Alveolar Extracellular Matrix Hydrogels Facilitate Proliferation of Induced Pluripotent Stem Cell-Derived Alveolar Epithelial Spheroids

Type 2 alveolar epithelial cells (AEC2s) are adult cells in the human lung that have a critical role in lung homeostasis. In addition to secreting surfactant that is necessary to prevent alveolar collapse, AEC2s are the facultative progenitors of the alveolus. Specifically, in response to injury AEC2s proliferate to replenish AEC2s and type 1 alveolar epithelial cells (AEC1s). However, despite the critical role of AEC2s in lung physiology and injury response, an inability to isolate an expandable culture of AEC2 in vitro has impeded a full understanding of AEC2 biology. Recently, a pure and expandable culture of induced-AEC2s (iAEC2s) spheroids (i.e. alveolospheres) has been derived using a directed differentiation method from induced pluripotent stem cells (iPSCs). These iAEC2s spheroids are amenable to prolonged culture periods, possess mature markers and functionality (e.g. surfactant secretion) of in vivo AEC2s, and can recapitulate AEC2 dysfunction. However, initial studies of iAEC2s were performed in Matrigel, an extracellular matrix (ECM) derived from a tumorigenic mouse cell line, and it is unknown how iAEC2s respond to a physiologically relevant lung matrix. Here, we employ a novel and innovative culture method to determine how iAEC2s respond to human alveolar ECM. Decellularized lungs from adult patients with a healthy lung history were used to produce 3-dimensional hydrogels from alveolar enriched ECM (aECM) for the culture of iAEC2s. Our preliminary results provide evidence that iAEC2s maintain alveolosphere formation and efficiently proliferate in aECM hydrogels in an aECM protein concentration dependent manner. Furthermore, gene expression analysis suggests that aECM promotes lamellar body associated AEC2 maturation markers (i.e. SFTPC, LAMP3, ABCA3) in iAEC2s. Importantly, the ability to culture iAEC2s in a physiologically relevant lung matrix could provide a unique model to determine the effects of normal and diseased lung ECM (e.g. idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease) on AEC2 behavior