Method Profile
An epithelial H9-derived lung organoid model develops injury and fibrosis-like markers after repeated irradiation and responds transcriptionally to pirfenidone.
Compare early radiation-associated molecular remodeling and compound effects in human lung epithelium.
H9 human embryonic stem-cell-derived lung organoids, passages 3–8; alveolar and airway epithelial populations without immune or fibroblast compartments.
Two irradiation cycles at 2, 4 or 8 Gy; two 4 Gy exposures selected for follow-up. Pirfenidone 0.5 mg/mL immediately after each exposure, with Figure 7 day 5 RNA assessment.
Controls and timing
Unirradiated and PBS-treated irradiated organoids. The narrative and methods place the second irradiation on day 3, whereas Figures 2 and 7 specify day 4.
Parallel animal arm
Separate female C57BL/6 mice received 14 Gy thoracic irradiation and were assessed at four and six months; histology used n = 5. The drug-response comparison was performed in organoids.
Repeated irradiation is compared with single and unirradiated conditions; pirfenidone is compared with irradiated vehicle. Recovered single-cell sequencing counts describe sampled cell populations. Epithelial fibrosis-like markers and delayed whole-lung pathology represent different model scales.
Dose/repetition comparison followed by one benchmark antifibrotic intervention.
Organoid size n = 60 per condition; molecular/drug measurements n = 3; single-cell profiles 10,265 control and 6,890 irradiated cells.
Single versus repeated irradiation, unirradiated control and pirfenidone versus PBS under selected repeated exposure.
Repeated irradiation reduced Ki-67 cells from approximately 30 to fewer than five per organoid and increased EMT-like/collagen-associated markers. Single irradiation did not significantly raise TGFβ; repetition did. Pirfenidone reduced TGFβ, α-SMA and COL1A2 RNA.
Repeated irradiation produced epithelial injury and fibrosis-like changes in H9-derived lung organoids. Pirfenidone given after each irradiation reduced TGFβ, α-SMA and COL1A2 transcripts; the drug experiment assessed an early epithelial response rather than recovery of established lung fibrosis.
