Gene Expression
Nebulas
Gene Expression NebulasSummary: Chronic obstructive pulmonary disease (COPD) is a serious global health problem characterized by chronic airway inflammation, progressive airflow limitation and destruction of lung parenchyma. Remodeling of the bronchial airways in COPD includes changes in both the bronchial epithelium and the subepithelial extracellular matrix (ECM). To explore the impact of an aberrant ECM on epithelial cell phenotype in COPD we developed a new ex vivo model, in which normal human bronchial epithelial (NHBE) cells repopulate and differentiate on decellularized human bronchial scaffolds derived from COPD patients and healthy individuals. By using transcriptomics, we show that bronchial ECM from COPD patients induces differential gene expression in primary NHBE cells when compared to normal bronchial ECM. The gene expression profile indicated altered activity of upstream mediators associated with COPD pathophysiology, including hepatocyte growth factor, transforming growth factor beta 1 and platelet-derived growth factor B, which suggests that COPD-related changes in the bronchial ECM contribute to the defective regenerative ability in the airways of COPD patients.
Overall Design: Normal bronchial epithelial (NHBE) cells were seeded on decellularized bronchial scaffolds from 3 COPD patients and 3 healthy donors. Differentiation was induced 4 days later and repopulated scaffolds were collected after 0, 7, 14, 21, 28 and 35 days of differentiation.
| Strategy: |
|
| Species: |
|
| Tissue: |
|
| Healthy Condition: |
|
| Cell Type: |
|
| Development Stage: |
|
| Growth Protocol: | Primary normal human bronchial epithelial (NHBE) cells were purchased from Lonza and cultured in bronchial epithelial growth medium (BEGM) (Lonza) to passage 3. The cells were harvested and seeded on decellularized bronchial scaffolds where they were allowed to attach and grow for 4 days in BEGM. Differentiation was induced after 4 days by exchanging the BEGM with a differentiation medium (50 % BEGM, 50% Dulbecco's Modified Eagle's Medium and 0.05 µM retinoic acid) and differentiation proceeded during 35 days. |
| Treatment Protocol: | Normal bronchial epithelial (NHBE) cells were seeded on decellularized bronchial scaffolds from 3 healthy donors. Differentiation was induced 4 days later and repopulated scaffolds were collected after 0 days of differentiation.; Normal bronchial epithelial (NHBE) cells were seeded on decellularized bronchial scaffolds from 3 healthy donors. Differentiation was induced 4 days later and repopulated scaffolds were collected after 7 days of differentiation.; Normal bronchial epithelial (NHBE) cells were seeded on decellularized bronchial scaffolds from 3 healthy donors. Differentiation was induced 4 days later and repopulated scaffolds were collected after 14 days of differentiation.; Normal bronchial epithelial (NHBE) cells were seeded on decellularized bronchial scaffolds from 3 healthy donors. Differentiation was induced 4 days later and repopulated scaffolds were collected after 21 days of differentiation.; Normal bronchial epithelial (NHBE) cells were seeded on decellularized bronchial scaffolds from 3 healthy donors. Differentiation was induced 4 days later and repopulated scaffolds were collected after 28 days of differentiation.; Normal bronchial epithelial (NHBE) cells were seeded on decellularized bronchial scaffolds from 3 healthy donors. Differentiation was induced 4 days later and repopulated scaffolds were collected after 35 days of differentiation.; Normal bronchial epithelial (NHBE) cells were seeded on decellularized bronchial scaffolds from 3 COPD patients. Differentiation was induced 4 days later and repopulated scaffolds were collected after 0 days of differentiation.; Normal bronchial epithelial (NHBE) cells were seeded on decellularized bronchial scaffolds from 3 COPD patients. Differentiation was induced 4 days later and repopulated scaffolds were collected after 7 days of differentiation.; Normal bronchial epithelial (NHBE) cells were seeded on decellularized bronchial scaffolds from 3 COPD patients. Differentiation was induced 4 days later and repopulated scaffolds were collected after 14 days of differentiation.; Normal bronchial epithelial (NHBE) cells were seeded on decellularized bronchial scaffolds from 3 COPD patients. Differentiation was induced 4 days later and repopulated scaffolds were collected after 21 days of differentiation.; Normal bronchial epithelial (NHBE) cells were seeded on decellularized bronchial scaffolds from 3 COPD patients. Differentiation was induced 4 days later and repopulated scaffolds were collected after 28 days of differentiation.; Normal bronchial epithelial (NHBE) cells were seeded on decellularized bronchial scaffolds from 3 COPD patients. Differentiation was induced 4 days later and repopulated scaffolds were collected after 35 days of differentiation. |
| Extract Protocol: | Total RNA was extracted using the RNeasy Mini kit (Qiagen). |
| Library Construction Protocol: | 560 ng total RNA/sample was used as input to create cDNA libraries using the TruSeq Stranded mRNA kit (Illumina) with dual indexing following standard instructions. |
| Molecule Type: | poly(A)+ RNA |
| Library Source: | |
| Library Layout: | SINGLE |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NextSeq 500 |
| Strand-Specific: | Specific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
|---|