RESUMO
Studying human fetal lungs can inform how developmental defects and disease states alter the function of the lungs. Here, we sequenced >150,000 single cells from 19 healthy human pseudoglandular fetal lung tissues ranging between gestational weeks 10-19. We capture dynamic developmental trajectories from progenitor cells that express abundant levels of the cystic fibrosis conductance transmembrane regulator (CFTR). These cells give rise to multiple specialized epithelial cell types. Combined with spatial transcriptomics, we show temporal regulation of key signalling pathways that may drive the temporal and spatial emergence of specialized epithelial cells including ciliated and pulmonary neuroendocrine cells. Finally, we show that human pluripotent stem cell-derived fetal lung models contain CFTR-expressing progenitor cells that capture similar lineage developmental trajectories as identified in the native tissue. Overall, this study provides a comprehensive single-cell atlas of the developing human lung, outlining the temporal and spatial complexities of cell lineage development and benchmarks fetal lung cultures from human pluripotent stem cell differentiations to similar developmental window.
Assuntos
Diferenciação Celular , Regulador de Condutância Transmembrana em Fibrose Cística , Células Epiteliais , Feto , Pulmão , Humanos , Pulmão/embriologia , Pulmão/citologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feto/citologia , Feto/embriologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Plasticidade Celular , Linhagem da Célula , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Análise de Célula Única , Transcriptoma , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Transdução de SinaisRESUMO
The lower respiratory tract is a hierarchical network of compliant tubular structures that are made from extracellular matrix proteins with a wall lined by an epithelium. While microfluidic airway-on-a-chip models incorporate the effects of shear and stretch on the epithelium, week-long air-liquid-interface culture at physiological shear stresses, the circular cross-section, and compliance of native airway walls have yet to be recapitulated. To overcome these limitations, a collagen tube-based airway model is presented. The lumen is lined with a confluent epithelium during two-week continuous perfusion with warm, humid air while presenting culture medium from the outside and compensating for evaporation. The model recapitulates human small airways in extracellular matrix composition and mechanical microenvironment, allowing for the first time dynamic studies of elastocapillary phenomena associated with regular breathing and mechanical ventilation, as well as their impacts on the epithelium. A case study reveales increasing damage to the epithelium during repetitive collapse and reopening cycles as opposed to overdistension, suggesting expiratory flow resistance to reduce atelectasis. The model is expected to promote systematic comparisons between different clinically used ventilation strategies and, more broadly, to enhance human organ-on-a-chip platforms for a variety of tubular tissues.
Assuntos
Colágeno , Células Epiteliais , Humanos , Células Epiteliais/citologia , Colágeno/química , Dispositivos Lab-On-A-ChipRESUMO
Introducing or correcting disease-causing mutations through genome editing in human pluripotent stem cells (hPSCs) followed by tissue-specific differentiation provide sustainable models of multiorgan diseases, such as cystic fibrosis (CF). However, low editing efficiency resulting in extended cell culture periods and the use of specialised equipment for fluorescence activated cell sorting (FACS) make hPSC genome editing still challenging. We aimed to investigate whether a combination of cell cycle synchronisation, single-stranded oligodeoxyribonucleotides, transient selection, manual clonal isolation, and rapid screening can improve the generation of correctly modified hPSCs. Here, we introduced the most common CF mutation, ΔF508, into the CFTR gene, using TALENs into hPSCs, and corrected the W1282X mutation using CRISPR-Cas9, in human-induced PSCs. This relatively simple method achieved up to 10% efficiency without the need for FACS, generating heterozygous and homozygous gene edited hPSCs within 3-6 weeks in order to understand genetic determinants of disease and precision medicine.
Assuntos
Edição de Genes , Células-Tronco Pluripotentes , Humanos , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , Células-Tronco Pluripotentes/metabolismo , Mutação , HeterozigotoRESUMO
Human embryonic stem cells (ES) and induced pluripotent stem cells (iPSC) are powerful tools that have the potential to generate in vitro human lung epithelial cells. However, challenges in efficiency and reproducibility remain in utilizing the cells for therapy discovery platforms. Here, we optimize our previously published protocols to efficiently generate three developmental stages of the lung model (fetal lung epithelial progenitors, fLEP; immature airway epithelial spheroid, AES; air-liquid interface culture, ALI), and demonstrate its potential for cystic fibrosis (CF) drug discovery platforms. The stepwise approach directs differentiation from hPSC to definitive endoderm, anterior ventral foregut endoderm, and fetal lung progenitor cells. The article also describes the generation of immature airway epithelial spheroids in Matrigel with epithelial cells sorted by a magnetic-activated cell sorting system, and the generation of adult-like airway epithelia through air-liquid interface conditions. We demonstrate that this optimized procedure generates remarkably higher cystic fibrosis transmembrane conductance regulator (CFTR) expression and function than our previous method, and thus is uniquely suitable for CF research applications. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: hESC/hiPSC differentiation to fetal lung progenitors Basic Protocol 2: Formation of airway epithelial spheroids Alternate Protocol 1: Cryopreservation of airway epithelial spheroids Basic Protocol 3: Differentiation and maturation in air-liquid interface culture Alternate Protocol 2: Differentiation and maturation of epithelial progenitors from airway epithelial spheroids in ALI culture.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Células-Tronco Pluripotentes , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Endoderma , Humanos , Pulmão , Reprodutibilidade dos TestesRESUMO
The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein is a cAMP-activated anion channel that is critical for regulating fluid and ion transport across the epithelium. This process is disrupted in CF epithelia, and patients harbouring CF-causing mutations experience reduced lung function as a result, associated with the increased rate of mortality. Much progress has been made in CF research leading to treatments that improve CFTR function, including small molecule modulators. However, clinical outcomes are not necessarily mutation-specific as individuals harboring the same genetic mutation may present with varying disease manifestations and responses to therapy. This suggests that the CFTR protein may have alternative functions that remain under-appreciated and yet can impact disease. In this mini review, we highlight some notable research implicating an important role of CFTR protein during early lung development and how mutant CFTR proteins may impact CF airway disease pathogenesis. We also discuss recent novel cell and animal models that can now be used to identify a developmental cause of CF lung disease.
RESUMO
The discovery of the Cystic fibrosis (CF) gene in 1989 has paved the way for incredible progress in treating the disease such that the mean survival age of individuals living with CF is now ~58 years in Canada. Recent developments in gene targeting tools and new cell and animal models have re-ignited the search for a permanent genetic cure for all CF. In this review, we highlight some of the more recent gene therapy approaches as well as new models that will provide insight into personalized therapies for CF.
Assuntos
Fibrose Cística , Animais , Fibrose Cística/genética , Fibrose Cística/terapia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Terapia Genética , Humanos , Pessoa de Meia-Idade , Mutação , Medicina de PrecisãoRESUMO
For those people with cystic fibrosis carrying rare CFTR mutations not responding to currently available therapies, there is an unmet need for relevant tissue models for therapy development. Here, we describe a new testing platform that employs patient-specific induced pluripotent stem cells (iPSCs) differentiated to lung progenitor cells that can be studied using a dynamic, high-throughput fluorescence-based assay of CFTR channel activity. Our proof-of-concept studies support the potential use of this platform, together with a Canadian bioresource that contains iPSC lines and matched nasal cultures from people with rare mutations, to advance patient-oriented therapy development. Interventions identified in the high-throughput, stem cell-based model and validated in primary nasal cultures from the same person have the potential to be advanced as therapies.
Assuntos
Diferenciação Celular/genética , Fibrose Cística/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Pulmão/metabolismo , Células-Tronco/metabolismo , Células Cultivadas , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Pulmão/citologia , Mutação , RNA-Seq/métodos , Células-Tronco/citologiaRESUMO
Cell lineage conversion of fibroblasts to specialized cell types through transdifferentiation may provide a fast and alternative cell source for regenerative medicine. Here we show that transient transduction of fibroblasts with the four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) in addition to the early lung transcription factor Nkx2-1 (also known as Ttf1), followed by directed differentiation of the cells, can convert mouse embryonic and human adult dermal fibroblasts into induced lung-like epithelial cells (iLEC). These iLEC differentiate into multiple lung cell types in air liquid interface cultures, repopulate decellularized rat lung scaffolds, and form lung epithelia composed of Ciliated, Goblet, Basal, and Club cells after transplantation into immune-compromised mice. As proof-of-concept, differentiated human iLEC harboring the Cystic Fibrosis mutation dF508 demonstrated pharmacological rescue of CFTR function using the combination of lumacaftor and ivacaftor. Overall, this is a promising alternative approach for generation of patient-specific lung-like progenitors to study lung function, disease and future regeneration strategies.
Assuntos
Transdiferenciação Celular , Reprogramação Celular , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Células Epiteliais/citologia , Fibroblastos/citologia , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Pulmão/citologia , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Medicina Regenerativa/métodos , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fator Nuclear 1 de Tireoide/genética , Fator Nuclear 1 de Tireoide/metabolismoRESUMO
BACKGROUND: Therapies targeting certain CFTR mutants have been approved, yet variations in clinical response highlight the need for in-vitro and genetic tools that predict patient-specific clinical outcomes. Toward this goal, the CF Canada-Sick Kids Program in Individual CF Therapy (CFIT) is generating a "first of its kind", comprehensive resource containing patient-specific cell cultures and data from 100 CF individuals that will enable modeling of therapeutic responses. METHODS: The CFIT program is generating: 1) nasal cells from drug naïve patients suitable for culture and the study of drug responses in vitro, 2) matched gene expression data obtained by sequencing the RNA from the primary nasal tissue, 3) whole genome sequencing of blood derived DNA from each of the 100 participants, 4) induced pluripotent stem cells (iPSCs) generated from each participant's blood sample, 5) CRISPR-edited isogenic control iPSC lines and 6) prospective clinical data from patients treated with CF modulators. RESULTS: To date, we have recruited 57 of 100 individuals to CFIT, most of whom are homozygous for F508del (to assess in-vitro: in-vivo correlations with respect to ORKAMBI response) or heterozygous for F508del and a minimal function mutation. In addition, several donors are homozygous for rare nonsense and missense mutations. Nasal epithelial cell cultures and matched iPSC lines are available for many of these donors. CONCLUSIONS: This accessible resource will enable development of tools that predict individual outcomes to current and emerging modulators targeting F508del-CFTR and facilitate therapy discovery for rare CF causing mutations.
Assuntos
Aminofenóis/uso terapêutico , Aminopiridinas/uso terapêutico , Benzodioxóis/uso terapêutico , Fibrose Cística/terapia , Terapia Genética/métodos , Medicina de Precisão/métodos , Desenvolvimento de Programas/métodos , Quinolonas/uso terapêutico , Canadá/epidemiologia , Criança , Fibrose Cística/epidemiologia , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Combinação de Medicamentos , Humanos , Incidência , Mutação de Sentido Incorreto , RNA/genéticaRESUMO
Pulmonary disease is the major cause of morbidity and mortality in patients with cystic fibrosis, a disease caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. Heterogeneity in CFTR genotype-phenotype relationships in affected individuals plus the escalation of drug discovery targeting specific mutations highlights the need to develop robust in vitro platforms with which to stratify therapeutic options using relevant tissue. Toward this goal, we adapted a fluorescence plate reader assay of apical CFTR-mediated chloride conductance to enable profiling of a panel of modulators on primary nasal epithelial cultures derived from patients bearing different CFTR mutations. This platform faithfully recapitulated patient-specific responses previously observed in the "gold-standard" but relatively low-throughput Ussing chamber. Moreover, using this approach, we identified a novel strategy with which to augment the response to an approved drug in specific patients. In proof of concept studies, we also validated the use of this platform in measuring drug responses in lung cultures differentiated from cystic fibrosis iPS cells. Taken together, we show that this medium throughput assay of CFTR activity has the potential to stratify cystic fibrosis patient-specific responses to approved drugs and investigational compounds in vitro in primary and iPS cell-derived airway cultures.
RESUMO
Cystic fibrosis (CF) is a fatal recessive genetic disorder caused by a mutation in the gene encoding CF transmembrane conductance regulator (CFTR) protein. Alteration in CFTR leads to thick airway mucus and bacterial infection. Cell therapy has been proposed for CFTR restoration, but efficacy has been limited by low engraftment levels. In our previous studies, we have shown that using a pre-conditioning regimen in combination with optimization of cell number and time of delivery, we could obtain greater bone marrow cell (BMC) retention in the lung. Here, we found that optimized delivery of wild-type (WT) BMC contributed to apical CFTR expression in airway epithelium and restoration of select ceramide species and fatty acids in CFTR-/- mice. Importantly, WT BMC delivery delayed Pseudomonas aeruginosa lung infection and increased survival of CFTR-/- recipients. Only WT BMCs had a beneficial effect beyond 6 months, suggesting a dual mechanism of BMC benefit: a non-specific effect early after cell delivery, possibly due to the recruitment of macrophages and neutrophils, and a late beneficial effect dependent on long-term CFTR expression. Taken together, our results suggest that BMC can improve overall lung function and may have potential therapeutic benefit for the treatment of CF.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/genética , Fibrose Cística/metabolismo , Animais , Células da Medula Óssea/metabolismo , Líquido da Lavagem Broncoalveolar , Ceramidas/metabolismo , Fibrose Cística/mortalidade , Fibrose Cística/terapia , Citocinas , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Feminino , Pulmão/metabolismo , Macrófagos/metabolismo , Camundongos , Neutrófilos/metabolismo , Mucosa Respiratória/metabolismoRESUMO
Airway epithelial cells are of great interest for research on lung development, regeneration and disease modeling. This protocol describes how to generate cystic fibrosis (CF) transmembrane conductance regulator protein (CFTR)-expressing airway epithelial cells from human pluripotent stem cells (PSCs). The stepwise approach from PSC culture to differentiation into progenitors and then mature epithelia with apical CFTR activity is outlined. Human PSCs that were inefficient at endoderm differentiation using our previous lung differentiation protocol were able to generate substantial lung progenitor cell populations. Augmented CFTR activity can be observed in all cultures as early as at 35 d of differentiation, and full maturation of the cells in air-liquid interface cultures occurs in <5 weeks. This protocol can be used for drug discovery, tissue regeneration or disease modeling.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células-Tronco Pluripotentes/citologia , Mucosa Respiratória/citologia , Humanos , Mucosa Respiratória/metabolismoRESUMO
The contribution of bone marrow cells (BMC) in lung repair is controversial. We previously reported a subpopulation of BMC that express Clara cell secretory protein (CCSP). To determine the contribution of endogenous CCSP(+) BMC to airway regeneration, we performed bone marrow transplantation studies using the CCtk mouse, which expresses a thymidine kinase suicide gene under regulation of the CCSP promoter. Mice were transplanted with wild-type or CCtk BMC and treated with ganciclovir to eliminate CCSP(+) cells. After airway injury using naphthalene, mice depleted of CCSP(+) BMC had more inflammatory cells in lung and decreased levels of oxygen in arterial blood. They also had reduced expression of airway epithelial genes and less Clara cells compared to control mice that had intact CCSP(+) BMC and bone marrow derived CCSP(+) cells in the airways. After naphthalene injury, administration of CCSP reproduced the beneficial effect of CCSP(+) BMC by improving recovery of airway epithelium, reducing lung inflammation and increasing oxygen in arterial blood from mice depleted of CCSP(+) BMC. Our data demonstrate that ablation of CCSP(+) BMC delays airway recovery and suggests the beneficial effect of CCSP(+) BMC in lung recovery is in part due to production of CCSP itself.
Assuntos
Lesão Pulmonar Aguda/genética , Células da Medula Óssea/metabolismo , Células Epiteliais/metabolismo , Pulmão/metabolismo , Regeneração/genética , Uteroglobina/genética , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/terapia , Animais , Células da Medula Óssea/patologia , Transplante de Medula Óssea , Células Epiteliais/patologia , Feminino , Regulação da Expressão Gênica , Genes Letais , Humanos , Pulmão/patologia , Masculino , Camundongos Transgênicos , Naftalenos , Oxigênio/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais , Timidina Quinase/genética , Timidina Quinase/metabolismo , Transgenes , Uteroglobina/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The understanding of key processes and signaling mechanisms in lung development has been mainly demonstrated through gain and loss of function studies in mice, while human lung development remains largely unexplored due to inaccessibility. Several recent reports have exploited the identification of key signaling mechanisms that regulate lineage commitment and restriction in mouse lung development, to direct differentiation of both mouse and human pluripotent stem cells towards lung epithelial cells. In this review, we discuss the recent advances in the generation of respiratory epithelia from pluripotent stem cells and the potential of these engineered cells for novel scientific discoveries in lung diseases and future translation into regenerative therapies.
RESUMO
We have previously reported a subpopulation of bone marrow cells (BMC) that express Clara cell secretory protein (CCSP), generally felt to be specific to lung Clara cells. Ablation of lung Clara cells has been reported using a transgenic mouse that expresses thymidine kinase under control of the CCSP promoter. Treatment with ganciclovir results in permanent elimination of CCSP(+) cells, failure of airway regeneration, and death. To determine if transtracheal delivery of wild-type bone marrow CCSP(+) cells is beneficial after ablation of lung CCSP(+) cells, transgenic mice were treated with ganciclovir followed by transtracheal administration of CCSP(+) or CCSP(-) BMC. Compared with mice administered CCSP(-) cells, mice treated with CCSP(+) cells had more donor cells lining the airway epithelium, where they expressed epithelial markers including CCSP. Although donor CCSP(+) cells did not substantially repopulate the airway, their administration resulted in increased host ciliated cells, better preservation of airway epithelium, reduction of inflammatory cells, and an increase in animal survival time. Administration of CCSP(+) BMC is beneficial after permanent ablation of lung Clara cells by increasing bronchial epithelial repair. Therefore, CCSP(+) BMC could be important for treatment of lung diseases where airways re-epithelialization is compromised.
Assuntos
Células da Medula Óssea/metabolismo , Bronquíolos/citologia , Células Epiteliais/metabolismo , Reepitelização , Uteroglobina/genética , Animais , Bronquíolos/metabolismo , Linhagem Celular , Proliferação de Células , Feminino , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Pneumopatias/terapia , Masculino , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Sistema Respiratório/metabolismo , Timidina Quinase/metabolismo , Uteroglobina/metabolismoRESUMO
Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene, which regulates chloride and water transport across all epithelia and affects multiple organs, including the lungs. Here we report an in vitro directed differentiation protocol for generating functional CFTR-expressing airway epithelia from human embryonic stem cells. Carefully timed treatment by exogenous growth factors that mimic endoderm developmental pathways in vivo followed by air-liquid interface culture results in maturation of patches of tight junctioncoupled differentiated airway epithelial cells that demonstrate active CFTR transport function. As a proof of concept, treatment of CF patient induced pluripotent stem cellderived epithelial cells with a small-molecule compound to correct for the common CF processing mutation resulted in enhanced plasma membrane localization of mature CFTR protein. Our study provides a method for generating patient-specific airway epithelial cells for disease modeling and in vitro drug testing.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/biossíntese , Células Epiteliais/citologia , Células-Tronco Pluripotentes/citologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Camundongos , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Mucosa Respiratória/citologia , Regulação para Cima/efeitos dos fármacosRESUMO
Cell replacement therapy is a promising approach for treatment of lung disease such as cystic fibrosis, although rates of engraftment need to be improved. We previously showed improved cell retention in the lung using transtracheal delivery compared to intravenous injection. Here, we optimized other parameters of cell delivery using 7-day cultured bone marrow cells (BMCs). Retention of BMC in the lung was dose-dependent. Naphthalene treatment had maximal effects on BMC retention when given 2 days before cell delivery. Naphthalene treatment of the donor amplified a CCSP(+) population and increased retention efficiency in the recipient. Repeated naphthalene treatment and repeated cell delivery both resulted in greater retention. The contribution of the second cell dose was minimal suggesting that a second delivery of BMC promotes proliferation of the first. Busulfan-induced myelosuppression augmented retention of exogenous BMC by up to 20-fold. These BMC helped CCSP reconstitution. Using the optimal delivery techniques and cytokeratin-18-driven green fluorescent protein (GFP) reporter mice, we detected threefold more GFP suggesting more BMC differentiated to epithelial cells. We propose that improved engraftment in the lung will increase cell replacement and thus be a more efficient therapeutic approach for various lung diseases.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Pneumopatias/terapia , Animais , Movimento Celular/fisiologia , Células Cultivadas , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Pneumopatias/induzido quimicamente , Pneumopatias/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Naftalenos/toxicidade , Reação em Cadeia da PolimeraseRESUMO
Inadequate invasion of the uterus by cytotrophoblasts is speculated to result in pregnancy-induced disorders such as preeclampsia. However, the molecular mechanisms that govern appropriate invasion of cytotrophoblasts are unknown. Here, we demonstrate that under low-oxygen conditions (2.5% oxygen), 2-methoxyestradiol (2-ME), which is a metabolite of estradiol and is generated by catechol-o-methyltransferase (COMT), induces invasion of cytotrophoblasts into a naturally-derived, extracellular matrix. Neither low-oxygen conditions nor 2-ME alone induces the invasion of cytotrophoblasts in this system; however, low-oxygen conditions combined with 2-ME result in the appropriate invasion of cytotrophoblasts into the extracellular matrix. Cytotrophoblast invasion under these conditions is also associated with a decrease in the expression of hypoxia-inducible factor-1alpha (HIF-1alpha), transforming growth factor-beta3 (TGF-beta3), and tissue inhibitor of metalloproteinases-2 (TIMP-2). Pregnant COMT-deficient mice with hypoxic placentas and preeclampsia-like features demonstrate an up-regulation of HIF-1alpha, TGF-beta3, and TIMP-2 when compared with wild-type mice; normal levels are restored on administration of 2-ME, which also results in the resolution of preeclampsia-like features in these mice. Indeed, placentas from patients with preeclampsia reveal lower levels of COMT and higher levels of HIF-1alpha, TGF-beta3, and TIMP-2 when compared with those from normal pregnant women. We demonstrate that low-oxygen conditions of the placenta are a critical co-stimulator along with 2-ME for the proper invasion of cytotrophoblasts to facilitate appropriate vascular development and oxygenation during pregnancy.
Assuntos
Vasos Sanguíneos/efeitos dos fármacos , Estradiol/análogos & derivados , Neovascularização Fisiológica/efeitos dos fármacos , Oxigênio/farmacologia , Pré-Eclâmpsia/etiologia , Trofoblastos/efeitos dos fármacos , 2-Metoxiestradiol , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Catecol O-Metiltransferase/genética , Catecol O-Metiltransferase/metabolismo , Adesão Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Sinergismo Farmacológico , Estradiol/metabolismo , Estradiol/farmacologia , Feminino , Humanos , Camundongos , Camundongos Knockout , Neovascularização Fisiológica/genética , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Gravidez , Trofoblastos/patologia , Trofoblastos/fisiologiaRESUMO
Clara cell secretory protein (CCSP) is one of the most abundant proteins in the airway surface fluid, and has many putative functions. Recent advances in the field of stem cells and lung regeneration have identified potentially new roles of CCSP and CCSP-expressing cell populations in airway maintenance, repair and regeneration. This review focuses on the airway regenerative potential of CCSP and the cells that express this protein. The use of this protein or CCSP-expressing cells as an indication of biologic processes that contribute to lung injury or repair is highlighted.
Assuntos
Citocinas/imunologia , Células Epiteliais/metabolismo , Inflamação/imunologia , Lesão Pulmonar/metabolismo , Pulmão/fisiologia , Regeneração , Células-Tronco/metabolismo , Uteroglobina/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea/fisiologia , Citocinas/metabolismo , Células Epiteliais/imunologia , Humanos , Inflamação/metabolismo , Lesão Pulmonar/imunologia , Células-Tronco/imunologia , Uteroglobina/química , Uteroglobina/genética , Uteroglobina/imunologiaRESUMO
The bone marrow compartment is enriched in stem and progenitor cells, and an unidentified subpopulation of these cells can contribute to lung epithelial repair. Here we identify this subpopulation and quantitate its relative contribution to injured airway epithelium. A subpopulation of adherent human and murine bone marrow cells that expresses Clara cell secretory protein (CCSP) was identified using flow cytometry. When cultured at the air-liquid interface in ex vivo cultures, Ccsp+ cells expressed type I and type II alveolar markers as well as basal cell markers and active epithelial sodium channels. Ccsp+ cells preferentially homed to naphthalene-damaged airways when delivered transtracheally or intravenously, with the former being more efficient than the latter. Interestingly, naphthalene-induced lung damage transiently increased Ccsp expression in bone marrow and peripheral circulation. Furthermore, lethally irradiated Ccsp-null mice that received tagged wild-type bone marrow contained donor-derived epithelium in both normal and naphthalene-damaged airways. This study therefore identifies what we believe to be a newly discovered cell in the bone marrow that might have airway reconstitution potential in the context of cell-based therapies for lung disease. Additionally, these data could reconcile previous controversies regarding the contribution of bone marrow to lung regeneration.