Extracellular Matrix-mediated Lung Epithelial Cell Differentiation

Differentiation of functional lung epithelial cells from pluripotent stem cells holds the potential for applications in regenerative medicine. However efficient differentiation to proximal and distal lung epithelial cell populations remains a challenging t ask. The three -dimensional extracellular matrix scaffold is a key component that regulates the interaction of secreted factors with cells during development by often binding to and limiting their diffusion within local gradients. The development of matrices that can recapitulate the in vivo environment is key for directing lung lineage -specific differentiation. Here we examined the role of the lung ECM in differentiation of pluripotent cells in vitro and demonstrate the robust inductive capacity of the native matrix alone using decellularized adult lung scaffolds. The decellularization procedure was optimized and the scaffolds generated were carefully characterized to ensure complete removal of resident cells and preservation of the ECM. Lung scaffolds were recellularized with mouse and rat embryonic stem cell -derived endoderm and maintained for up to three weeks of culture at air liquid interface, in defined, serum -free medium conditions. Recellularization of lung scaffolds with endodermal cells resulted in differentiation to early NKX2 -1+/SOX2+ proximal lung progenitor cells and a heterogeneous basal epithelial cell population, within seven days of culture. Extended culture resulted in robust differentiation to mature airway epithelia, complete with FOXJ1+/TUBB4A+ ciliated cells and SCGB1A1+ secretory club cells, with morphological and functional similarities to native airways. Differentiated day 21 cells contained beating ciliated cells in culture and exhibited functional CFTR protein expression. Heparitinase I, but not chondroitinase ABC, treatment of scaffolds revealed that the differentiation achieved is dependent on heparan sulfate proteoglycans (HSPG) and its bound factors on the ECM. This work demonstrates the importance of a 3-dimensional matrix environment and the role of site-specific cues for directing differentiation of pluripotent stem cells to lung epithelial cells. This is a valuable step towards uncovering ECM-mediated signaling during lung specification and offers a platform for modeling lung development and airway-related diseases using pluripotent stem cells.