| 1. |
- Almendros, Isaac, et al.
(författare)
-
Early Career Members at the ERS Lung Science Conference: cell-matrix interactions in lung disease and regeneration: Early career forum
- 2018
-
Ingår i: Breathe. - : European Respiratory Society (ERS). - 1810-6838 .- 2073-4735. ; 14:2, s. 78-83
-
Tidskriftsartikel (refereegranskat)abstract
- The 16th ERS Lung Science Conference (LSC) took place on March 8–11, 2018, in Estoril, Portugal, with around 200 delegates from all over the world. This year’s topic was “Cell-matrix interactions in lung disease and regeneration” and involved excellent presentations by leading experts in the field covering everything from exploratory studies on how the matrix functions, matrix remodelling and biomarkers in disease, to more technical knowledge described in the field of lung bioengineering. As in previous years, the Saturday afternoon was reserved for a programme dedicated to early career delegates, which this year focussed on “Maximising your publication output”. In this article, we summarise the Early Career Member highlights of this year’s LSC.
|
|
| 2. |
- Alsafadi, Hani N, et al.
(författare)
-
Applications and Approaches for Three-Dimensional Precision-Cut Lung Slices. Disease Modeling and Drug Discovery
- 2020
-
Ingår i: American Journal of Respiratory Cell and Molecular Biology. - 1044-1549. ; 62:6, s. 681-691
-
Forskningsöversikt (refereegranskat)abstract
- Chronic lung diseases (CLDs), such as chronic obstructive pulmonary disease, interstitial lung disease, and lung cancer, are among the leading causes of morbidity globally and impose major health and financial burdens on patients and society. Effective treatments are scarce, and relevant human model systems to effectively study CLD pathomechanisms and thus discover and validate potential new targets and therapies are needed. Precision-cut lung slices (PCLS) from healthy and diseased human tissue represent one promising tool that can closely recapitulate the complexity of the lung's native environment, and recently, improved methodologies and accessibility to human tissue have led to an increased use of PCLS in CLD research. Here, we discuss approaches that use human PCLS to advance our understanding of CLD development, as well as drug discovery and validation for CLDs. PCLS enable investigators to study complex interactions among different cell types and the extracellular matrix in the native three-dimensional architecture of the lung. PCLS further allow for high-resolution (live) imaging of cellular functions in several dimensions. Importantly, PCLS can be derived from diseased lung tissue upon lung surgery or transplantation, thus allowing the study of CLDs in living human tissue. Moreover, CLDs can be modeled in PCLS derived from normal lung tissue to mimic the onset and progression of CLDs, complementing studies in end-stage diseased tissue. Altogether, PCLS are emerging as a remarkable tool to further bridge the gap between target identification and translation into clinical studies, and thus open novel avenues for future precision medicine approaches.
|
|
| 3. |
- Alsafadi, Hani N.
(författare)
-
Role of the co-transcriptional regulators Yap/Taz in the normal and fibrotic lung epithelia
- 2023
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Idiopathic pulmonary fibrosis (IPF) is a fatal disease that exhibits patterns of usual interstitial pneumonia with honeycombing. IPF is characterized by damaged distal lung epithelium with excessive tissue scarring and extracellular matrix remodeling. The etiology of IPF is unknown and current therapies cannot end or reverse disease progression. Aberrant reactivation of developmental pathways is evident in IPF. Among these developmental actors are the co-transcriptional regulators Yap and Taz (YT). YT modulate processes such as proliferation, differentiation, and organ size and are regulated by the Hippo pathway. YT do not have a DNA binding domain but act through interaction with other transcription factors (TFs). YT play a role in fibrotic fibroblasts, but their role is not yet known in the fibrotic lung epithelium. The aim of this thesis project is to develop the tools needed to explore the role of Hippo-YT in fibrotic lung epithelium and to identify the TFs that YT interact with to exert their various functions.We first developed a method to simultaneously isolate proximal and distal lung progenitor cells from an individual mouse with the aid of a 3D printed surgical guide and found that the precision of dissecting the lung lobes affects the purity of the isolated distal progenitors and how they behave in organoid assays. We further found the Hippo pathway to be dysregulated in the fibrotic lung epithelium which led to increases in nuclear YT as well as known downstream targets. Interestingly, we found epithelial YT signaling to be actively involved in extracellular matrix remodeling in the fibrotic lung epithelium through modulation of lysyl oxidase expression, a collagen crosslinking enzyme. Targeting YT in vivo using an FDA approved drug ameliorated the fibrotic phenotype, indicating that YT targeting may be an option to treat fibrosis. We further used cleavage under target and release using nuclease (CUT&RUN) to identify the exact motif sequences on the genome where complexes containing YT bind in the normal and fibrotic lung epithelial. We further identified putative TFs that are known to bind the motif sequences identified. We found that YT have different interaction partners in the proximal and distal lung epithelium and further identified specific YT interactions in the human fibrotic lung epithelium.This current research project sets the basis for the identification of exact targeting mechanisms for finding therapeutics for IPF. YT are known to be responsible for a wide range of biologic processes and targeting YT’s profibrotic activity and promoting their pro-regenerative activities may result in beneficial effects for IPF patients.
|
|
| 4. |
- Alsafadi, Hani N, et al.
(författare)
-
Simultaneous isolation of proximal and distal lung progenitor cells from individual mice using a 3D printed guide reduces proximal cell contamination of distal lung epithelial cell isolations
- 2022
-
Ingår i: Stem Cell Reports. - : Elsevier BV. - 2213-6711. ; 17:12, s. 2718-2731
-
Tidskriftsartikel (refereegranskat)abstract
- The respiratory epithelium consists of multiple, functionally distinct cell types and is maintained by regionally specific progenitor populations that repair the epithelium following injury. Several in vitro methods exist for studying lung epithelial repair using primary murine lung cells, but isolation methods are hampered by a lack of surface markers distinguishing epithelial progenitors along the respiratory epithelium. Here, we developed a 3D printed lobe divider (3DLD) to aid in simultaneous isolation of proximal versus distal lung epithelial progenitors from individual mice that give rise to differentiated epithelia in multiple in vitro assays. In contrast to 3DLD-isolated distal progenitor cells, commonly used manual tracheal ligation methods followed by lobe removal resulted in co-isolation of rare proximal cells with distal cells, which altered the transcriptional landscape and size distribution of distal organoids. The 3DLD aids in reproducible isolation of distal versus proximal progenitor populations and minimizes the potential for contaminating populations to confound in vitro assays.
|
|
| 5. |
- Bölükbas, Deniz A., et al.
(författare)
-
The Preparation of Decellularized Mouse Lung Matrix Scaffolds for Analysis of Lung Regenerative Cell Potential
- 2019
-
Ingår i: Methods in molecular biology (Clifton, N.J.). - New York, NY : Springer New York. - 1940-6029. ; 1940, s. 275-295
-
Tidskriftsartikel (refereegranskat)abstract
- Lung transplantation is the only option for patients with end-stage lung disease, but there is a shortage of available lung donors. Furthermore, efficiency of lung transplantation has been limited due to primary graft dysfunction. Recent mouse models mimicking lung disease in humans have allowed for deepening our understanding of disease pathomechanisms. Moreover, new techniques such as decellularization and recellularization have opened up new possibilities to contribute to our understanding of the regenerative mechanisms involved in the lung. Stripping the lung of its native cells allows for unprecedented analyses of extracellular matrix and sets a physiologic platform to study the regenerative potential of seeded cells. A comprehensive understanding of the molecular pathways involved for lung development and regeneration in mouse models can be translated to regeneration strategies in higher organisms, including humans. Here we describe and discuss several techniques used for murine lung de- and recellularization, methods for evaluation of efficacy including histology, protein/RNA isolation at the whole lung, as well as lung slices level.
|
|
| 6. |
- De Santis, Martina M, et al.
(författare)
-
Extracellular-Matrix-Reinforced Bioinks for 3D Bioprinting Human Tissue
- 2021
-
Ingår i: Advanced Materials. - : Wiley. - 1521-4095 .- 0935-9648. ; 33:3
-
Tidskriftsartikel (refereegranskat)abstract
- Recent advances in 3D bioprinting allow for generating intricate structures with dimensions relevant for human tissue, but suitable bioinks for producing translationally relevant tissue with complex geometries remain unidentified. Here, a tissue-specific hybrid bioink is described, composed of a natural polymer, alginate, reinforced with extracellular matrix derived from decellularized tissue (rECM). rECM has rheological and gelation properties beneficial for 3D bioprinting while retaining biologically inductive properties supporting tissue maturation ex vivo and in vivo. These bioinks are shear thinning, resist cell sedimentation, improve viability of multiple cell types, and enhance mechanical stability in hydrogels derived from them. 3D printed constructs generated from rECM bioinks suppress the foreign body response, are pro-angiogenic and support recipient-derived de novo blood vessel formation across the entire graft thickness in a murine model of transplant immunosuppression. Their proof-of-principle for generating human tissue is demonstrated by 3D bioprinting human airways composed of regionally specified primary human airway epithelial progenitor and smooth muscle cells. Airway lumens remained patent with viable cells for one month in vitro with evidence of differentiation into mature epithelial cell types found in native human airways. rECM bioinks are a promising new approach for generating functional human tissue using 3D bioprinting.
|
|
| 7. |
- Gerckens, Michael, et al.
(författare)
-
Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling
- 2019
-
Ingår i: Journal of visualized experiments : JoVE. - : MyJove Corporation. - 1940-087X. ; :144
-
Tidskriftsartikel (refereegranskat)abstract
- Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue's stiffness, which is crucial for subsequent slicing. 500 µm thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.
|
|
| 8. |
- Gerckens, Michael, et al.
(författare)
-
Phenotypic drug screening in a human fibrosis model identified a novel class of antifibrotic therapeutics
- 2021
-
Ingår i: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 7:52, s. 1-19
-
Tidskriftsartikel (refereegranskat)abstract
- Fibrogenic processes instigate fatal chronic diseases leading to organ failure and death. Underlying biological processes involve induced massive deposition of extracellular matrix (ECM) by aberrant fibroblasts. We subjected diseased primary human lung fibroblasts to an advanced three-dimensional phenotypic high-content assay and screened a repurposing drug library of small molecules for inhibiting ECM deposition. Fibrotic Pattern Detection by Artificial Intelligence identified tranilast as an effective inhibitor. Structure-activity relationship studies confirmed N-(2-butoxyphenyl)-3-(phenyl)acrylamides (N23Ps) as a novel and highly potent compound class. N23Ps suppressed myofibroblast transdifferentiation, ECM deposition, cellular contractility, and altered cell shapes, thus advocating a unique mode of action. Mechanistically, transcriptomics identified SMURF2 as a potential therapeutic target network. Antifibrotic activity of N23Ps was verified by proteomics in a human ex vivo tissue fibrosis disease model, suppressing profibrotic markers SERPINE1 and CXCL8. Conclusively, N23Ps are a novel class of highly potent compounds inhibiting organ fibrosis in patients.
|
|
| 9. |
|
|
| 10. |
- Lehmann, Mareike, et al.
(författare)
-
Differential effects of Nintedanib and Pirfenidone on lung alveolar epithelial cell function in ex vivo murine and human lung tissue cultures of pulmonary fibrosis 11 Medical and Health Sciences 1102 Cardiorespiratory Medicine and Haematology 06 Biological Sciences 0601 Biochemistry and Cell Biology
- 2018
-
Ingår i: Respiratory Research. - : Springer Science and Business Media LLC. - 1465-9921 .- 1465-993X. ; 19:1
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease. Repetitive injury and reprogramming of the lung epithelium are thought to be critical drivers of disease progression, contributing to fibroblast activation, extracellular matrix remodeling, and subsequently loss of lung architecture and function. To date, Pirfenidone and Nintedanib are the only approved drugs known to decelerate disease progression, however, if and how these drugs affect lung epithelial cell function, remains largely unexplored. Methods: We treated murine and human 3D ex vivo lung tissue cultures (3D-LTCs; generated from precision cut lung slices (PCLS)) as well as primary murine alveolar epithelial type II (pmATII) cells with Pirfenidone or Nintedanib. Murine 3D-LTCs or pmATII cells were derived from the bleomycin model of fibrosis. Early fibrotic changes were induced in human 3D-LTCs by a mixture of profibrotic factors. Epithelial and mesenchymal cell function was determined by qPCR, Western blotting, Immunofluorescent staining, and ELISA. Results: Low μM concentrations of Nintedanib (1 μM) and mM concentrations of Pirfenidone (2.5 mM) reduced fibrotic gene expression including Collagen 1a1 and Fibronectin in murine and human 3D-LTCs as well as pmATII cells. Notably, Nintedanib stabilized expression of distal lung epithelial cell markers, especially Surfactant Protein C in pmATII cells as well as in murine and human 3D-LTCs. Conclusions: Pirfenidone and Nintedanib exhibit distinct effects on murine and human epithelial cells, which might contribute to their anti-fibrotic action. Human 3D-LTCs represent a valuable tool to assess anti-fibrotic mechanisms of potential drugs for the treatment of IPF patients.
|
|
| 11. |
- Nowakowska, Joanna, et al.
(författare)
-
Increased expression of ORMDL3 in allergic asthma : a case control and in vitro study
- 2023
-
Ingår i: Journal of Asthma. - : Informa UK Limited. - 0277-0903 .- 1532-4303. ; 60:3, s. 458-467
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Asthma is the most frequent chronic disease in children. One of the most replicated genetic findings in childhood asthma is the ORMDL3 gene confirmed in several GWA studies in several paediatric populations.OBJECTIVES: The purpose of this study was to analyse ORMDL3 variants and expression in childhood asthma in the Polish population.MATERIALS AND METHODS: In the study we included 416 subject, 223 asthmatic children and 193 healthy control subjects. The analysis of two SNPs (rs3744246 and rs8076131) was performed using genotyping with TaqMan probes. The methylation of the ORMDL3 promoter was examined with Methylation Sensitive HRM (MS-HRM), covering 9 CpG sites. The expression of ORMDL3 was analysed in PBMCs from paediatric patients diagnosed with allergic asthma and primary human bronchial epithelial cells derived from healthy subjects treated with IL-13, IL-4, or co-treatment with both cytokines to model allergic airway inflammation.RESULTS: We found that ORMDL3 expression was increased in allergic asthma both in PBMCs from asthmatic patients as well as in human bronchial epithelial cells stimulated with the current cytokines. We did not observe significant differences between cases and controls either in the genotype distribution of analysed SNPs (rs3744246 and rs8076131) nor in the level of promoter methylation.CONCLUSIONS: Increased ORMDL3 expression is associated with paediatric allergic asthma and upregulated in the airways upon Th2-cytokines stimulation, but further functional studies are required to fully understand its role in this disease.
|
|
| 12. |
- Ota, Chiharu, et al.
(författare)
-
Dynamic expression of HOPX in alveolar epithelial cells reflects injury and repair during the progression of pulmonary fibrosis
- 2018
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8
-
Tidskriftsartikel (refereegranskat)abstract
- Mechanisms of injury and repair in alveolar epithelial cells (AECs) are critically involved in the progression of various lung diseases including idiopathic pulmonary fibrosis (IPF). Homeobox only protein x (HOPX) contributes to the formation of distal lung during development. In adult lung, alveolar epithelial type (AT) I cells express HOPX and lineage-labeled Hopx+ cells give rise to both ATI and ATII cells after pneumonectomy. However, the cell function of HOPX-expressing cells in adult fibrotic lung diseases has not been investigated. In this study, we have established a flow cytometry-based method to evaluate HOPX-expressing cells in the lung. HOPX expression in cultured ATII cells increased over culture time, which was accompanied by a decrease of proSP-C, an ATII marker. Moreover, HOPX expression was increased in AECs from bleomycin-instilled mouse lungs in vivo. Small interfering RNA-based knockdown of Hopx resulted in suppressing ATII-ATI trans-differentiation and activating cellular proliferation in vitro. In IPF lungs, HOPX expression was decreased in whole lungs and significantly correlated to a decline in lung function and progression of IPF. In conclusion, HOPX is upregulated during early alveolar injury and repair process in the lung. Decreased HOPX expression might contribute to failed regenerative processes in end-stage IPF lungs.
|
|
| 13. |
- Ptasinski, Victoria, et al.
(författare)
-
Modeling fibrotic alveolar transitional cells with pluripotent stem cell-derived alveolar organoids
- 2023
-
Ingår i: Life Science Alliance. - 2575-1077. ; 6:8
-
Tidskriftsartikel (refereegranskat)abstract
- Repeated injury of the lung epithelium is proposed to be the main driver of idiopathic pulmonary fibrosis (IPF). However, available therapies do not specifically target the epithelium and human models of fibrotic epithelial damage with suitability for drug discovery are lacking. We developed a model of the aberrant epithelial reprogramming observed in IPF using alveolar organoids derived from human-induced pluripotent stem cells stimulated with a cocktail of pro-fibrotic and inflammatory cytokines. Deconvolution of RNA-seq data of alveolar organoids indicated that the fibrosis cocktail rapidly increased the proportion of transitional cell types including the KRT5-/KRT17+ aberrant basaloid phenotype recently identified in the lungs of IPF patients. We found that epithelial reprogramming and extracellular matrix (ECM) production persisted after removal of the fibrosis cocktail. We evaluated the effect of the two clinically approved compounds for IPF, nintedanib and pirfenidone, and found that they reduced the expression of ECM and pro-fibrotic mediators but did not completely reverse epithelial reprogramming. Thus, our system recapitulates key aspects of IPF and is a promising system for drug discovery.
|
|
| 14. |
- Stegmayr, John, et al.
(författare)
-
Isolation of high yield and quality RNA from human precision-cut lung slices for RNA-sequencing and computational integration with larger patient cohorts
- 2021
-
Ingår i: American Journal of Physiology: Lung Cellular and Molecular Physiology. - : American Physiological Society. - 1522-1504 .- 1040-0605. ; 320:2, s. 232-240
-
Tidskriftsartikel (refereegranskat)abstract
- Precision-cut lung slices (PCLS) have gained increasing interest as a model to study lung biology/disease and screening novel therapeutics. In particular, PCLS derived from human tissue can better recapitulate some aspects of lung biology/disease as compared to animal models. Several experimental readouts have been established for use with PCLS, but obtaining high yield and quality RNA for downstream analysis has remained challenging. This is particularly problematic for utilizing the power of next-generation sequencing techniques, such as RNA-sequencing (RNA-seq), for non-biased and high through-put analysis of PCLS human cohorts. In the current study, we present a novel approach for isolating high quality RNA from a small amount of tissue, including diseased human tissue, such as idiopathic pulmonary fibrosis (IPF). We show that the RNA isolated using this method has sufficient quality for RT-qPCR and RNA-seq analysis. Furthermore, the RNA-seq data from human PCLS could be used in several established computational pipelines, including deconvolution of bulk RNA-seq data using publicly available single-cell RNA-seq data. Deconvolution using Bisque revealed a diversity of cell populations in human PCLS, including several immune cell populations, which correlated with cell populations known to be present and aberrant in human disease.
|
|
| 15. |
- Wagner, Darcy E, et al.
(författare)
-
Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Disease 2019
- 2020
-
Ingår i: ERJ Open Research. - : European Respiratory Society (ERS). - 2312-0541. ; 6:4
-
Forskningsöversikt (refereegranskat)abstract
- A workshop entitled "Stem Cells, Cell Therapies and Bioengineering in Lung Biology and Diseases" was hosted by the University of Vermont Larner College of Medicine in collaboration with the National Heart, Lung and Blood Institute, the Alpha-1 Foundation, the Cystic Fibrosis Foundation, the International Society for Cell and Gene Therapy and the Pulmonary Fibrosis Foundation. The event was held from July 15 to 18, 2019 at the University of Vermont, Burlington, Vermont. The objectives of the conference were to review and discuss the current status of the following active areas of research: 1) technological advancements in the analysis and visualisation of lung stem and progenitor cells; 2) evaluation of lung stem and progenitor cells in the context of their interactions with the niche; 3) progress toward the application and delivery of stem and progenitor cells for the treatment of lung diseases such as cystic fibrosis; 4) progress in induced pluripotent stem cell models and application for disease modelling; and 5) the emerging roles of cell therapy and extracellular vesicles in immunomodulation of the lung. This selection of topics represents some of the most dynamic research areas in which incredible progress continues to be made. The workshop also included active discussion on the regulation and commercialisation of regenerative medicine products and concluded with an open discussion to set priorities and recommendations for future research directions in basic and translation lung biology.
|
|
