Pulmonary Fibrosis (PF) has a median survival from diagnosis of 2-4 years. Lung transplant remains the only cure. Mutations causing shortened telomere length in type II alveolar epithelial cells (AT2) result in severe, early-onset PF. We hypothesize that pro-fibrotic signalling is activated by telomere attrition and that AT2 cells generated from induced pluripotent stem cells (iPSC) (iAT2) can be used as a preclinical model.

Genomic DNA samples were acquired on Days 0, 3, 200 and 400 during directed differentiation of iPSCs towards iAT2s. Telomere length analysis was performed using high-throughput quantitative fluorescent in-situ hybridization (HT-Q-FISH), real-time quantitative PCR and Southern Blot. The expression of pro-fibrotic, inflammatory and senescence genes at the different timepoints was measured by qPCR.

Analogous to the telomere attrition occurring in vivo, median telomere length decreases from 10.2Kbp to 8.2Kbp from day 0 to day 200 and further to 3.8Kbp at day 400 in our model measured by HT-Q-FISH and supported by the other methods. The percentage of very short telomeres (<3Kbp) measured by HT-Q-FISH increased from 2.75% on day 0 to 6.65% on day 200 to 40.25% on day 400. This telomere shortening was associated with an increased expression of senescence, fibrotic and anti-apoptotic genes and decreased expression of pro-apoptotic genes.

This is the first reported model of telomere shortening in human iAT2 cells and explores the relationship of telomere length and pro-fibrotic pathways. Our preliminary work suggests a negative association between telomere shortening and pro-fibrotic signalling, indicating an important role in the onset of fibrosis.