RESUMEN
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.
Asunto(s)
Neoplasias Hepáticas , FN-kappa B , Humanos , FN-kappa B/metabolismo , Proteínas Quinasas/metabolismo , Necroptosis , Inflamación/patología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , ApoptosisRESUMEN
Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.
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Células Epiteliales Alveolares/metabolismo , COVID-19/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , SARS-CoV-2/metabolismo , Células Epiteliales Alveolares/patología , Células Epiteliales Alveolares/virología , Animales , Antivirales , COVID-19/genética , COVID-19/patología , Chlorocebus aethiops , Efecto Citopatogénico Viral , Citoesqueleto , Evaluación Preclínica de Medicamentos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Células Madre Pluripotentes Inducidas/virología , Fosfoproteínas/genética , Transporte de Proteínas , Proteoma/genética , SARS-CoV-2/genética , Transducción de Señal , Células Vero , Tratamiento Farmacológico de COVID-19RESUMEN
Emphysema and chronic obstructive pulmonary disease (COPD) most commonly result from the effects of environmental exposures in genetically susceptible individuals. Genome-wide association studies have implicated ADGRG6 in COPD and reduced lung function, and a limited number of studies have examined the role of ADGRG6 in cells representative of the airway. However, the ADGRG6 locus is also associated with DLCO/VA, an indicator of gas exchange efficiency and alveolar function. Here, we sought to evaluate the mechanistic contributions of ADGRG6 to homeostatic function and disease in type 2 alveolar epithelial cells. We applied an inducible CRISPR interference (CRISPRi) human induced pluripotent stem cell (iPSC) platform to explore ADGRG6 function in iPSC-derived AT2s (iAT2s). We demonstrate that ADGRG6 exerts pleiotropic effects on iAT2s including regulation of focal adhesions, cytoskeleton, tight junctions, and proliferation. Moreover, we find that ADGRG6 knockdown in cigarette smoke-exposed iAT2s alters cellular responses to injury, downregulating apical complexes in favor of proliferation. Our work functionally characterizes the COPD GWAS gene ADGRG6 in human alveolar epithelium.
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Células Madre Pluripotentes Inducidas , Enfermedad Pulmonar Obstructiva Crónica , Receptores Acoplados a Proteínas G , Humanos , Células Epiteliales Alveolares/metabolismo , Células Epiteliales/metabolismo , Estudio de Asociación del Genoma Completo , Células Madre Pluripotentes Inducidas/metabolismo , Pulmón/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Cancer testis antigens (CTAs) are an extensive gene family with a unique expression pattern restricted to germ cells, but aberrantly reactivated in cancer tissues. Studies indicate that the expression (or re-expression) of CTAs within the MAGE-A family is common in hepatocellular carcinoma (HCC). However, no systematic characterization has yet been reported. The aim of this study is to perform a comprehensive profile of CTA de-regulation in HCC and experimentally evaluate the role of MAGEA3 as a driver of HCC progression. The transcriptomic analysis of 44 multi-regionally sampled HCCs from 12 patients identified high intra-tumor heterogeneity of CTAs. In addition, a subset of CTAs was significantly overexpressed in histologically poorly differentiated regions. Further analysis of CTAs in larger patient cohorts revealed high CTA expression related to worse overall survival and several other markers of poor prognosis. Functional analysis of MAGEA3 was performed in human HCC cell lines by gene silencing and in a genetic mouse model by overexpression of MAGEA3 in the liver. Knockdown of MAGEA3 decreased cell proliferation, colony formation and increased apoptosis. MAGEA3 overexpression was associated with more aggressive tumors in vivo. In conclusion MAGEA3 enhances tumor progression and should be considered as a novel therapeutic target in HCC.
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Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Proteínas de Neoplasias/genética , Testículo/inmunología , Transcriptoma , Apoptosis/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/inmunología , Proliferación Celular/genética , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/inmunología , Masculino , Pronóstico , Regulación hacia ArribaRESUMEN
There is an urgent need to understand how SARS-CoV-2 infects the airway epithelium and in a subset of individuals leads to severe illness or death. Induced pluripotent stem cells (iPSCs) provide a near limitless supply of human cells that can be differentiated into cell types of interest, including airway epithelium, for disease modeling. We present a human iPSC-derived airway epithelial platform, composed of the major airway epithelial cell types, that is permissive to SARS-CoV-2 infection. Subsets of iPSC-airway cells express the SARS-CoV-2 entry factors angiotensin-converting enzyme 2 (ACE2), and transmembrane protease serine 2 (TMPRSS2). Multiciliated cells are the primary initial target of SARS-CoV-2 infection. On infection with SARS-CoV-2, iPSC-airway cells generate robust interferon and inflammatory responses, and treatment with remdesivir or camostat mesylate causes a decrease in viral propagation and entry, respectively. In conclusion, iPSC-derived airway cells provide a physiologically relevant in vitro model system to interrogate the pathogenesis of, and develop treatment strategies for, COVID-19 pneumonia.
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COVID-19 , Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Células Epiteliales , Humanos , SARS-CoV-2RESUMEN
Alpha-1 antitrypsin deficiency (AATD) is most commonly caused by the Z mutation, a single-base substitution that leads to AAT protein misfolding and associated liver and lung disease. In this study, we apply adenine base editors to correct the Z mutation in patient induced pluripotent stem cells (iPSCs) and iPSC-derived hepatocytes (iHeps). We demonstrate that correction of the Z mutation in patient iPSCs reduces aberrant AAT accumulation and increases its secretion. Adenine base editing (ABE) of differentiated iHeps decreases ER stress in edited cells, as demonstrated by single-cell RNA sequencing. We find ABE to be highly efficient in iPSCs and do not identify off-target genomic mutations by whole-genome sequencing. These results reveal the feasibility and utility of base editing to correct the Z mutation in AATD patient cells.
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Adenina , Sistemas CRISPR-Cas , Edición Génica , Hepatocitos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Deficiencia de alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/terapia , alfa 1-Antitripsina/genética , Biomarcadores , Diferenciación Celular/genética , Células Cultivadas , Estrés del Retículo Endoplásmico , Expresión Génica , Hepatocitos/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Mutación , alfa 1-Antitripsina/químicaRESUMEN
Alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, are typically identified through the use of the canonical markers, SFTPC and ABCA3. Self-renewing AEC2-like cells have been generated from human induced pluripotent stem cells (iPSCs) through the use of knock-in SFTPC fluorochrome reporters. However, developmentally, SFTPC expression onset begins in the fetal distal lung bud tip and thus is not specific to mature AEC2s. Furthermore, SFTPC reporters appear to identify only those iPSC-derived AEC2s (iAEC2s) expressing the highest SFTPC levels. Here, we generate an ABCA3 knock-in GFP fusion reporter (ABCA3:GFP) that enables the purification of iAEC2s while allowing visualization of lamellar bodies, organelles associated with AEC2 maturation. Using an SFTPCtdTomato and ABCA3:GFP bifluorescent line for in vitro distal lung-directed differentiation, we observe later onset of ABCA3:GFP expression and broader identification of the subsequently emerging iAEC2 population based on ABCA3:GFP expression compared with SFTPCtdTomato expression. Comparing ABCA3:GFP/SFTPCtdTomato double-positive with ABCA3:GFP single-positive (SP) cells by RNA sequencing and functional studies reveals iAEC2 cellular heterogeneity with both populations functionally processing surfactant proteins but the SP cells exhibiting faster growth kinetics, increased clonogenicity, increased expression of progenitor markers, lower levels of SFTPC expression, and lower levels of AEC2 maturation markers. Over time, we observe that each population (double-positive and SP) gives rise to the other and each can serve as the parents of indefinitely self-renewing iAEC2 progeny. Our results indicate that iAEC2s are a heterogeneous population of cells with differing proliferation versus maturation properties, the majority of which can be tracked and purified using the ABCA3:GFP reporter or surrogate cell surface proteins, such as SLC34A2 and CPM.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Células Epiteliales Alveolares/citología , Células Madre Pluripotentes Inducidas/citología , Alveolos Pulmonares/citología , Proteína C Asociada a Surfactante Pulmonar/metabolismo , Diferenciación Celular/fisiología , Células Epiteliales/metabolismo , Humanos , Pulmón/metabolismo , Proteínas Asociadas a Surfactante Pulmonar/metabolismoRESUMEN
BACKGROUND & AIMS: Cholangiocarcinoma (CCA), a deadly malignancy of the bile ducts, can be classified based on its anatomical location into either intrahepatic (iCCA) or extrahepatic (eCCA), each with different pathogenesis and clinical management. There is limited understanding of the molecular landscape of eCCA and no targeted therapy with clinical efficacy has been approved. We aimed to provide a molecular classification of eCCA and identify potential targets for molecular therapies. METHODS: An integrative genomic analysis of an international multicenter cohort of 189 eCCA cases was conducted. Genomic analysis included whole-genome expression, targeted DNA-sequencing and immunohistochemistry. Molecular findings were validated in an external set of 181 biliary tract tumors from the ICGC. RESULTS: KRAS (36.7%), TP53 (34.7%), ARID1A (14%) and SMAD4 (10.7%) were the most prevalent mutations, with â¼25% of tumors having a putative actionable genomic alteration according to OncoKB. Transcriptome-based unsupervised clustering helped us define 4 molecular classes of eCCA. Tumors classified within the Metabolic class (19%) showed a hepatocyte-like phenotype with activation of the transcription factor HNF4A and enrichment in gene signatures related to bile acid metabolism. The Proliferation class (23%), more common in patients with distal CCA, was characterized by enrichment of MYC targets, ERBB2 mutations/amplifications and activation of mTOR signaling. The Mesenchymal class (47%) was defined by signatures of epithelial-mesenchymal transition, aberrant TGFß signaling and poor overall survival. Finally, tumors in the Immune class (11%) had a higher lymphocyte infiltration, overexpression of PD-1/PD-L1 and molecular features associated with a better response to immune checkpoint inhibitors. CONCLUSION: An integrative molecular characterization identified distinct subclasses of eCCA. Genomic traits of each class provide the rationale for exploring patient stratification and novel therapeutic approaches. LAY SUMMARY: Targeted therapies have not been approved for the treatment of extrahepatic cholangiocarcinoma. We performed a multi-platform molecular characterization of this tumor in a cohort of 189 patients. These analyses revealed 4 novel transcriptome-based molecular classes of extrahepatic cholangiocarcinoma and identified â¼25% of tumors with actionable genomic alterations, which has potential prognostic and therapeutic implications.
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Neoplasias de los Conductos Biliares , Colangiocarcinoma , Terapia Molecular Dirigida/métodos , Análisis de Secuencia de ADN/métodos , Transducción de Señal/genética , Anciano , Antígeno B7-H1/genética , Neoplasias de los Conductos Biliares/tratamiento farmacológico , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/patología , Colangiocarcinoma/tratamiento farmacológico , Colangiocarcinoma/genética , Colangiocarcinoma/patología , Estudios de Cohortes , Descubrimiento de Drogas , Europa (Continente)/epidemiología , Femenino , Estudio de Asociación del Genoma Completo/métodos , Factor Nuclear 4 del Hepatocito/genética , Humanos , Inmunohistoquímica , Masculino , Pronóstico , Receptor de Muerte Celular Programada 1/genética , Receptor ErbB-2/genética , Estados Unidos/epidemiologíaRESUMEN
The growing burden of liver fibrosis and lack of effective antifibrotic therapies highlight the need for identification of pathways and complementary model systems of hepatic fibrosis. A rare, monogenic disorder in which children with mutations in mannose phosphate isomerase (MPI) develop liver fibrosis led us to explore the function of MPI and mannose metabolism in liver development and adult liver diseases. Herein, analyses of transcriptomic data from three human liver cohorts demonstrate that MPI gene expression is down-regulated proportionate to fibrosis in chronic liver diseases, including nonalcoholic fatty liver disease and hepatitis B virus. Depletion of MPI in zebrafish liver in vivo and in human hepatic stellate cell (HSC) lines in culture activates fibrotic responses, indicating that loss of MPI promotes HSC activation. We further demonstrate that mannose supplementation can attenuate HSC activation, leading to reduced fibrogenic activation in zebrafish, culture-activated HSCs, and in ethanol-activated HSCs. Conclusion: These data indicate the prospect that modulation of mannose metabolism pathways could reduce HSC activation and improve hepatic fibrosis.
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Células Estrelladas Hepáticas/fisiología , Cirrosis Hepática/etiología , Manosa-6-Fosfato Isomerasa/fisiología , Manosa/farmacología , Animales , Células Cultivadas , Glicosilación , Humanos , Masculino , Factor de Crecimiento Derivado de Plaquetas/fisiología , Transducción de Señal/fisiología , Pez CebraRESUMEN
AIMS: Access to tissue in patients with hepatocellular carcinoma (HCC) is limited compared to other malignancies, particularly at advanced stages. This has precluded a thorough characterisation of molecular drivers of HCC dissemination, particularly in relation to distant metastases. Biomarker assessment is restricted to early stages, and paired primary-metastatic comparisons between samples from the same patient are difficult. METHODS AND RESULTS: We report the evaluation of 88 patients with HCC who underwent autopsy, including multiregional sampling of primary and metastatic sites totalling 230 nodules analysed. The study included morphological assessment, immunohistochemistry and mutation status of the TERT promoter, the most frequently mutated gene in HCC. We confirm a strong predilection of HCC for lung dissemination, including subclinical micrometastases (unrecognised during imaging and macroscopic examinations) in 30% of patients with disseminated disease. Size of dominant tumour nodule; multinodularity; macrovascular invasion; high histological, nuclear and architectural grades; and cellular crowding were associated with the presence of extrahepatic metastasis. Among the immunohistochemistry markers tested, metastatic nodules had significantly higher K19 and EpCAM expression than primary liver tumours. Morphological and immunohistochemical features showed that metastatic HCC could be traced back to the primary tumour, sometimes to a specific hepatic nodule. CONCLUSIONS: This study suggests limited heterogeneity in metastatic sites compared to primary tumour sites.
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Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/secundario , Heterogeneidad Genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Anciano , Autopsia , Biomarcadores de Tumor/análisis , Brasil , Diferenciación Celular , Estudios de Cohortes , Molécula de Adhesión Celular Epitelial/biosíntesis , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Modelos Logísticos , Neoplasias Pulmonares/secundario , Masculino , Persona de Mediana Edad , Mutación , Metástasis de la Neoplasia , Fenotipo , Telomerasa/genéticaRESUMEN
BACKGROUND & AIMS: Agents that induce an immune response against tumors by altering T-cell regulation have increased survival times of patients with advanced-stage tumors, such as melanoma or lung cancer. We aimed to characterize molecular features of immune cells that infiltrate hepatocellular carcinomas (HCCs) to determine whether these types of agents might be effective against liver tumors. METHODS: We analyzed HCC samples from 956 patients. We separated gene expression profiles from tumor, stromal, and immune cells using a non-negative matrix factorization algorithm. We then analyzed the gene expression pattern of inflammatory cells in HCC tumor samples. We correlated expression patterns with the presence of immune cell infiltrates and immune regulatory molecules, determined by pathology and immunohistochemical analyses, in a training set of 228 HCC samples. We validated the correlation in a validation set of 728 tumor samples. Using data from 190 tumors in the Cancer Genome Atlas, we correlated immune cell gene expression profiles with numbers of chromosomal aberrations (based on single-nucleotide polymorphism array) and mutations (exome sequence data). RESULTS: We found approximately 25% of HCCs to have markers of an inflammatory response, with high expression levels of the CD274 molecule (programmed death-ligand 1) and programmed cell death 1, markers of cytolytic activity, and fewer chromosomal aberrations. We called this group of tumors the Immune class. It contained 2 subtypes, characterized by markers of an adaptive T-cell response or exhausted immune response. The exhausted immune response subclass expressed many genes regulated by transforming growth factor beta 1 that mediate immunosuppression. We did not observe any differences in numbers of mutations or expression of tumor antigens between the immune-specific class and other HCCs. CONCLUSIONS: In an analysis of HCC samples from 956 patients, we found almost 25% to express markers of an inflammatory response. We identified 2 subclasses, characterized by adaptive or exhausted immune responses. These findings indicate that some HCCs might be susceptible to therapeutic agents designed to block the regulatory pathways in T cells, such as programmed death-ligand 1, programmed cell death 1, or transforming growth factor beta 1 inhibitors.
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Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/inmunología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/inmunología , Factor de Crecimiento Transformador beta1/genética , Inmunidad Adaptativa/genética , Anciano , Antígeno B7-H1/metabolismo , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/patología , Metilación de ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Tolerancia Inmunológica/genética , Inmunohistoquímica , Inmunofenotipificación , Neoplasias Hepáticas/química , Neoplasias Hepáticas/patología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Masculino , Persona de Mediana Edad , Mutación , Receptor de Muerte Celular Programada 1/metabolismo , Transducción de Señal , Transcriptoma , Microambiente Tumoral/inmunología , beta Catenina/genéticaRESUMEN
BACKGROUND: Adventitious root (AR) formation is a critical step in vegetative propagation of most ornamental plants, such as carnation. AR formation from stem cuttings is usually divided into several stages according to physiological and metabolic markers. Auxin is often applied exogenously to promote the development of ARs on stem cuttings of difficult-to-root genotypes. RESULTS: By whole transcriptome sequencing, we identified the genes involved in AR formation in carnation cuttings and in response to exogenous auxin. Their expression profiles have been analysed through RNA-Seq during a time-course experiment in the stem cutting base of two cultivars with contrasting efficiencies of AR formation. We explored the kinetics of root primordia formation in these two cultivars and in response to exogenously-applied auxin through detailed histological and physiological analyses. CONCLUSIONS: Our results provide, for the first time, a number of molecular, histological and physiological markers that characterize the different stages of AR formation in this species and that could be used to monitor adventitious rooting on a wide collection of carnation germplasm with the aim to identify the best-rooting cultivars for breeding purposes.
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Dianthus/genética , Perfilación de la Expresión Génica/métodos , Raíces de Plantas/genética , Transcriptoma/genética , Dianthus/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Análisis por Micromatrices/métodos , Proteínas de Plantas/biosíntesis , Raíces de Plantas/crecimiento & desarrollo , Tallos de la Planta/genética , Tallos de la Planta/crecimiento & desarrolloRESUMEN
BACKGROUND: Vernalization is an obligatory requirement of extended exposure to low temperatures to induce flowering in certain plants. It is the most important factor affecting flowering time and quality in Easter lily (Lilium longiflorum). Exposing the bulbs to 4 °C gradually decreases flowering time up to 50% compared to non-vernalized plants. We aim to understand the molecular regulation of vernalization in Easter lily, for which we characterized the global expression in lily bulb meristems after 0, 2, 5, 7 and 9 weeks of incubation at 4 °C. RESULTS: We assembled de-novo a transcriptome which, after filtering, yielded 121,572 transcripts and 42,430 genes which hold 15,414 annotated genes, with up to 3,657 GO terms. This extensive annotation was mapped to the more general GO slim plant with a total of 94 terms. The response to cold exposure was summarized in 6 expression clusters, providing useful patterns for dissecting the dynamics of vernalization in lily. The functional annotation (GO and GO slim plant) was used to group transcripts in gene sets. Analysis of these gene sets and profiles revealed that most of the enriched functions among genes up-regulated by cold exposure were related to epigenetic processes and chromatin remodeling. Candidate vernalization genes in lily were selected based on their sequence similarity to known regulators of flowering in other species. CONCLUSIONS: We present a detailed analysis of gene expression dynamics during vernalization in Lilium, covering several time points and accounting for biological variation by the use of replicates. The resulting collection of transcripts and novel isoforms provides a useful resource for studying the changes occurring during vernalization at a fine level. The selected potential candidate genes can shed light on the regulation of this process.
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Flores/crecimiento & desarrollo , Perfilación de la Expresión Génica/métodos , Genes de Plantas , Lilium/genética , Frío , Epigénesis Genética , Flores/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Ontología de Genes , Lilium/fisiología , Anotación de Secuencia MolecularRESUMEN
Huge research effort has been invested over many years to determine the phenotypes of natural or artificial mutations in HIV proteins--interpretation of mutation phenotypes is an invaluable source of new knowledge. The results of this research effort are recorded in the scientific literature, but it is difficult for virologists to rapidly find it. Manually locating data on phenotypic variation within the approximately 270,000 available HIV-related research articles, or the further 1,500 articles that are published each month is a daunting task. Accordingly, the HIV research community would benefit from a resource cataloguing the available HIV mutation literature. We have applied computational text-mining techniques to parse and map mutagenesis and polymorphism information from the HIV literature, have enriched the data with ancillary information and have developed a public, web-based interface through which it can be intuitively explored: the HIV mutation browser. The current release of the HIV mutation browser describes the phenotypes of 7,608 unique mutations at 2,520 sites in the HIV proteome, resulting from the analysis of 120,899 papers. The mutation information for each protein is organised in a residue-centric manner and each residue is linked to the relevant experimental literature. The importance of HIV as a global health burden advocates extensive effort to maximise the efficiency of HIV research. The HIV mutation browser provides a valuable new resource for the research community. The HIV mutation browser is available at: http://hivmut.org.
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Biología Computacional/métodos , Bases de Datos Genéticas , Infecciones por VIH/virología , VIH-1/genética , Mutación/genética , Secuencia de Aminoácidos , Humanos , Datos de Secuencia MolecularRESUMEN
Mutations in ATP-binding cassette A3 (ABCA3), a phospholipid transporter critical for surfactant homeostasis in pulmonary alveolar type II epithelial cells (AEC2s), are the most common genetic causes of childhood interstitial lung disease (chILD). Treatments for patients with pathological variants of ABCA3 mutations are limited, in part due to a lack of understanding of disease pathogenesis resulting from an inability to access primary AEC2s from affected children. Here, we report the generation of AEC2s from affected patient induced pluripotent stem cells (iPSCs) carrying homozygous versions of multiple ABCA3 mutations. We generated syngeneic CRISPR/Cas9 gene-corrected and uncorrected iPSCs and ABCA3-mutant knockin ABCA3:GFP fusion reporter lines for in vitro disease modeling. We observed an expected decreased capacity for surfactant secretion in ABCA3-mutant iPSC-derived AEC2s (iAEC2s), but we also found an unexpected epithelial-intrinsic aberrant phenotype in mutant iAEC2s, presenting as diminished progenitor potential, increased NFκB signaling, and the production of pro-inflammatory cytokines. The ABCA3:GFP fusion reporter permitted mutant-specific, quantifiable characterization of lamellar body size and ABCA3 protein trafficking, functional features that are perturbed depending on ABCA3 mutation type. Our disease model provides a platform for understanding ABCA3 mutation-mediated mechanisms of alveolar epithelial cell dysfunction that may trigger chILD pathogenesis.
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Transportadoras de Casetes de Unión a ATP , Enfermedades Pulmonares Intersticiales , Células Madre Pluripotentes , Humanos , Células Epiteliales Alveolares/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Pulmón/patología , Enfermedades Pulmonares Intersticiales/genética , Enfermedades Pulmonares Intersticiales/metabolismo , Enfermedades Pulmonares Intersticiales/patología , Mutación , Células Madre Pluripotentes/metabolismo , Tensoactivos/metabolismoRESUMEN
Alveolar epithelial type I cells (AT1s) line the gas exchange barrier of the distal lung and have been historically challenging to isolate or maintain in cell culture. Here, we engineer a human in vitro AT1 model system via directed differentiation of induced pluripotent stem cells (iPSCs). We use primary adult AT1 global transcriptomes to suggest benchmarks and pathways, such as Hippo-LATS-YAP/TAZ signaling, enriched in these cells. Next, we generate iPSC-derived alveolar epithelial type II cells (AT2s) and find that nuclear YAP signaling is sufficient to promote a broad transcriptomic shift from AT2 to AT1 gene programs. The resulting cells express a molecular, morphologic, and functional phenotype reminiscent of human AT1 cells, including the capacity to form a flat epithelial barrier producing characteristic extracellular matrix molecules and secreted ligands. Our results provide an in vitro model of human alveolar epithelial differentiation and a potential source of human AT1s.
Asunto(s)
Células Epiteliales Alveolares , Diferenciación Celular , Células Madre Pluripotentes Inducidas , Humanos , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Transducción de Señal , Células Cultivadas , Transcriptoma/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismoRESUMEN
While the generation of many lineages from pluripotent stem cells has resulted in basic discoveries and clinical trials, the derivation of tissue-specific mesenchyme via directed differentiation has markedly lagged. The derivation of lung-specific mesenchyme is particularly important since this tissue plays crucial roles in lung development and disease. Here we generate a mouse induced pluripotent stem cell (iPSC) line carrying a lung-specific mesenchymal reporter/lineage tracer. We identify the pathways (RA and Shh) necessary to specify lung mesenchyme and find that mouse iPSC-derived lung mesenchyme (iLM) expresses key molecular and functional features of primary developing lung mesenchyme. iLM recombined with engineered lung epithelial progenitors self-organizes into 3D organoids with juxtaposed layers of epithelium and mesenchyme. Co-culture increases yield of lung epithelial progenitors and impacts epithelial and mesenchymal differentiation programs, suggesting functional crosstalk. Our iPSC-derived population thus provides an inexhaustible source of cells for studying lung development, modeling diseases, and developing therapeutics.
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Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Animales , Ratones , Diferenciación Celular , Tórax , MesodermoRESUMEN
Hemogenic endothelial cells (HECs) are specialized cells that undergo endothelial-to-hematopoietic transition (EHT) to give rise to the earliest precursors of hematopoietic progenitors that will eventually sustain hematopoiesis throughout the lifetime of an organism. Although HECs are thought to be primarily limited to the aorta-gonad-mesonephros (AGM) during early development, EHT has been described in various other hematopoietic organs and embryonic vessels. Though not defined as a hematopoietic organ, the lung houses many resident hematopoietic cells, aids in platelet biogenesis, and is a reservoir for hematopoietic stem and progenitor cells (HSPCs). However, lung HECs have never been described. Here, we demonstrate that the fetal lung is a potential source of HECs that have the functional capacity to undergo EHT to produce de novo HSPCs and their resultant progeny. Explant cultures of murine and human fetal lungs display adherent endothelial cells transitioning into floating hematopoietic cells, accompanied by the gradual loss of an endothelial signature. Flow cytometric and functional assessment of fetal-lung explants showed the production of multipotent HSPCs that expressed the EHT and pre-HSPC markers EPCR, CD41, CD43, and CD44. scRNA-seq and small molecule modulation demonstrated that fetal lung HECs rely on canonical signaling pathways to undergo EHT, including TGFß/BMP, Notch, and YAP. Collectively, these data support the possibility that post-AGM development, functional HECs are present in the fetal lung, establishing this location as a potential extramedullary site of de novo hematopoiesis.
Asunto(s)
Hemangioblastos , Hematopoyesis , Animales , Ratones , Humanos , Células Madre Hematopoyéticas/metabolismo , Diferenciación Celular , Endotelio , Hemangioblastos/metabolismoRESUMEN
Durable reconstitution of the distal lung epithelium with pluripotent stem cell (PSC) derivatives, if realized, would represent a promising therapy for diseases that result from alveolar damage. Here, we differentiate murine PSCs into self-renewing lung epithelial progenitors able to engraft into the injured distal lung epithelium of immunocompetent, syngeneic mouse recipients. After transplantation, these progenitors mature in the distal lung, assuming the molecular phenotypes of alveolar type 2 (AT2) and type 1 (AT1) cells. After months in vivo, donor-derived cells retain their mature phenotypes, as characterized by single-cell RNA sequencing (scRNA-seq), histologic profiling, and functional assessment that demonstrates continued capacity of the engrafted cells to proliferate and differentiate. These results indicate durable reconstitution of the distal lung's facultative progenitor and differentiated epithelial cell compartments with PSC-derived cells, thus establishing a novel model for pulmonary cell therapy that can be utilized to better understand the mechanisms and utility of engraftment.