Human apical-out nasal organoids reveal an essential role of matrix metalloproteinases in airway epithelial differentiation
Extracellular matrix (ECM) assembly/disassembly is a critical regulator for airway epithelial development and remodeling. Airway organoid is widely used in respiratory research, yet there is limited study to indicate the roles and mechanismsof ECM organization in epithelial growth and differentiation by using in vitro organoid system. Moreover, most of current Matrigel-based airway organoids are in basal-out orientation where accessing the apical surface is challenging. We present a novel human apical-out airway organoid using a biochemically defined hybrid hydrogel system. During human nasal epithelial progenitor cells (hNEPCs) differentiation, thegel gradually degraded, leading to the organoid apical surfaces facing outward. The expression and activity of ECM-degrading enzymes, matrix metalloproteinases (MMP7, MMP9, MMP10 and MMP13) increased during organoid differentiation,where inhibition of MMPs significantly suppressed the normal ciliation, resulting in increased goblet cell proportion. Moreover, a decrease of MMPs was found in goblet cell hyperplastic epithelium in inflammatory mucosa. This system reveals essential roles of epithelial-derived MMPs on epithelial cell fate determination, and provides an applicable platform enabling further study for ECM in regulating airway development in health and diseases. Overall design: To investige the role of matrix metallo proteinases(MMP) in airway epithelial differentiation accompanied with gel degradation on the surface,we cultured untreated organoids and act-treated organoids in which MMP activity were  inhibited by actinonin(MMP inhibitor). In addition,comparison of molecular characteristics between hANO and ALI model was performed to  to demonstrate their similarity. We then performed gene expression profiling analysis using data obtained from RNA-seq of organoids  from 3 donors' nasal polyps tissue and ALI of other 3 donors'  nasal polyps at three time points. Comperative gene expression profiling analysis of RNA-seq data for untreated organoids vs act-treated organoids and untreated organoids vs ALI model.