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

Abstract

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 to investigate the effect of specific culture conditions on the differentiation of hESC into lung epithelial cells.

Methods: Undifferentiated hESC, grown on a porous membrane in hESC medium for four days, were switched to a differentiation medium for four days; this was followed by culture in air-liquid interface conditions during another 20 days. Expression of several lung markers was measured by immunohistochemistry and by quantitative real-time RT-PCR at four different time points throughout the differentiation and compared to appropriate controls.

Results: Expression of CC16 and NKX2.1 showed a 1,000- and 10,000- 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.

Conclusion: These results suggest that by using our novel culture protocol hESC can be differentiated into the major cell types of lung epithelial tissue.

Figure 1

Relative quantification of CC16 and NKX2.1 mRNA levels in four different hESC lines at day 5, 10, 15 and 20 of ALI culture. Relative quantification of CC16 (A) and NKX2.1 (B) mRNA levels in samples from human embryonic stem cells (hESC) cultured on porous membranes according to the ALI differentiation protocol. Samples were collected on days 5, 10, 15, and 20 of ALI culture. A peak expression is seen at day 10, with a 1000 and 10000- fold expression, followed by a decrease further over time. The NO ALI control samples were considered as reference. (Note that the y-axis is on a logarithmic scale). Two endogenous controls were used (GAPDH and Ubiquitin C). These experiments were repeated at least three times with VUB03_DM1, VUB04_CF, VUB07 and VUB14 hESC lines and data are presented here as means.

Figure 2

Quantitative RT-PCR analysis of Aquaporin 5, FOXJ1, β-tubulin IV and SP-C mRNA levels at day 20. Aquaporin 5, FOXJ1, β-tubulin IV and SP-C mRNA levels were measured in samples from hESC cultured on porous membranes with the ALI differentiation protocol and in NO ALI control samples. After 20 days of ALI culture Aquaporin 5, FOXJ1 and β-tubulin IV are all significantly upregulated (p < 0.05) as is SP-C compared to NO ALI control samples. Note that the y-axis is on a logarithmic scale. The samples from hESC spontaneous differentiation control samples were considered as a reference and data represent the mean ± SD. Experiments were performed at least three times and at least two different samples were taken for each measurement to provide biological replicates. Data are shown for VUB07. These experiments were also carried out with VUB03_DM1, VUB04_CF and VUB14 and showed similar results (data not shown). Two endogenous controls were used (GAPDH and Ubiquitin C).

Figure 3

Relative quantification of NANOG and POU5F1 mRNA levels. Relative quantification of NANOG and POU5F1 mRNA levels in samples from human embryonic stem cells (hESC) cultured on porous membranes with the ALI differentiation protocol (ALI), compared to the NO ALI control samples. Samples were collected on days 5, 10, 15, and 20, and data are represented as means ± SD. NANOG and POU5F1 mRNA levels decreased over time in both conditions and no statistical difference could be observed. Two endogenous controls were used (GAPDH and Ubiquitin C). Results of VUB04_CF are shown.

Figure 4

Quantitative RT-PCR analysis of Vimentin. Relative mRNA quantification of Vimentin in samples from human embryonic stem cells (hESC) cultured on porous membranes with the ALI differentiation protocol compared to the NO ALI control samples. Samples were collected on days 5, 10, 15, and 20, and data are represented as means ± SD. In both conditions an increase in expression levels could be observed. Control cultures showed a slighter increase in expression level, though no statistical difference could be measured. Results of VUB04_CF are shown.

Figure 5

Immunostaining of CC16. Representative image of CC16 immunostaining (green) in combination with nuclear DAPI staining (blue) in undifferentiated hESC control samples (A), in samples cultured with the ALI differentiation protocol (VUB03_DM1)(B) and in fetal lung (20 weeks old)(C). Staining was found in the cytoplasm of the cells lining the bronchiolar epithelium (C) and in the cells lining the cavities (B) of the tissue formed by ALI culture. No staining was seen in undifferentiated hESC controls (A). Original magnification 200× (A) and 400× (B-C) These experiments were also carried out with VUB04_CF and with VUB09_FSHD hESC lines and showed similar results (data not shown).

Figure 6

Immunostaining of SP-C. hESC were grown with the ALI differentiation protocol for 20 days (B-D). Images of two different cell lines (VUB03_DM1 (B-C) and VUB04_CF (D) were scanned by confocal microscopy after immunostaining with SP-C antibody (green) and nuclear DAPI staining (blue). Figure 6A shows immunostaining of SP-C in fetal lung (20 weeks old). Original magnification 200× (A,B) and 600× (C-D).

Figure 7

Immunostaining of SP-A. Fluorescence microscopic pictures of sections immunostained with SP-A antibody (green) and nuclear DAPI staining (blue). For fetal lung tissue (22 weeks old) (magnification 200×) (A) and hESC cultured with the ALI differentiation protocol at day 20 (VUB03_DM1 (B (magnification 400×) Pictures of sections in a perpendicular direction are shown on three different cell lines: VUB07 (C), VUB03_DM1 (D) and VUB04_CF (E).

Figure 8

Immunofluorescence double staining for SP-A and SP-C. hESC (VUB03_DM1) were cultured with the ALI differentiation protocol for 20 days. Original magnification 600×. Nuclear DAPI staining in blue. Staining for SP-A (red) and SP-C (green) were combined. SP-A was found on one side of the cells, while SP-C can be seen in the cytoplasm. Similar findings were obtained for VUB07 (not shown).

Figure 9

Immunofluorescence staining for three stem cell markers: TRA1-60 (A,D), TRA1-81 (B,E) and SSEA4 (C,F). 4 μm thick sections of hESC grown with the ALI differentiation protocol for 20 days (D-F) were compared to sections of undifferentiated hESC control samples (A-C). Undifferentiated hESC showed extensive staining for all three markers. After 20 days of differentiation in ALI conditions only sporadic areas of staining could be observed. Representative results of VUB07 are shown. Original magnification: 200× (A, D-F) and 400× (B-C).

Figure 10

Light microscopy images of Vimentin staining. hESC (VUB03_DM1) were cultured with the ALI differentiation protocol for 20 days (B) and compared with staining in fetal lung tissue (A). Original magnification 100× (A), 200× (B). Staining for Vimentin was found in most cells except the epithelial-like cells. In fetal lung tissue (22 weeks old) staining can be seen in most cells except for bronchiolar epithelial cells.

Figure 11

Secreted CC16 protein was measured by ELISA. CC16 secretion of three different hESC lines was compared. Secretion could be detected starting from two to four days after the induction of the ALI conditions. A similar secretion pattern over time could be measured in all three lines, with a peak in secretion level that is measured around day 8 followed by a decrease and stabilization further over time. (Figure 11A) CC16 secretion in samples from human embryonic stem cells (hESC) VUB07 cultured on porous membranes with the ALI differentiation protocol were compared to the NO ALI control samples and the "undifferentiated hESC control" (Figure 11B). All samples were measured in triplicate. No protein secretion could be detected in "No ALI control samples" till 20 days of culture. In the "spontaneous differentiation control" no secretion could be measured at any time. The minimum detectable concentration was <50 pg/mL.