Stepwise differentiation of airway epithelial cells from human tonsil-derived mesenchymal stem cells

Background: Airway defects, often resulting from tumor resection, trauma, or infection, pose significant treatment challenges owing to the intricate, multilayered structure of the airway. Successful recovery depends on reconstructing the respiratory epithelium, the lining next to the cartilage. Although autologous epithelial or progenitor cells are used to reconstruct the epithelium, they are not ideal for regeneration because of difficulties in expansion and differentiation in vitro. In this study, we developed an alternative approach to respiratory epithelial regeneration using human tonsil-derived mesenchymal stem cells (TMSCs) to induce epithelial cell differentiation through a stepwise process. Methods: TMSCs were isolated from the human tonsillar tissues of patients undergoing tonsillectomy and differentiated into airway epithelial cells following human embryonic development. To generate airway epithelial cells, TMSCs were exposed to various chemical agents or protein combinations during a 4-step process. Results: We observed that TMSCs can be induced into the definitive endoderm (DE) with a low concentration of activin A, activating the Nodal/TGF-β signaling pathway. Subsequently, a combination of growth factors regulating BMP, TGF-β, and Wnt signaling induces the differentiation of TMSC-derived DE cells into anterior foregut endoderm, identified by upregulating PAX9, SOX2, and GATA3 gene expression. In the final 3–4 steps, an environment rich in Wnt and FGFs differentiated TMSCs into airway epithelial cells through lung progenitor cells, as evidenced by the increased gene expression of lung progenitor cell markers (NKX2-1), airway cell markers (KRT5), and ciliated cell markers (FoxJ1). Specifically, TMSC-derived airway epithelial cells exhibited a columnar epithelial structure resembling an F-actin filament structure. Conclusions: Our results demonstrated that TMSC-derived airway epithelial cells can be generated through stepwise differentiation and represent a potential alternative for the functional recovery of respiratory defects.