(36) Lung Organoids: Preserving Age Signature Reveals Potential Pediatric Application

Background: In recent years, three-dimensional (3D) organoid cultures have significantly advanced biomedical research by providing a unique model system to investigate the complexity of various organs. Organoids, which are miniaturized organs derived from stem cells, are known to preserve their parental genome, morphology, and physiology. However, little is known about whether organoid systems preserve cell-intrinsic features related to the age of the parental organism from which they were derived, limiting their use in pediatric-focused research.

Objectives: We hypothesized that lung organoids grown from alveolar type II epithelial (AT2) cells isolated from neonatal, juvenile, and adult mice would retain age-related differences in gene expression.

Design/Methods: To test this hypothesis, we employed a tamoxifen-inducible transgenic mouse model (SPC-CreERT2; R26RTdtomato) to fluorescently label our lung stem cell population (AT2 cells). We then used fluorescence-activated cell sorting (FACS) to isolate these labeled cells for co-culture with either age-matched primary fibroblasts or immortalized fibroblast cell line.

Results: We found that AT2 cells isolated from neonatal, juvenile, and adult mice formed organoids at a similar rate. Interestingly, neonatal and juvenile organoids formed a greater number of colony forming units and had a larger diameter compared to the adult group. Moreover, we found that the age and origin of the fibroblast affected colony forming efficiency. Gene expression and DNA methylation patterns in isolated AT2 cells before and after co-culture with fibroblasts were analyzed to assess the retention of age-related signatures.

Conclusion: Our study combines DNA and RNA sequencing with in vitro analysis to provide a comprehensive transcriptome-wide characterization of alveolar organoids as well as insights into the applications, advantages, and limitations of utilizing lung organoids. Additionally, we explore the potential pediatric applications of our findings, shedding light on how age-preserved organoid systems could contribute to pediatric research and clinical practice.