventional DC2s were enriched in the vicinity of HRSCs, but plasmacytoid DCs and activated DCs were excluded. Unexpectedly, cDCs and monocytes expressed immunoregulatory checkpoints PD-L1, TIM-3, and the tryptophan-catabolizing protein IDO, at the same level as macrophages. Expression of these molecules increased with age. We also found that classical monocytes are important signaling hubs, potentially controlling the retention of cDC2 and ThExh via CCR1-, CCR4-, CCR5-, and CXCR3-dependent signaling. Enrichment of the cDC2-monocyte-macrophage network in diagnostic biopsies is associated with early treatment failure. These results reveal unanticipated complexity and spatial polarization within the MNP compartment, further demonstrating their potential roles in immune evasion by cHL.
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Enfermedad de Hodgkin , Humanos , Enfermedad de Hodgkin/diagnóstico , Células de Reed-Sternberg/metabolismo , Macrófagos/metabolismo , Monocitos/metabolismo , Inmunosupresores , Microambiente TumoralRESUMEN
Despite the recent advances in our understanding of the role of lipids, metabolites, and related enzymes in mediating kidney injury, there is limited integrated multi-omics data identifying potential metabolic pathways driving impaired kidney function. The limited availability of kidney biopsies from living donors with acute kidney injury has remained a major constraint. Here, we validated the use of deceased transplant donor kidneys as a good model to study acute kidney injury in humans and characterized these kidneys using imaging and multi-omics approaches. We noted consistent changes in kidney injury and inflammatory markers in donors with reduced kidney function. Neighborhood and correlation analyses of imaging mass cytometry data showed that subsets of kidney cells (proximal tubular cells and fibroblasts) are associated with the expression profile of kidney immune cells, potentially linking these cells to kidney inflammation. Integrated transcriptomic and metabolomic analysis of human kidneys showed that kidney arachidonic acid metabolism and seven other metabolic pathways were upregulated following diminished kidney function. To validate the arachidonic acid pathway in impaired kidney function we demonstrated increased levels of cytosolic phospholipase A2 protein and related lipid mediators (prostaglandin E2) in the injured kidneys. Further, inhibition of cytosolic phospholipase A2 reduced injury and inflammation in human kidney proximal tubular epithelial cells in vitro. Thus, our study identified cell types and metabolic pathways that may be critical for controlling inflammation associated with impaired kidney function in humans.
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Lesión Renal Aguda , Fenotipo , Humanos , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Lesión Renal Aguda/etiología , Masculino , Persona de Mediana Edad , Metabolómica/métodos , Femenino , Trasplante de Riñón/efectos adversos , Adulto , Citometría de Imagen/métodos , Riñón/patología , Riñón/metabolismo , Fosfolipasas A2/metabolismo , Ácido Araquidónico/metabolismo , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Transcriptoma , Dinoprostona/metabolismo , Dinoprostona/análisis , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Células Epiteliales/metabolismo , Células Epiteliales/patología , Biopsia , MultiómicaRESUMEN
Rare hematopoietic stem and progenitor cell (HSPC) pools outside the bone marrow (BM) contribute to blood production in stress and disease but remain ill-defined. Although nonmobilized peripheral blood (PB) is routinely sampled for clinical management, the diagnosis and monitoring potential of PB HSPCs remain untapped, as no healthy PB HSPC baseline has been reported. Here we comprehensively delineate human extramedullary HSPC compartments comparing spleen, PB, and mobilized PB to BM using single-cell RNA-sequencing and/or functional assays. We uncovered HSPC features shared by extramedullary tissues and others unique to PB. First, in contrast to actively dividing BM HSPCs, we found no evidence of substantial ongoing hematopoiesis in extramedullary tissues at steady state but report increased splenic HSPC proliferative output during stress erythropoiesis. Second, extramedullary hematopoietic stem cells/multipotent progenitors (HSCs/MPPs) from spleen, PB, and mobilized PB share a common transcriptional signature and increased abundance of lineage-primed subsets compared with BM. Third, healthy PB HSPCs display a unique bias toward erythroid-megakaryocytic differentiation. At the HSC/MPP level, this is functionally imparted by a subset of phenotypic CD71+ HSCs/MPPs, exclusively producing erythrocytes and megakaryocytes, highly abundant in PB but rare in other adult tissues. Finally, the unique erythroid-megakaryocytic-skewing of PB is perturbed with age in essential thrombocythemia and ß-thalassemia. Collectively, we identify extramedullary lineage-primed HSPC reservoirs that are nonproliferative in situ and report involvement of splenic HSPCs during demand-adapted hematopoiesis. Our data also establish aberrant composition and function of circulating HSPCs as potential clinical indicators of BM dysfunction.
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Hematopoyesis , Células Madre Hematopoyéticas , Adulto , Médula Ósea , Células de la Médula Ósea/fisiología , Eritropoyesis , Humanos , MegacariocitosRESUMEN
OBJECTIVES: To summarise the causes of ureteric damage and the current standard of care, discussing the risks and benefits of available therapeutic options. We then focus on the current and future solutions that can be provided by ureteric bioengineering and provide a description of the ideal characteristics of a bioengineered product. METHODS: We performed a literature search in February 2021 in: Google Scholar, Medline, and Web of Science. Three searches were conducted, investigating: (a) the epidemiology of ureteric pathology, (b) the current standard of care, and (c) the state of the art in ureteric bioengineering. RESULTS: The most-common causes of ureteric damage are iatrogenic injury and external trauma. Current approaches to treatment include stent placement or surgical reconstruction. Reconstruction can be done using either urological tissue or segments of the gastrointestinal tract. Limitations include scarring, strictures, and infections. Several bioengineered alternatives have been explored in animal studies, with variations in the choice of scaffold material, cellular seeding populations, and pre-implantation processing. Natural grafts and hybrid material appear to be associated with superior outcomes. Furthermore, seeding of the scaffold material with stem cells or differentiated urothelial cells allows for better function compared to acellular scaffolds. Some studies have attempted to pre-implant the graft in the omentum prior to reconstruction, but this has yet to prove any definitive benefits. CONCLUSION: There is an unmet clinical need for safer and more effective treatment for ureteric injuries. Urological bioengineering is a promising solution in preclinical studies. However, substantial scientific, logistic, and economic challenges must be addressed to harness its transformative potential in improving outcomes.
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Uréter , Enfermedades Urológicas , Animales , Bioingeniería , Constricción Patológica , Resultado del Tratamiento , Uréter/cirugíaRESUMEN
Neutrophils play a key role in innate immunity. As the dominant circulating phagocyte, they are rapidly recruited from the bloodstream to sites of infection or injury to internalize and destroy microbes. More recently, neutrophils have been identified in uninfected organs, challenging the classical view of their function. Here we show that neutrophils were present in lymph nodes (LNs) in homeostasis. Using flow cytometry and confocal imaging, we identified neutrophils within LNs in naive, unchallenged mice, including LNs draining the skin, lungs, and gastrointestinal tract. Neutrophils were enriched within specific anatomical regions, in the interfollicular zone, a site of T cell activation. Intravital two-photon microscopy demonstrated that LN neutrophils were motile, trafficked into LNs from both blood and tissues via high endothelial venules and afferent lymphatics, respectively, and formed interactions with dendritic cells in LNs. Murine and human LN neutrophils had a distinct phenotype compared with circulating neutrophils, with higher major histocompatibility complex II (MHCII) expression, suggesting a potential role in CD4 T cell activation. Upon ex vivo stimulation with IgG immune complex (IC), neutrophils up-regulated expression of MHCII and costimulatory molecules and increased T cell activation. In vivo, neutrophils were capable of delivering circulating IC to LNs, suggesting a broader functional remit. Overall, our data challenge the perception that neutrophil patrol is limited to the circulation in homeostasis, adding LNs to their routine surveillance territory.
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Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/inmunología , Ganglios Linfáticos/inmunología , Vasos Linfáticos/inmunología , Activación de Linfocitos/inmunología , Neutrófilos/inmunología , Animales , Movimiento Celular , Femenino , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Interferón gamma , Ratones , Ratones Endogámicos C57BL , FenotipoRESUMEN
Chinese hamster ovary (CHO) cell lines are the most widely used in vitro cells for research and production of recombinant proteins such as rhGH, tPA, and erythropoietin. We aimed to investigate changes in protein profiles after cryopreservation using 2D-DIGE MALDI-TOF MS and network pathway analysis. The proteome changes that occur in CHO cells between freshly prepared cells and cryopreserved cells with and without Me2SO were compared to determine the key proteins and pathways altered during recovery from cryopreservation. A total of 54 proteins were identified and successfully matched to 37 peptide mass fingerprints (PMF). 14 protein spots showed an increase while 23 showed decrease abundance in the Me2SO free group compared to the control. The proteins with increased abundance included vimentin, heat shock protein 60 kDa, mitochondrial, heat shock 70 kDa protein 9, protein disulfide-isomerase A3, voltage-dependent anion-selective channel protein 2. Those with a decrease in abundance were myotubularin, glutathione peroxidase, enolase, phospho glyceromutase, chloride intracellular channel protein 1. The main canonical functional pathway affected involved the unfolded protein response, aldosterone Signaling in Epithelial Cells, 14-3-3-mediated signaling. 2D-DIGE MALDI TOF mass spectrometry and network pathway analysis revealed the differential proteome expression of FreeStyle CHO cells after cryopreservation with and without 5% Me2SOto involve pathways related to post-translational modification, protein folding and cell death and survival (score = 56, 22 focus molecules). This study revealed, for the first time to our knowledge the proteins and their regulated pathways involved in the cryoprotective action of 5% Me2SO. The use of 5% Me2SO as a cryoprotectant maintained the CHO cell proteome in the cryopreserved cells, similar to that of fresh CHO cells.
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Criopreservación , Crioprotectores/farmacología , Dimetilsulfóxido/farmacología , Proteoma/efectos de los fármacos , Animales , Células CHO , Cricetulus , Proteoma/metabolismo , ProteómicaRESUMEN
As the dimensionality, throughput and complexity of cytometry data increases, so does the demand for user-friendly, interactive analysis tools that leverage high-performance machine learning frameworks. Here we introduce FlowAtlas: an interactive web application that enables dimensionality reduction of cytometry data without down-sampling and that is compatible with datasets stained with non-identical panels. FlowAtlas bridges the user-friendly environment of FlowJo and computational tools in Julia developed by the scientific machine learning community, eliminating the need for coding and bioinformatics expertise. New population discovery and detection of rare populations in FlowAtlas is intuitive and rapid. We demonstrate the capabilities of FlowAtlas using a human multi-tissue, multi-donor immune cell dataset, highlighting key immunological findings. FlowAtlas is available at https://github.com/gszep/FlowAtlas.jl.git.
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Biología Computacional , Citometría de Flujo , Inmunofenotipificación , Programas Informáticos , Humanos , Inmunofenotipificación/métodos , Citometría de Flujo/métodos , Biología Computacional/métodos , Aprendizaje AutomáticoRESUMEN
Aberrant vascular smooth muscle cell (VSMC) homeostasis and proliferation characterize vascular diseases causing heart attack and stroke. Here we elucidate molecular determinants governing VSMC proliferation by reconstructing gene regulatory networks from single-cell transcriptomics and epigenetic profiling. We detect widespread activation of enhancers at disease-relevant loci in proliferation-predisposed VSMCs. We compared gene regulatory network rewiring between injury-responsive and nonresponsive VSMCs, which suggested shared transcription factors but differing target loci between VSMC states. Through in silico perturbation analysis, we identified and prioritized previously unrecognized regulators of proliferation, including RUNX1 and TIMP1. Moreover, we showed that the pioneer transcription factor RUNX1 increased VSMC responsiveness and that TIMP1 feeds back to promote VSMC proliferation through CD74-mediated STAT3 signaling. Both RUNX1 and the TIMP1-CD74 axis were expressed in human VSMCs, showing low levels in normal arteries and increased expression in disease, suggesting clinical relevance and potential as vascular disease targets.
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The complex and dynamic cellular composition of the human endometrium remains poorly understood. Previous endometrial single-cell atlases profiled few donors and lacked consensus in defining cell types. We introduce the Human Endometrial Cell Atlas (HECA), a high-resolution single-cell reference atlas (313,527 cells) combining published and new endometrial single-cell transcriptomics datasets of 63 women with and without endometriosis. HECA assigns consensus and identifies previously unreported cell types, mapped in situ using spatial transcriptomics and validated using a new independent single-nuclei dataset (312,246 nuclei, 63 donors). In the functionalis, we identify intricate stromal-epithelial cell coordination via transforming growth factor beta (TGFß) signaling. In the basalis, we define signaling between fibroblasts and an epithelial population expressing progenitor markers. Integration of HECA with large-scale endometriosis genome-wide association study data pinpoints decidualized stromal cells and macrophages as most likely dysregulated in endometriosis. The HECA is a valuable resource for studying endometrial physiology and disorders, and for guiding microphysiological in vitro systems development.
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The immune system comprises multiple cell lineages and heterogeneous subsets found in blood and tissues throughout the body. While human immune responses differ between sites and over age, the underlying sources of variation remain unclear as most studies are limited to peripheral blood. Here, we took a systems approach to comprehensively profile RNA and surface protein expression of over 1.25 million immune cells isolated from blood, lymphoid organs, and mucosal tissues of 24 organ donors aged 20-75 years. We applied a multimodal classifier to annotate the major immune cell lineages (T cells, B cells, innate lymphoid cells, and myeloid cells) and their corresponding subsets across the body, leveraging probabilistic modeling to define bases for immune variations across donors, tissue, and age. We identified dominant tissue-specific effects on immune cell composition and function across lineages for lymphoid sites, intestines, and blood-rich tissues. Age-associated effects were intrinsic to both lineage and site as manifested by macrophages in mucosal sites, B cells in lymphoid organs, and T and NK cells in blood-rich sites. Our results reveal tissue-specific signatures of immune homeostasis throughout the body and across different ages. This information provides a basis for defining the transcriptional underpinnings of immune variation and potential associations with disease-associated immune pathologies across the human lifespan.
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Skeletal muscle aging is a key contributor to age-related frailty and sarcopenia with substantial implications for global health. Here we profiled 90,902 single cells and 92,259 single nuclei from 17 donors to map the aging process in the adult human intercostal muscle, identifying cellular changes in each muscle compartment. We found that distinct subsets of muscle stem cells exhibit decreased ribosome biogenesis genes and increased CCL2 expression, causing different aging phenotypes. Our atlas also highlights an expansion of nuclei associated with the neuromuscular junction, which may reflect re-innervation, and outlines how the loss of fast-twitch myofibers is mitigated through regeneration and upregulation of fast-type markers in slow-twitch myofibers with age. Furthermore, we document the function of aging muscle microenvironment in immune cell attraction. Overall, we present a comprehensive human skeletal muscle aging resource ( https://www.muscleageingcellatlas.org/ ) together with an in-house mouse muscle atlas to study common features of muscle aging across species.
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Envejecimiento , Músculo Esquelético , Humanos , Envejecimiento/fisiología , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Animales , Ratones , Adulto , Anciano , Sarcopenia/patología , Sarcopenia/metabolismo , Masculino , Unión Neuromuscular/metabolismo , Persona de Mediana Edad , FemeninoRESUMEN
Spatially resolved omics technologies reveal context-dependent cellular regulatory networks in tissues of interest. Beyond transcriptome analysis, information on epigenetic traits and chromatin accessibility can provide further insights on gene regulation in health and disease. Nevertheless, compared to the enormous advancements in spatial transcriptomics technologies, the field of spatial epigenomics is much younger and still underexplored. In this study, we report laser capture microdissection coupled to ATAC-seq (LCM-ATAC-seq) applied to fresh frozen samples for the spatial characterization of chromatin accessibility. We first demonstrate the efficient use of LCM coupled to in situ tagmentation and evaluate its performance as a function of cell number, microdissected areas, and tissue type. Further, we demonstrate its use for the targeted chromatin accessibility analysis of discrete contiguous or scattered cell populations in tissues via single-nuclei capture based on immunostaining for specific cellular markers.
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Secuenciación de Inmunoprecipitación de Cromatina , Cromatina , Cromatina/genética , Captura por Microdisección con Láser , Perfilación de la Expresión Génica , CongelaciónRESUMEN
Single-cell transcriptomics has allowed unprecedented resolution of cell types/states in the human lung, but their spatial context is less well defined. To (re)define tissue architecture of lung and airways, we profiled five proximal-to-distal locations of healthy human lungs in depth using multi-omic single cell/nuclei and spatial transcriptomics (queryable at lungcellatlas.org ). Using computational data integration and analysis, we extend beyond the suspension cell paradigm and discover macro and micro-anatomical tissue compartments including previously unannotated cell types in the epithelial, vascular, stromal and nerve bundle micro-environments. We identify and implicate peribronchial fibroblasts in lung disease. Importantly, we discover and validate a survival niche for IgA plasma cells in the airway submucosal glands (SMG). We show that gland epithelial cells recruit B cells and IgA plasma cells, and promote longevity and antibody secretion locally through expression of CCL28, APRIL and IL-6. This new 'gland-associated immune niche' has implications for respiratory health.