Figure 1.
SAGM-cultured hAECs express CFTR gene and protein.
CFTR expression in hAECs cultured in DMEM:F12 (white bars) and hAECs cultured in Small Airway Epithelial Growth Medium (SAGM) (black bars) for 7, 14, 21 and 28 days (A) (n = 3). hAECs cultured in SAGM for 28 days had a 389.4±70.0 fold increase in CFTR gene expression (p≤0.001), (error bars represent SD). hAECs cultured in DMEM:F12 did not show a significant increase in CFTR gene expression. Human lung total RNA was utilized as a positive control for this study (grey bar). Western blot analysis of CFTR protein expression of freshly isolated hAECs or hAECs cultured in SAGM for 7, 14, 21 and 28 days (B). Both the immature (band A/B) and glycosylated (band C) forms of CFTR were detected in hAEC samples after culture in SAGM for 21 and 28 days (Figure 1B). Only the glycosylated band C was detected in our human lung positive control sample. Flow cytometry analysis of CFTR expression in SAGM cultured hAECs demonstrated that approximately 65% of hAECs expressed the CFTR protein (C) (n = 3).
Figure 2.
SAGM-cultured hAECs form 3-dimensional structures positive for CFTR.
hAECs cultured in DMEM/F12 visualized with fluorescent dyes (CFSE (green) and DAPI (blue)) remained as a confluent, plastic-adherent monolayer (A) (n = 3). hAECs cultured in SAGM for 28 days formed layers of cells clustered into a honeycomb-like morphology growing above a plastic-adherent hAEC monolayer (B) (n = 3). Scanning Electron Microscopy (SEM) analysis of the honeycomb-like 3D structures reveal close interactions between the plastic adherent hAECs and hAECs within the 3D honeycomb-like structures (C (100x) and D (500x)) (n = 3). CFTR expressing cells were found within all 3D structures observed in culture (E–G) and appeared to localize to the edges of the 3D cell clusters (H–J) (n = 3). X-ray tomographic image of the subcellular distribution of CFTR protein using X-ray tomography (K) (n = 4). Shown is a single hAEC from a culture of hAECs after 28 days in SAGM. We were able to visualize cell nuclei (blue), and localize the gold-labeled, silver-enhanced CFTR antibodies (red). CFTR was abundant on the cell membrane, and localized to an area covering approximately half of the plasma membrane.
Figure 3.
SAGM-cultured hAECs produce functional CFTR ion channels.
Supernatant iodine concentration was measured and CFTR channel function was calculated and plotted as supernatant iodine concentration vs forskolin concentration (A) (error bars represent SD, n = 9). hAECs cultured in SAGM demonstrated forskolin-activated CFTR function. Treatment of cultures with CFTR-inhibitor CFTR-172 reversed forskolin activation of CFTR function. Fetal rat lung epithelial (FRLE) cells were utilized as a positive control in this study and displayed similar CFTR stimulation and inhibition as observed in SAGM-cultured hAECs. We did not detect any significant forskolin-activated CFTR function in undifferentiated hAECs. 2D elemental mapping of the forskolin-activated flux of iodine across the cell membrane as well as 2D elemental maps for sulfur, potassium and calcium (B) supported the observed forskolin-activated ion flux across the membrane (n = 3).