A novel method for expansion and differentiation of mouse tracheal epithelial cells in culture

Abstract

Air-liquid interface (ALI) cultures of mouse tracheal epithelial cells (MTEC) are a well-established model to study airway epithelial cells, but current methods require large numbers of animals which is unwanted in view of the 3R principle and introduces variation. Moreover, stringent breeding schemes are frequently needed to generate sufficient numbers of genetically modified animals. Current protocols do not incorporate expansion of MTEC, and therefore we developed a protocol to expand MTEC while maintaining their differentiation capacity. MTEC were isolated and expanded using the ROCK inhibitor Y-27632 in presence or absence of the γ-secretase inhibitor DAPT, a Notch pathway inhibitor. Whereas MTEC proliferated without DAPT, growth rate and cell morphology improved in presence of DAPT. ALI-induced differentiation of expanded MTEC resulted in an altered capacity of basal cells to differentiate into ciliated cells, whereas IL-13-induced goblet cell differentiation remained unaffected. Ciliated cell differentiation improved by prolonging the ALI differentiation or by adding DAPT, suggesting that basal cells retain their ability to differentiate. This technique using expansion of MTEC and subsequent ALI differentiation drastically reduces animal numbers and costs for in vitro experiments, and will reduce biological variation. Additionally, we provide novel insights in the dynamics of basal cell populations in vitro.

Figure 1

Expanding MTEC in KSFM expansion medium with Notch signaling inhibitor. (a) MTEC are expanded to passage 1 in KSFM expansion medium followed by passaging and culturing on transwell inserts. MTEC are grown to full confluence in MTEC proliferation medium, followed by air-liquid interface differentiation in MTEC differentiation medium. (b) Serial expansion of MTEC in different medium conditions. Epithelial cells are cultured in proliferation medium with ROCK inhibitor (Y27632), in KSFM medium with ROCK inhibitor or KSFM medium with ROCK inhibitor (Y27632) and γ-secretase inhibitor (DAPT). Phase-contrast images of the MTEC at various passages showing the morphology of the cells. Scale bar, 100 µm. (c) The graph represents the number of cells obtained after passaging when cultured in KSFM with ROCK inhibitor alone or in the presence of DAPT (mean ± SEM). *p < 0.05 by unpaired t-test (n = 5). The table shows the number of 12-well inserts that can be obtained after each passage.

Figure 2

Expansion of MTEC in vitro leads to decreased ciliary and club cell differentiation. (a) Schematic representation of culture protocol. (b) Co-staining of different epithelial markers on MTEC passage 2 after 8 days of air liquid interface (ALI) culture. From left to right, inserts were stained with basal cell marker KRT5 and luminal marker KRT8, cilia cell TUBB4B with tight junction protein ZO-1, secretory club cell marker SCGB1A1 with cilia marker TUBB4B and the last panel shows TRP63 positive basal cells with ciliated cell marker FOXJ1. Nuclei are stained with DAPI (blue). Scale bar, 30 µm. (c) Resistance measurements of MTEC after different passages and 8 days of ALI (mean ± SEM). *p < 0.05 by one-way ANOVA (n = 3). (d) Quantification of the percentage of FOXJ1 positive ciliated cells after 8 days of ALI comparing MTEC in passage (P) 0, 1 and 2 (mean ± SEM). *p < 0.05 by one-way ANOVA (n = 3). (e) qRT-PCR analysis of Foxj1 in MTEC in passage (P) 0, 1 and 2 (mean ± SEM). *p < 0.05 by one-way ANOVA (n = 4). (f) Representative images and quantification of ciliated cells (FOXJ1) after 8 days or 21 days ALI and with or without the presence of DAPT during differentiation (mean ± SEM). *p < 0.05, ***p < 0.001 by two-way ANOVA (n = 6). Scale bar, 30 µm.

Figure 3

IL-13 exposure during MTEC differentiation induces goblet cells. (a) Hematoxylin and eosin on tracheal sections of control or house dust mite (HDM) treated mice. Immunofluorescence staining with Mucin 5AC (MUC5AC) shows the presence of goblet cells. MUC5AC expressing cells are indicated by the white arrows. Scale bar, 200 µm and 30 µm. (b) A schematic representation of the culture protocol. (c) Immunofluorescence staining of MUC5AC of MTEC passage 0 and 2 after 8 days of ALI culture with or without IL-13. Scale bar, 30 µM. Graph shows the percentage of MUC5AC expressing goblet cells in culture (mean ± SEM). *p < 0.05 by Student two-tailed t-test (n = 3). (d) Resistance measurements after 8 days of ALI culture with or without IL-13 (5 ng/ml) treatment (mean ± SEM). *p < 0.05 by Student two-tailed t-test (n = 3). Scale bar: 30 µm. (e) Immunofluorescence staining of FOXJ1 positive ciliated cells in a passage 0 (P0) and 2 (P2) after 8 days of ALI culture with or without IL-13 (5 ng/ml). Scale bar, 30 µm. Graph show the percentage of FOXJ1 expressing ciliated cells in culture (mean ± SEM). *p < 0.05 by Student two-tailed t-test (n = 3).

Figure 4

The population of TRP63 positive basal cells change during expansion. (a) Schematic representation of culture protocol. (b) Representative images of the number of TRP63 positive basal cells at ALI day 0. Scale bar, 30 µM. Graph shows the percentage of TRP63 positive basal cells at day 0 of ALI culture in a passage 0 (P0), passage 1 (P1) and passage 2 (P2) (mean ± SEM). One-way ANOVA (n = 3). (c) Immunofluorescence of basal cell marker KRT5, and luminal cell maker KRT8 at day 0 of ALI. The overlay picture and the one-channel pictures are shown. The boxes indicate the area of which an enlarged image is presented. Scale bar: 30 µm. (d) Western blot analysis of KRT5 at day 0 of ALI in a passage (P) 0 and 2. Beta-actin (ACTB) is used as loading control. *p < 0.05 by Student two-tailed t-test (n = 7). (e) Immunofluorescence of basal cell marker, P75-nerve growth factor (NGFR) and basal cell maker keratin 14 (KRT14) at day 0 of ALI. Scale bar: 30 µm. (f) Western blot analysis of KRT14 at day 0 of ALI in a passage (P) 0 and 2. Beta-actin (ACTB) is used as loading control. *p < 0.05 by Student two-tailed t-test (n = 9).