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

Abstract

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.

FIG. 1.

Procedure for the creation of the 3D substrate system. 3D, three-dimensional; PBS, phosphate-buffered saline.

FIG. 2.

(A) A 10 × photograph of the chitosan–collagen matrix. (B) A 10 × optical micrograph of epithelial cell growth from ES cells on 250 μm grooves after 6 weeks of seeding of the cells. (C) Ciliogenesis started at 2 weeks after seeding of the cells. Cilia started beating at the edge of the gel (top view) (see Supplementary Movie S1; available online at www.liebertonline.com). Black arrow shows the position of the beating cilia. (D) Anti-β-tubulin staining of cilia after 2 weeks of culture. White arrow shows the anti-β-tubulin stain of the cilia. (E) Scanning electron micrographs of epithelial cell growth from ES cells on matrix after 6 weeks. (F) An enlarged top view showing cilia. (G) Mature cilia reached a length of approximately 6–7 μm. (H) Over 80% of the cultured cell surface was covered with mature cilia after 10 weeks of culture. ES, embryonic stem. Color images available online at www.liebertonline.com/ten.

FIG. 3.

Basal CBF of three avian ES-derived ciliated cell samples measured from day 1 to 40. An average basal CBF value was derived from each of the 5 min measurement intervals for each sample at each day. At each day, a mean CBF value was obtained from the average basal CBF values of the three samples. They were presented as the basal CBF (mean ± standard deviation) plotted against the day. CBF, ciliary beat frequency.

FIG. 4.

Cumulative stimulatory responses of CBF by 1 and 10 μM terbutaline of the three avian ES-derived ciliated cell samples measured at days 21, 30, and 40. At each measurement day, an average CBF value was derived from each of the 5 min measurement intervals per experimental condition for each sample. A mean CBF value was then obtained from the CBF averages of each of the experimental conditions of the three samples. They are the basal CBFs and the CBF stimulatory responses induced by 1 and 10 μM terbutatine. They were presented as the CBFs (mean ± standard deviation) of each experimental condition. *p < 0.5, compared with basal CBF; **p < 0.1, compared with basal CBF.