Derivation and characterization of alveolar epithelial cells from murine embryonic stem cells in vitro.

We present a protocol that has been developed for induction of the differentiation of murine embryonic stem (ES) cells to alveolar type II cells. With this protocol, undifferentiated murine ES cells are induced to form embryoid bodies (EBs). The 10-d-old EBs are transferred to adherent culture conditions and are fed with high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% v/v fetal bovine serum, 2 mM L-glutamine, and 0.1 mM 2-mercaptoethanol for 20 d without splitting. Then, the cells are fed with a medium designed for the maintenance and growth of mature distal airway epithelial cells (small airway growth medium, SAGM) for 3 d. Characterization of the alveolar type II cells was done using real-time reverse transcriptase polymerase chain reaction detection of surfactant protein C mRNA and immunocytochemical detection of prosurfactant protein C. Real-time reverse transcriptase polymerase chain reaction revealed that SAGM increases the mRNA expression level of SPC by a factor of 8 when compared to that of cells grown in supplemented high-glucose DMEM (p < 0.05, Student t-test). Immunocytochemistry revealed that proSPC-expressing cells comprised 2.8 +/- 0.23% of the total cell population in SAGM-treated samples and 0.5 +/- 0.1% in samples treated with supplemented high-glucose DMEM (p < 0.05, chi2 test).