ABSTRACT
Objective: The ability to generate lung alveolar epithelial type II [ATII] cells from pluripotent stem cells [PSCs] enables the study of lung development, regenerative medicine, and modeling of lung diseases. The establishment of defined, scalable differentiation methods is a step toward this goal. This study intends to investigate the competency of small molecule induced mouse embryonic stem cell-derived definitive endoderm [mESC-DE] cells towards ATII cells
Materials and Methods: In this experimental study, we designed a two-step differentiation protocol. mESC line Royan B20 [RB20] was induced to differentiate into DE [6 days] and then into ATII cells [9 days] by using an adherent culture method. To induce differentiation, we treated the mESCs for 6 days in serum-free differentiation [SFD] media and induced them with 200 nM small molecule inducer of definitive endoderm 2 [IDE2]. For days 7-15 [9 days] of induction, we treated the resultant DE cells with new differentiation media comprised of 100 ng/ml fibroblast growth factor [FGF2] [group F], 0.5 micro g/ml hydrocortisone [group H], and A549 conditioned medium [A549 CM] [group CM] in SFD media. Seven different combinations of factors were tested to assess the efficiencies of these factors to promote differentiation. The expressions of DE- and ATII-specific markers were investigated during each differentiation step
Results: Although both F and H [alone and in combination] promoted differentiation through ATII-like cells, the highest percentage of surfactant protein C [SP-C] expressing cells [37%] were produced in DE-like cells treated by F+H+CM. Ultrastructural analyses also confirmed the presence of lamellar bodies [LB] in the ATII-like cells
Conclusion: These results suggest that hydrocortisone can be a promoting factor in alveolar fate differentiation of IDE2- induced mESC-DE cells. These cells have potential for drug screening and cell-replacement therapies