A 3D aging human ex-vivo lung model to decode the role of mesenchyme in epithelial senescence and repair - TITLE: A 3D aging human ex-vivo lung model to decode the role of mesenchyme in epithelial senescence and repair PROJECT SUMMARY/ABSTRACT The alveolar sac is the terminal unit of the lung responsible for gas exchange, maintained by coordinated epithelial-mesenchymal (EM) interactions. Within this niche, the epithelial compartment is composed of alveolar epithelial type II cells (AT2s), which serve as a putative plastic progenitors for type I cells (AT1s), which majorly constitute the native sac. On the other hand, from the mesenchymal compartment, lung resident mesenchymal stromal cells (LR-MSCs) regulate epithelial differentiation, extracellular matrix (ECM) remodeling, and mechanical homeostasis needed for respiration. With aging, this dynamic crosstalk becomes disrupted, resulting in epithelial dysfunction, matrix remodeling, and impaired tissue mechanics. However, the mechanisms by which aging alter EM signaling and alveolar homeostasis remain poorly understood, in part due to the lack of human- relevant models that can functionally and structurally mimic the alveolar sac and be capable of physiological ventilation. To this end, having successfully engineered a ventilatable, bioprinted 3D alveolar sac derived from induced-pluripotent stem cell-derived AT2s that recapitulates native-like structural morphology and function, such as discrete compartmentalized, well-established apico-basal polarity for epithelial barrier integrity, surfactant production and mechano-responsiveness; we unexpectedly found that extended culture (> 40 weeks) drives these otherwise stable AT2s into a senescence-like state (aging) marked by slower growth, upregulation of EM signaling, and aberrant ECM remodeling - an observation that now underpins this proposed study. Here, we propose to incorporate primary human LR-MSCs, classified by donor age, into our platform to create heterocellular alveolar sacs and investigate how aging in either or both compartments alters epithelial plasticity and differentiation, ECM dynamics, and mechanical properties in a 3D aging human ex-vivo lung model. Our central hypothesis is that LR-MSCs support alveolar integrity and function under homeostatic conditions but promote dysfunction and remodeling when aged or exposed to senescent cues. We will test this through two aims: (1) develop and characterize a static 3D alveolar sac model, incorporating young and aged AT2s and LR- MSCs to investigate EM crosstalk in aging, and (2) evaluate how ventilation modulates epithelial and mesenchymal responses, focusing on mechano-transductive signaling and structural decline in the aged models. Using the functional ex-vivo lung model, we can dissect how aging disrupts lung regeneration and primes for fibrotic remodeling.
