Constrained differentiation of human pluripotent stem cells into homogeneous bronchial epithelium on a fibroblast seeded matrix

Rationale Derived iPSCs airway epithelium are challenging given their dependency on the mesenchymal compartment. We hypothesized that growing vAFE cells on well-organized stiff matrix precolonized by adult pulmonary fibroblasts would improve epithelial differentiation yield and maturity. Methods Collagen-1/chitosan matrix were engineered to reach stiffness and scaffolding characteristics of subepithelial compartments. Primary fibroblasts derived from human lung samples were seeded for 45 days before the addition of vAFE cells differentiated from iPSCs, and comparisons made with iPSC-derived fibroblasts. Beads tracking was used to assess cilia beating efficiency. Results Primary human bronchial fibroblasts were able to enrich the CC Matrix with extracellular matrix components such as collagen, decorin and vimentin. In turn, iALI cultures performed in primary fibroblasts enriched CC matrix successfully led to high level of epithelial differentiation including rare cells (club, basal, neuroendocrine, ciliated, secretory). Large apical surfaces were covered by approximately 60% of ciliated cells able to generate mucociliary vortex. Conclusion Primary human bronchial fibroblasts seeded into collagen-chitosan matrix dramatically improved iALI epithelial differentiation from vAFE cells, related to highly specific transcriptomic signatures when compared to iPSC derived fibroblasts.