RESUMO
The RB1 gene is frequently mutated in human cancers but its role in tumorigenesis remains incompletely defined. Using an induced pluripotent stem cell (iPSC) model of hereditary retinoblastoma (RB), we report that the spliceosome is an up-regulated target responding to oncogenic stress in RB1-mutant cells. By investigating transcriptomes and genome occupancies in RB iPSCderived osteoblasts (OBs), we discover that both E2F3a, which mediates spliceosomal gene expression, and pRB, which antagonizes E2F3a, coregulate more than one-third of spliceosomal genes by cobinding to their promoters or enhancers. Pharmacological inhibition of the spliceosome in RB1-mutant cells leads to global intron retention, decreased cell proliferation, and impaired tumorigenesis. Tumor specimen studies and genome-wide TCGA (The Cancer Genome Atlas) expression profile analyses support the clinical relevance of pRB and E2F3a in modulating spliceosomal gene expression in multiple cancer types including osteosarcoma (OS). High levels of pRB/E2F3aregulated spliceosomal genes are associated with poor OS patient survival. Collectively, these findings reveal an undiscovered connection between pRB, E2F3a, the spliceosome, and tumorigenesis, pointing to the spliceosomal machinery as a potentially widespread therapeutic vulnerability of pRB-deficient cancers.
Assuntos
Neoplasias Ósseas , Carcinogênese , Fator de Transcrição E2F3 , Regulação Neoplásica da Expressão Gênica , Células-Tronco Pluripotentes Induzidas , Osteossarcoma , Proteínas de Ligação a Retinoblastoma , Spliceossomos , Ubiquitina-Proteína Ligases , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Carcinogênese/genética , Fator de Transcrição E2F3/genética , Fator de Transcrição E2F3/metabolismo , Genes do Retinoblastoma , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , Osteossarcoma/genética , Osteossarcoma/patologia , Neoplasias da Retina/genética , Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The entire lung epithelium arises from SRY box 9 (SOX9)-expressing progenitors that form the respiratory tree and differentiate into airway and alveolar cells. Despite progress in understanding their initial specification within the embryonic foregut, how these progenitors are subsequently maintained is less clear. Using inducible, progenitor-specific genetic mosaic mouse models, we showed that ß-catenin (CTNNB1) maintains lung progenitors by promoting a hierarchical lung progenitor gene signature, suppressing gastrointestinal (GI) genes, and regulating NK2 homeobox 1 (NKX2.1) and SRY box 2 (SOX2) in a developmental stage-dependent manner. At the early, but not later, stage post-lung specification, CTNNB1 cell-autonomously maintained normal NKX2.1 expression levels and suppressed ectopic SOX2 expression. Genetic epistasis analyses revealed that CTNNB1 is required for fibroblast growth factor (Fgf)/Kirsten rat sarcoma viral oncogene homolog (Kras)-mediated promotion of the progenitors. In silico screening of Eurexpress and translating ribosome affinity purification (TRAP)-RNAseq identified a progenitor gene signature, a subset of which depends on CTNNB1. Wnt signaling also maintained NKX2.1 expression and suppressed GI genes in cultured human lung progenitors derived from embryonic stem cells.
Assuntos
Linhagem da Célula/genética , Células-Tronco Embrionárias/metabolismo , Células Epiteliais/citologia , Pulmão/embriologia , Mucosa Respiratória/citologia , Mucosa Respiratória/embriologia , beta Catenina/fisiologia , Animais , Células Cultivadas , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Pulmão/citologia , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Knockout , Gravidez , Mucosa Respiratória/metabolismo , Transcriptoma , beta Catenina/genéticaRESUMO
We review recent progress in the stem cell biology of the respiratory system, and discuss its scientific and translational ramifications. Several studies have defined novel stem cells in postnatal lung and airways and implicated their roles in tissue homeostasis and repair. In addition, significant advances in the generation of respiratory epithelium from pluripotent stem cells (PSCs) now provide a novel and powerful platform for understanding lung development, modeling pulmonary diseases, and implementing drug screening. Finally, breakthroughs have been made in the generation of decellularized lung matrices that can serve as a scaffold for repopulation with respiratory cells derived from either postnatal or PSCs. These studies are a critical step forward towards the still distant goal of stem cell-based regenerative medicine for diseases of lung and airways.
Assuntos
Diferenciação Celular , Células Epiteliais/citologia , Epitélio/crescimento & desenvolvimento , Sistema Respiratório/citologia , Células-Tronco/citologia , Animais , HumanosRESUMO
Human small airway epithelial cells (SAECs) immortalized with human telomerase reverse transcriptase were exposed to either a single or multiple doses of α-particles. Irradiated cells showed a dose-dependent cytotoxicity and progressive neoplastic transformation phenotype. These included an increase in saturation density of growth, a greater resistance to N-phosphonoacetyl-L-aspartate, faster anchorage-independent growth, reinforced cell invasion and c-Myc expression. In addition, the transformed cells formed progressively growing tumors upon inoculation into athymic nude mice. Specifically, α-irradiation induced damage to both mitochondrial DNA (mtDNA) and mitochondrial functions in transformed cells as evidenced by increased mtDNA copy number and common deletion, decreased oxidative phosphorylation activity as measured by cytochrome C oxidase (COX) activity and oxygen consumption. There was a linear correlation between mtDNA copy number, common deletion, COX activity and cellular transformation represented by soft agar colony formation and c-Myc expression. These results suggest that mitochondria are associated with neoplastic transformation of SAEC cells induced by α-particles, and that the oncogenesis process may depend not only on the genomes inside the nucleus, but also on the mitochondrial DNA outside the nucleus.
Assuntos
Partículas alfa , Transformação Celular Neoplásica/efeitos da radiação , Células Epiteliais/efeitos da radiação , Mitocôndrias/fisiologia , Mitocôndrias/efeitos da radiação , Animais , Asparagina/análogos & derivados , Asparagina/farmacologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células Cultivadas , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Masculino , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Organofosfonatos/farmacologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
Mitochondrial DNA depleted (ρ(0)) human skin fibroblasts (HSF) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes, encoding numerous protein classes, in NF-κB and STAT3 signaling pathways, and by decreased activity of mitochondrial death pathway, compared to the parental ρ(+) HSF. In contrast, the extrinsic TRAIL/TRAIL-Receptor mediated death pathway remained highly active, and exogenous TRAIL in a combination with cycloheximide (CHX) induced higher levels of apoptosis in ρ(0) cells compared to ρ(+) HSF. Global gene expression analysis using microarray and qRT-PCR demonstrated that mRNA expression levels of many growth factors and their adaptor proteins (FGF13, HGF, IGFBP4, IGFBP6, and IGFL2), cytokines (IL6, ΙL17Β, ΙL18, ΙL19, and ΙL28Β) and cytokine receptors (IL1R1, IL21R, and IL31RA) were substantially decreased after mitochondrial DNA depletion. Some of these genes were targets of NF-κB and STAT3, and their protein products could regulate the STAT3 signaling pathway. Alpha-irradiation further induced expression of several NF-κB/STAT3 target genes, including IL1A, IL1B, IL6, PTGS2/COX2 and MMP12, in ρ(+) HSF, but this response was substantially decreased in ρ(0) HSF. Suppression of the IKK-NF-κB pathway by the small molecular inhibitor BMS-345541 and of the JAK2-STAT3 pathway by AG490 dramatically increased TRAIL-induced apoptosis in the control and irradiated ρ(+) HSF. Inhibitory antibodies against IL6, the main activator of JAK2-STAT3 pathway, added into the cell media, also increased TRAIL-induced apoptosis in HSF, especially after alpha-irradiation. Collectively, our results indicated that NF-κB activation was partially lost in ρ(0) HSF resulting in downregulation of the basal or radiation-induced expression of numerous NF-κB targets, further suppressing IL6-JAK2-STAT3 that in concert with NF-κB regulated protection against TRAIL-induced apoptosis.
Assuntos
Apoptose , Cicloeximida/farmacologia , DNA Mitocondrial/metabolismo , Raios gama , Perfilação da Expressão Gênica , NF-kappa B/metabolismo , Fator de Transcrição STAT3/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Biomarcadores/metabolismo , Western Blotting , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Citometria de Fluxo , Imunofluorescência , Humanos , Luciferases/metabolismo , NF-kappa B/genética , Análise de Sequência com Séries de Oligonucleotídeos , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/efeitos da radiação , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/genética , Pele/citologia , Pele/efeitos dos fármacos , Pele/efeitos da radiaçãoRESUMO
Recent synergistic advances in organ-on-chip and tissue engineering technologies offer opportunities to create in vitro-grown tissue or organ constructs that can faithfully recapitulate their in vivo counterparts. Such in vitro tissue or organ constructs can be utilized in multiple applications, including rapid drug screening, high-fidelity disease modeling, and precision medicine. Here, we report an imaging-guided bioreactor that allows in situ monitoring of the lumen of ex vivo airway tissues during controlled in vitro tissue manipulation and cultivation of isolated rat trachea. Using this platform, we demonstrated partial removal of the rat tracheal epithelium (i.e., de-epithelialization) without disrupting the underlying subepithelial cells and extracellular matrix. Through different tissue evaluation assays, such as immunofluorescent staining, DNA/protein quantification, and electron beam microscopy, we showed that the epithelium of the tracheal lumen can be effectively removed with negligible disruption in the underlying tissue layers, such as cartilage and blood vessel. Notably, using a custom-built micro-optical imaging device integrated with the bioreactor, the trachea lumen was visualized at the cellular level, and removal of the endogenous epithelium and distribution of locally delivered exogenous cells were demonstrated in situ. Moreover, the de-epithelialized trachea supported on the bioreactor allowed attachment and growth of exogenous cells seeded topically on its denuded tissue surface. Collectively, the results suggest that our imaging-enabled rat trachea bioreactor and localized cell replacement method can facilitate creation of bioengineered in vitro airway tissue that can be used in different biomedical applications.
Assuntos
Engenharia Tecidual , Traqueia , Animais , Reatores Biológicos , Cartilagem , Ratos , Reepitelização , Engenharia Tecidual/métodos , Alicerces TeciduaisRESUMO
Injured or diseased airway epithelium due to repeated environmental insults or genetic mutations can lead to a functional decline of the lung and incurable lung diseases. Bioengineered airway tissue constructs can facilitate in vitro investigation of human lung diseases and accelerate the development of effective therapeutics. Here, we report robust tissue manipulation modalities that allow: (i) selective removal of the endogenous epithelium of in vitro cultured airway tissues and (ii) spatially uniform distribution and prolonged cultivation of exogenous cells that are implanted topically onto the denuded airway lumen. Results obtained highlight that our approach to airway tissue manipulation can facilitate controlled removal of the airway epithelium and subsequent homogeneous distribution of newly implanted cells. This study can contribute to the creation of innovative tissue engineering methodologies that can facilitate the treatment of lung diseases, such as cystic fibrosis, primary ciliary dyskinesia, and chronic obstructive pulmonary disease.
Assuntos
Hidrogéis , Traqueia , Animais , Células Epiteliais , Pulmão , Ratos , Engenharia TecidualRESUMO
Melanoma is the most lethal form of human skin cancer. However, only limited chemotherapy is currently available for the metastatic stage of the disease. Since chemotherapy, radiation and sodium arsenite treatment operate mainly through induction of the intrinsic mitochondrial pathway, a strongly decreased mitochondrial function in metastatic melanoma cells, could be responsible for low efficacy of the conventional therapy of melanoma. Another feature of metastatic melanoma cells is their proinflammatory phenotype, linked to endogenous expression of the inflammatory cytokines, such as TNFα IL6 and IL8, their receptors, and constitutive NF-κB- and STAT3-dependent gene expression, including cyclooxygenase-2 (PTGS2/COX2). In the present study, we treated melanoma cells with immunological (monoclonal antibody against TNFα or IL6), pharmacological (small molecular inhibitors of IKKß-NF-κB and JAK2-STAT3) or genetic (specific RNAi for COX-2) agents that suppressed the inflammatory response in combination with induction of apoptosis via TRAIL. As a result of these combined treatments, exogenous TRAIL via interactions with TRAIL-R2/R1 strongly increased levels of apoptosis in resistant melanoma cells. The present study provides new understanding of the regulation of TRAIL-mediated apoptosis in melanoma and will serve as the foundation for the potential development of a novel approach for a therapy of resistant melanomas.
Assuntos
Apoptose/efeitos dos fármacos , Melanoma/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Western Blotting , Linhagem Celular , Ciclo-Oxigenase 2/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , NF-kappa B/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Reação em Cadeia da Polimerase , Fator de Transcrição STAT3/metabolismoRESUMO
The cellular and molecular mechanisms of how asbestos fibers induce cancers and other diseases are not well understood. Both serpentine and amphibole asbestos fibers have been shown to induce oxidative stress, inflammatory responses, cellular toxicity and tissue injuries, genetic changes, and epigenetic alterations in target cells in vitro and tissues in vivo. Most of these mechanisms are believe to be shared by both fiber-induced cancers and noncancerous diseases. This article summarizes the findings from existing literature with a focus on genetic changes, specifically, mutagenicity of asbestos fibers. Thus far, experimental evidence suggesting the involvement of mutagenesis in asbestos carcinogenicity is more convincing than asbestos-induced fibrotic diseases. The potential contributions of mutagenicity to asbestos-induced diseases, with an emphasis on carcinogenicity, are reviewed from five aspects: (1) whether there is a mutagenic mode of action (MOA) in fiber-induced carcinogenesis; (2) mutagenicity/carcinogenicity at low dose; (3) biological activities that contribute to mutagenicity and impact of target tissue/cell type; (4) health endpoints with or without mutagenicity as a key event; and finally, (5) determinant factors of toxicity in mutagenicity. At the end of this review, a consensus statement of what is known, what is believed to be factual but requires confirmation, and existing data gaps, as well as future research needs and directions, is provided.
Assuntos
Amianto/toxicidade , Carcinógenos Ambientais/toxicidade , Fibras Minerais/toxicidade , Neoplasias/induzido quimicamente , Animais , Amianto/administração & dosagem , Amianto/química , Asbestose/metabolismo , Carcinógenos Ambientais/administração & dosagem , Carcinógenos Ambientais/química , Fenômenos Químicos , Dano ao DNA , Humanos , Fibras Minerais/análise , Mitose/efeitos dos fármacos , Mutação/efeitos dos fármacos , Neoplasias/metabolismo , Doenças Pleurais/induzido quimicamente , Doenças Pleurais/metabolismoRESUMO
In living tissues, mechanical stiffness and biological function are intrinsically linked. Alterations in the stiffness of tissues can induce pathological interactions that affect cellular activity and tissue function. Underlying connections between tissue stiffness and disease highlights the importance of accurate quantitative characterizations of soft tissue mechanics, which can improve our understanding of disease and inform therapeutic development. In particular, accurate measurement of lung mechanical properties has been especially challenging due to the anatomical and mechanobiological complexities of the lung. Discrepancies between measured mechanical properties of dissected lung tissue samples and intact lung tissues in vivo has limited the ability to accurately characterize integral lung mechanics. Here, we report a non-destructive vacuum-assisted method to evaluate mechanical properties of soft biomaterials, including intact tissues and hydrogels. Using this approach, we measured elastic moduli of rat lung tissue that varied depending on stress-strain distribution throughout the lung. We also observed that the elastic moduli of enzymatically disrupted lung parenchyma increased by at least 64%. The reported methodology enables assessment of the nonlinear viscoelastic characteristics of intact lungs under normal and abnormal (i.e., injured, diseased) conditions and allows measurement of mechanical properties of tissue-mimetic biomaterials for use in therapeutics or in vitro models. STATEMENT OF SIGNIFICANCE: Accurate quantification of tissue stiffness is critical for understanding mechanisms of disease and developing effective therapeutics. Current modalities to measure tissue stiffness are destructive and preclude accurate assessment of lung mechanical properties, as lung mechanics are determined by complex features of the intact lung. To address the need for alternative methods to assess lung mechanics, we report a non-destructive vacuum-based approach to quantify tissue stiffness. We applied this method to correlate lung tissue mechanics with tissue disruption, and to assess the stiffness of biomaterials. This method can be used to inform the development of tissue-mimetic materials for use in therapeutics and disease models, and could potentially be applied for in-situ evaluation of tissue stiffness as a diagnostic or prognostic tool.
Assuntos
Hidrogéis , Pulmão , Animais , Módulo de Elasticidade , RatosRESUMO
The derivation of tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would have broad reaching implications for regenerative medicine. Here, we report the directed differentiation of human iPSCs into airway basal cells ("iBCs"), a population resembling the stem cell of the airway epithelium. Using a dual fluorescent reporter system (NKX2-1GFP;TP63tdTomato), we track and purify these cells as they first emerge as developmentally immature NKX2-1GFP+ lung progenitors and subsequently augment a TP63 program during proximal airway epithelial patterning. In response to primary basal cell medium, NKX2-1GFP+/TP63tdTomato+ cells display the molecular and functional phenotype of airway basal cells, including the capacity to self-renew or undergo multi-lineage differentiation in vitro and in tracheal xenografts in vivo. iBCs and their differentiated progeny model perturbations that characterize acquired and genetic airway diseases, including the mucus metaplasia of asthma, chloride channel dysfunction of cystic fibrosis, and ciliary defects of primary ciliary dyskinesia.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Diferenciação Celular , Células Epiteliais , Humanos , Pulmão , TraqueiaRESUMO
One of the long-term objectives of the research in our laboratory was to determine whether mitochondrial DNA (mtDNA) mutations were generated in cell lines exposed to a variety of known mutagens. Many of these mutagens are known to increase oxidative stress in the cell, and one potential outcome of this would be an increased incidence of point mutations in mtDNA. Recently, there has been some controversy regarding the validity of point mutations in the regulatory region of mtDNA as a predictive or causative marker for carcinogenesis. Studies were undertaken to assess whether nuclear mutagens such as arsenic (As), asbestos, and ultraviolet (UV) and gamma-radiation, induced both heteroplasmic and homoplasmic point mutations in mtDNA. A direct sequencing approach was used to reduce the occurrence of experimental errors and cross-checked all base changes with databases of known polymorphisms. Our results showed that, while base changes did occur, there was no marked difference between the number of changes in treated and untreated cells. Furthermore, in human lymphocyte samples from subjects exposed to As, most of these base changes were previously reported. Interestingly, there was an increase in the number of transversions (purine ( pyrimidine) in smokers from a human population study, but as with the findings in cell culture samples, there was no difference in the total number of base changes. Data suggest that only a change in the number of rare transversions would be indicative of an increase in point mutations in mtDNA after exposure to mutagens.
Assuntos
DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/efeitos da radiação , Poluentes Ambientais/toxicidade , Raios gama , Regulação da Expressão Gênica/efeitos dos fármacos , Mutagênicos/toxicidade , Raios Ultravioleta , Animais , Arsênio/toxicidade , Amianto/toxicidade , Células CHO , Carcinógenos/toxicidade , Linhagem Celular , Cricetinae , Cricetulus , Dano ao DNA , Exposição Ambiental/efeitos adversos , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Linfócitos/efeitos da radiação , Mutação/genética , Espécies Reativas de Oxigênio , Fumar/genéticaRESUMO
Arsenic is a well-established human carcinogen that is chronically consumed in drinking water by millions of people worldwide. Recent evidence has suggested that arsenic is a genotoxic carcinogen. Furthermore, we have shown that mitochondria mediate the mutagenic effects of arsenic in mammalian cells, as arsenic did not induce nuclear mutations in mitochondrial DNA (mtDNA)-depleted cells. Using the human-hamster hybrid A(L) cells, we show here that arsenic alters mitochondrial function by decreasing cytochrome c oxidase function and oxygen consumption but increasing citrate synthase function. These alterations correlated with depletion in mtDNA copy number and increase in large heteroplasmic mtDNA deletions. In addition, mtDNA isolated periodically from cultures treated continuously with arsenic did not consistently display the same deletion pattern, indicating that the mitochondrial genome was subjected to repeated and continuous damage. These data support the theory that the mitochondria, and particularly mtDNA, are important targets of the mutagenic effects of arsenic in mammalian cells.
Assuntos
Arsênio/toxicidade , Dano ao DNA , DNA Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Animais , Cricetinae , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Relação Dose-Resposta a Droga , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Células Híbridas , Mitocôndrias/genética , Mitocôndrias/metabolismo , Testes de Mutagenicidade , Oxirredução , Consumo de Oxigênio/efeitos dos fármacosRESUMO
Autosomal recessive IRF7 and IRF9 deficiencies impair type I and III IFN immunity and underlie severe influenza pneumonitis. We report three unrelated children with influenza A virus (IAV) infection manifesting as acute respiratory distress syndrome (IAV-ARDS), heterozygous for rare TLR3 variants (P554S in two patients and P680L in the third) causing autosomal dominant (AD) TLR3 deficiency. AD TLR3 deficiency can underlie herpes simplex virus-1 (HSV-1) encephalitis (HSE) by impairing cortical neuron-intrinsic type I IFN immunity to HSV-1. TLR3-mutated leukocytes produce normal levels of IFNs in response to IAV. In contrast, TLR3-mutated fibroblasts produce lower levels of IFN-ß and -λ, and display enhanced viral susceptibility, upon IAV infection. Moreover, the patients' iPSC-derived pulmonary epithelial cells (PECs) are susceptible to IAV. Treatment with IFN-α2b or IFN-λ1 rescues this phenotype. AD TLR3 deficiency may thus underlie IAV-ARDS by impairing TLR3-dependent, type I and/or III IFN-mediated, PEC-intrinsic immunity. Its clinical penetrance is incomplete for both IAV-ARDS and HSE, consistent with their typically sporadic nature.
Assuntos
Influenza Humana/genética , Padrões de Herança/genética , Pneumonia/genética , Receptor 3 Toll-Like/deficiência , Alelos , Criança , Pré-Escolar , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Evolução Fatal , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Heterozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lactente , Recém-Nascido , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/fisiologia , Interferons/metabolismo , Mutação com Perda de Função/genética , Pulmão/patologia , Masculino , Mutação de Sentido Incorreto/genética , Poli I-C/farmacologia , Transporte ProteicoRESUMO
End-stage lung disease is the third leading cause of death worldwide, accounting for 400,000 deaths per year in the United States alone. To reduce the morbidity and mortality associated with lung disease, new therapeutic strategies aimed at promoting lung repair and increasing the number of donor lungs available for transplantation are being explored. Because of the extreme complexity of this organ, previous attempts at bioengineering functional lungs from fully decellularized or synthetic scaffolds lacking functional vasculature have been largely unsuccessful. An intact vascular network is critical not only for maintaining the blood-gas barrier and allowing for proper graft function but also for supporting the regenerative cells. We therefore developed an airway-specific approach to removing the pulmonary epithelium, while maintaining the viability and function of the vascular endothelium, using a rat model. The resulting vascularized lung grafts supported the attachment and growth of human adult pulmonary cells and stem cell-derived lung-specified epithelial cells. We propose that de-epithelialization of the lung with preservation of intact vasculature could facilitate cell therapy of pulmonary epithelium and enable bioengineering of functional lungs for transplantation.
Assuntos
Bioengenharia , Transplante de Pulmão , Pulmão/irrigação sanguínea , Pulmão/fisiologia , Regeneração , Animais , Bioengenharia/métodos , Sobrevivência Celular , Matriz Extracelular , Imunofluorescência , Pulmão/ultraestrutura , Músculo Liso , Ratos , Medicina Regenerativa , Mucosa Respiratória , Alicerces TeciduaisRESUMO
Although the U.S. Environmental Protection Agency has restricted the industrial use of regulated forms of asbestos in the United States since the early 1970s, environmental exposure to asbestos remains a health concern in the United States and is a significant health issue among developing countries. Exposure to asbestos is associated with chronic pulmonary diseases and cancer of the lung, pleura, and peritoneum. The mechanism of fiber carcinogenesis is far from clear and is likely to be complex, depending on fiber dimensions, surface properties, and physical durability. The induction of reactive oxygen and nitrogen species upon phagocytosis of fibers plays an important role in fiber genotoxicity. The beta igH3, a secreted protein induced by the transforming growth factor-beta and essential for cell adhesion, is downregulated in asbestos-induced tumorigenic human bronchial epithelial cells. Ectopic expression of the beta igH3 gene abrogates the tumorigenic phenotype and suggests that the gene plays a causal role in fiber carcinogenesis. A better understanding of the carcinogenic mechanism of asbestos and other mineral fibers will provide useful information on interventional and preventive measures for asbestos-mediated diseases such as human pleural and peritoneal mesotheliomas.
Assuntos
Amianto/efeitos adversos , Carcinógenos Ambientais/efeitos adversos , Proteínas da Matriz Extracelular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mutagênicos/efeitos adversos , Doenças Respiratórias/genética , Fator de Crescimento Transformador beta/genética , Células Cultivadas , Cocarcinogênese , Exposição Ambiental/efeitos adversos , Proteínas da Matriz Extracelular/metabolismo , Fibras Minerais/efeitos adversos , Doenças Respiratórias/patologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Lung and airway epithelial cells generated in vitro from human pluripotent stem cells (hPSCs) have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. Here we describe a strategy for directed differentiation of hPSCs into developmental lung progenitors, and their subsequent differentiation into predominantly distal lung epithelial cells. The protocol entails four stages that recapitulate lung development, and it takes â¼50 d. First, definitive endoderm (DE) is induced in the presence of high concentrations of activin A. Subsequently, lung-biased anterior foregut endoderm (AFE) is specified by sequential inhibition of bone morphogenetic protein (BMP), transforming growth factor-ß (TGF-ß) and Wnt signaling. AFE is then ventralized by applying Wnt, BMP, fibroblast growth factor (FGF) and retinoic acid (RA) signaling to obtain lung and airway progenitors. Finally, these are further differentiated into more mature epithelial cells types using Wnt, FGF, cAMP and glucocorticoid agonism. This protocol is conducted in defined conditions, it does not involve genetic manipulation of the cells and it results in cultures in which the majority of the cells express markers of various lung and airway epithelial cells, with a predominance of cells identifiable as functional type II alveolar epithelial cells.
Assuntos
Diferenciação Celular/fisiologia , Células Epiteliais/citologia , Células-Tronco Pluripotentes/citologia , Sistema Respiratório/citologia , Células-Tronco/citologia , Engenharia Tecidual/métodos , Humanos , Técnicas In Vitro/métodosRESUMO
Severe influenza disease strikes otherwise healthy children and remains unexplained. We report compound heterozygous null mutations in IRF7, which encodes the transcription factor interferon regulatory factor 7, in an otherwise healthy child who suffered life-threatening influenza during primary infection. In response to influenza virus, the patient's leukocytes and plasmacytoid dendritic cells produced very little type I and III interferons (IFNs). Moreover, the patient's dermal fibroblasts and induced pluripotent stem cell (iPSC)-derived pulmonary epithelial cells produced reduced amounts of type I IFN and displayed increased influenza virus replication. These findings suggest that IRF7-dependent amplification of type I and III IFNs is required for protection against primary infection by influenza virus in humans. They also show that severe influenza may result from single-gene inborn errors of immunity.
Assuntos
Heterozigoto , Vírus da Influenza A Subtipo H1N1 , Influenza Humana/imunologia , Fator Regulador 7 de Interferon/genética , Interferon Tipo I/biossíntese , Síndrome do Desconforto Respiratório/imunologia , Criança , Células Dendríticas/imunologia , Feminino , Fibroblastos/imunologia , Genes Recessivos , Humanos , Células-Tronco Pluripotentes Induzidas/imunologia , Influenza Humana/complicações , Influenza Humana/genética , Interferon Tipo I/genética , Leucócitos/imunologia , Pulmão/imunologia , Mutação , Síndrome do Desconforto Respiratório/genética , Síndrome do Desconforto Respiratório/virologia , Mucosa Respiratória/imunologiaRESUMO
The ability to generate lung and airway epithelial cells from human pluripotent stem cells (hPSCs) would have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. We have established, based on developmental paradigms, a highly efficient method for directed differentiation of hPSCs into lung and airway epithelial cells. Long-term differentiation of hPSCs in vivo and in vitro yielded basal, goblet, Clara, ciliated, type I and type II alveolar epithelial cells. The type II alveolar epithelial cells were capable of surfactant protein-B uptake and stimulated surfactant release, providing evidence of specific function. Inhibiting or removing retinoic acid, Wnt and BMP-agonists to signaling pathways critical for early lung development in the mouse-recapitulated defects in corresponding genetic mouse knockouts. As this protocol generates most cell types of the respiratory system, it may be useful for deriving patient-specific therapeutic cells.