Generation of lung epithelial-like tissue from human embryonic stem cells

BACKGROUND: Human embryonic stem cells (hESC) have the capacity to differentiate in vivo and in vitro into cells from all three germ lineages. The aim of the present study was an investigation on the effect of specific culture conditions on differentiation of hESC into lung epithelial cells. METHODS: Undifferentiated hESC were grown on a porous membrane in a differentiation medium for four days followed by culture for further 25 days in air-liquid interface conditions. Expression of several lung markers was measured by quantitative real-time RT-PCR at four different time points throughout the differentiation and compared to spontaneously differentiated cells. RESULTS: Expression of CC16 and NKX2.1 showed a 1000 and 10000 fold increase at day 10 of differentiation. Other lung markers such as SP-C and Aquaporin 5 had the highest expression after twenty days of culture, as well as two markers for ciliated cells, FOXJ1 and beta-tubulin IV. The results from qRT-PCR were confirmed by immunohistochemistry on paraffin-embedded samples. Antibodies against CC16, SP-A and SP-C were chosen as specific markers for Clara Cells and alveolar type II cells. The functionality was tested by measuring the secretion of CC16 in the medium using an enzyme immunoassay. This showed a peak in secretion level after ten days of culture, which is comparable to the real-time PCR results. CONCLUSIONS: These results suggest that by using our novel culture protocol hESC can be differentiated into the major cell types of lung epithelial tissue.

Publication year:2009

Keywords:human embryonic stem cells, lung epithelial-like tissue