Abstract Rationale We sought to develop a human preclinical model of idiopathic pulmonary fibrosis (IPF) to study epithelial-intrinsic dysfunction and epithelial-mesenchymal crosstalk using patient-specific induced pluripotent stem cells (iPSCs) carrying pathogenic telomerase mutations. Methods We generated iPSCs from two patients with IPF harboring distinct pathogenic variants in telomerase, TERT R951W and L55Q, and engineered corrected syngeneic controls. TERT R951W mutant and corrected iPSCs were differentiated in parallel into alveolar type 2 cells (iAT2s). Telomerase activity was quantified using telomerase repeated amplification protocol (TRAP) and telomere length measured using quantitative telomere fluorescent in situ hybridization (FISH). Single cell RNA sequencing was performed to identify differentially expressed genes, which were validated by RT-qPCR. Gene ontology (GO) enrichment and transcription factor inference analyses were applied to identify candidate pathways and regulatory networks. Self-renewal capacity was assessed by cell yields and EdU-incorporation. Western blots (WB) were used to examine markers of mitochondrial biogenesis and autophagy. Mutant and corrected iAT2s were cocultured with WT iPSC-derived lung mesenchyme. Epithelial-mesenchymal crosstalk was assessed using immunostaining, flow cytometry, and RT-qPCR in FACS-purified epithelia (EPCAM+) and mesenchymal (EPCAM-) fractions. Results Mutant iAT2s displayed markedly reduced telomerase activity and shorter telomeres (Figure A and B). Top upregulated genes included aberrant transitional state markers (PRSS2, GDF15, CYR61, CTGF, KRT7) and mesenchymal-like genes (COL2A1, COL11A1). Mutant iAT2s exhibited a higher KRT5-/KRT17+ gene module score. GO analysis showed enrichment in genes associated with tissue remodeling and collagen fibril organization pathways. p53 was inferred as the top differentially activated transcription factor (Figure C and D). Functionally, mutant iAT2s displayed impaired self-renewal capacity with decreased cell yields and proliferation. WB analysis revealed reduced mitochondrial biogenesis (lower pPGC1a and TOM20) and defective autophagy (increased P62). In epithelial-mesenchymal cocultures, mutant iAT2s induced elevated fibrotic markers in the mesenchymal fraction including CTHRC1 and increased KRT17 and CDKN1A expression in the epithelial fraction (Figure E). Immunostaining confirmed more KRT17+ cells and flow cytometry demonstrated a higher proportion of ACTA2+ mesenchymal cells. Conclusions Telomerase mutations in iAT2s recapitulate disease-relevant epithelial signatures observed in IPF lungs. Dysfunctional epithelium is characterized by shortened telomeres, impaired self-renewal, and defective mitochondrial biogenesis, and becomes further dysregulated in response to mesenchymal interactions, inducing fibrotic mesenchymal activation. Ongoing studies aim to define the mechanisms linking telomerase dysfunction to epithelial dysfunction, determine whether distinct telomerase mutations converge on similar phenotypes, and relate these findings to in vivo TERT-mutant IPF lung tissue. This abstract is funded by: Pulmonary Fibrosis Foundation
Chang et al. (Fri,) studied this question.