Zhiying Liao

Guangzhou National Laboratory, School of Biomedical Engineering, Guangzhou Medical University, China

Regional epithelial lineages of the human respiratory system reside within an extracellular matrix (ECM) whose mechanics vary along the airway-alveolar  axis,  yet  how  ECM  directs  epithelial fates  remains unclear.  Here,  utilizing  human  pluripotent  stem  cells  derived  lung organoids   embedded    in   stiffness-tunable    gelatin    methacryloyl (GelMA) hydrogels as an in vitro model, we show that extracellular

stiffness governs region-specific epithelial differentiation. This study discovers    that     increased-stiffness     enhances     lung     progenitors     generation.     During proximal-to-distal  airway  specification,   by   modulating  ECM  stiffness  from   high  to   low,  a sequential emergence of proximal airway cells (e.g. goblet and ciliated cells), followed by cells existed in the proximal-to-distal transitional zone, and finally enriched with distal secretory cells.

During alveolar differentiation, increased stiffness promotes alveolar type 2 and type 1 cells maturation  and  drives  type  2  to  type   1  transition.   Furthermore,   bulk  and  single   cell RNA-sequencing  reveals  extracellular  stiffness   primarily  regulates  epithelial  fates  through mechanical downstream pathways (e.g., Hippo, hypoxia, Wnt). Finally, infection assays with the Omicron BA.1.1 and Delta variants on mechanical stiffness-derived site-specific lung organoids successfully recapitulated their distinct infection tropism. This research elucidates extracellular matrix stiffness as a critical factor of epithelial cell fate determination and region-specific lung generation,  and  also  offers  a  valuable  in  vitro  model  for  studying  region-specific  lung development, diseases pathogenesis, and drug screening.