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| Status |
Public on Aug 17, 2020 |
| Title |
GLI1+ mesenchymal stromal cells modulate epithelial metaplasia in lung fibrosis |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
A single-cell transcriptional analysis was performed on GLI1+ stromal cells from the adult murine lung during homeostasis and after fibrotic injury. The goal is to understand the role of GLI1+ stromal cells in lung fibrosis and repair. Whole adult murine lung tissue from two samples were separately dissociated to single cells and subjected to fluorescence activated cell sorting (FACS) to select all live GLI1+ cells. One sample was treated with bleomycin to induce fibrosis and the other was treated with saline as a control. The single cell RNA-sequencing library was generated separately for the bleomycin and saline-treated samples. Cells were sequenced at a depth of ~70,000 reads/cell. We captured approximately 17,700 cells with a median of 2,400 genes detected per cell utilizing a droplet-based barcoding approach to capture single cells for RNA sequencing. After bleomycin-induced fibrosis, we identified a novel "myofibroblast" subset of GLI1 cells that contribute to injury and repair. Injured GLI1 cells also reveal dysregulation in key developmental pathways, including BMP signaling, that contribute to metaplastic repair after fibrotic injury.
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| Overall design |
Two Gli1-CreERT2/R26R-YFP adult murine lungs were analyzed. Both animals were injected with tamoxifen on five consecutive days to induce YFP expression in GLI1 cells. One week after tamoxifen induction, one lung was treated intranasally with bleomycin every 7 days for four weeks to induce fibrotic injury in the lung. The other lung was treated with saline at the same timepoints as a control. Seven days after the last treatment, the whole lung from each Gli1-CreERT2/R26R-YFP animal was separately dissociated to single cells, stained with DAPI, and subjected to FACS to select live GLI1+ cells. Doublets were excluded based on forward and side scatter, and live cells were selected based on DAPI fluorescent exclusion. Then the GLI1+ cells were sorted by endogenous YFP fluorescence. The live GLI1+ cells were prepared separately for the bleomycin and saline-treated samples using the 10X single cell instrument and v2 10X single cell kits according to standard protocol. Single cell sequencing of adult murine lung GLI1+ population with and without fibrotic injury was performed separately using the HiSeq2500.
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| Contributor(s) |
Cassandras M, Wang C, Peng T |
| Citation(s) |
33046884 |
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| Submission date |
Nov 06, 2019 |
| Last update date |
Nov 16, 2020 |
| Contact name |
Peng Tien |
| E-mail(s) |
tien.peng@ucsf.edu
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| Phone |
4155144180
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| Organization name |
University of California San Francisco
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| Department |
Department of Pulmonary Medicine
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| Lab |
Dr. Tien Peng
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| Street address |
513 Parnassus Ave, Health Sciences East 1350
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94143 |
| Country |
USA |
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| Platforms (1) |
| GPL17021 |
Illumina HiSeq 2500 (Mus musculus) |
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| Samples (2) |
| GSM4151704 |
adult murine lung GLI1+ stromal cells treated with PBS |
| GSM4151705 |
adult murine lung GLI1+ stromal cells treated with bleomycin |
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| Relations |
| BioProject |
PRJNA588064 |
| SRA |
SRP228805 |
