Induction of ciliated cells from avian embryonic stem cells using three-dimensional matrix.

We have devised a simple three-dimensional (3D) tissue-culturing method to induce ciliogenesis from avian embryonic stem (ES) cells by using avian fertilized eggs. Unlike the previous reported techniques, this method does not require trypsinization, which would reduce the viability of the cells; it also does not require an air-liquid interface to induce ciliogenesis and to maintain the growth of the induced ciliated cells. ES cells seeded and attached on this collagen-coated chitosan 3D gel grew spontaneously and robustly. Following 2 weeks in culture with inhibition of embryoid body formation, cells with noticeable and vigorous beating cilia were observed. We measured the ciliary beat frequencies of these ES-differentiated ciliated cells for 40 days. These results were consistent with all reported measurements made for other species of ciliated cells, including human, from our previous study. These data imply that the cilia of these ES-derived ciliated cells, beating at their intrinsic basal autorhythmic rate, preserve the integrity of the regulatory mechanisms of ciliary beat frequency. In conclusion, we have shown that ES cells cultured in a 3D tissue-engineered scaffold is a promising approach for developing an in vitro cell model that closely mimics the in vivo ciliated cell natural milieu. This cell model can potentially be the source of ciliated cells for cell-based high-throughput screening and discovery of pulmonary drugs.