Document Type : Original Article
Department of Internal Medicine, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran;Institute of Biomedical Research, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
Department of Internal Medicine, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
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 μg/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.
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.
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.