Generation of multiciliated cells in functional airway epithelia from human induced pluripotent stem cells

Abstract

Despite therapeutic advancement, pulmonary disease still remains a major cause of morbidity and mortality around the world. Opportunities to study human lung disease either in vivo or in vitro are currently limited. Using induced pluripotent stem cells (iPSCs), we generated mature multiciliated cells in a functional airway epithelium. Robust multiciliogenesis occurred when notch signaling was inhibited and was confirmed by (i) the assembly of multiple pericentrin-stained centrioles at the apical surface, (ii) expression of transcription factor forkhead box protein J1, and (iii) presence of multiple acetylated tubulin-labeled cilia projections in individual cells. Clara, goblet, and basal cells were all present, confirming the generation of a complete polarized epithelial-cell layer. Additionally, cAMP-activated and cystic fibrosis transmembrane regulator inhibitor 172-sensitive cystic fibrosis transmembrane regulator currents were recorded in isolated epithelial cells. Our report demonstrating the generation of mature multiciliated cells in respiratory epithelium from iPSCs is a significant advance toward modeling a number of human respiratory diseases in vitro.

Keywords: bronchi; definitive endoderm; differentiation.

Fig. 1.

Differentiation to definitive endoderm. (A) Summary of the differentiation protocol indicating the days of media changes (media are in SI Appendix, Table SII). (B) Up-regulation of FOXA2, GATA6, and SOX17 mRNA at day 4 of differentiation; data are normalized to iPSC. (C) Colocalization of SOX17 and FOXA2 at day 4 and day 11 of differentiation. (Scale bar: 100 μm.) (D) mRNA expression of DE markers over the time course of differentiation; data are normalized to whole adult lung cDNA. Red and blue bars represent two independent iPSC lines. (E) Quantification of the % of cells also expressing FOXA2 or SOX17 (mean ± SEM). n = 6 independent experiments. ***P < 0.01, ****P < 0.001.

Fig. 2.

Anterior foregut endoderm specification. Representative immunofluorescent images of day 9 of differentiation indicating the colocalization of FOXA2, NKx2.1, and EpCAM (A) or PAX8 (B). (C) The same time point stained with CK18, NKx2.1, and SOX2. (D) The % of the DAPI cells stained with SOX2, NKx2.1, or both and (E) the % of the DAPI cells stained with FOXA2 or FOXA2 and NKx2.1 at day 9 of differentiation (mean ± SEM). n = 7 independent experiments. (Scale bar: 100 μm.)

Fig. 3.

Generation of pulmonary epithelium. (A) Differentiated normal human bronchial epithelial (NHBE) cells stained with ZO-1 (red). iPSCs at day 28 of differentiation stained with ZO-1 (red), epithelial cadherin (ECAD, red), and epithelial calmodulin (EpCAM, green). Images shown at 63x as labeled. (Scale bar: 50 μm.) Images shown at 20x as labeled. (Scale bar: 100 μm.) (B) H&E-stained histological samples showing a cuboidal layer of epithelial cells on the apical surface. (C) Staining of the apical cell layer with CK18 and ECAD (red). (Scale bar: 50 μm.) Staining of the apical cell layer with EpCAM (green). (Scale bar: 100 μm.) (D) Images showing the mesenchymal layer stained by CD90 and vimentin (red) and the epithelial layer stained with EpCAM (green). (Scale bars: 100 μm.) Nuclei are counterstained with DAPI (blue) in all images.

Fig. 4.

Generation of MCCs with Inhibition of notch signaling. (A) Representative images showing colocalization of FOXJ1 and DAPI at days 16 and 20 of differentiation. (Scale bar: 100 μm.) (B) qPCR for FOXJ1 expression during differentiation. Data are shown for two primer sets (gray/black) and are corrected for internal control GAPDH and normalized to whole-lung cDNA. Data are expressed as mean ± SEM and represent n ≥ 2 repeats per cell line. (C) Expression of transcription factor FOXJ1 (cyan); nuclei are counterstained with DAPI (blue) and cells indicated with ZO-1 (red). (Scale bar: 50 μm.) Representative images of cilia in the absence (Control) (D) and in the presence (DAPT-treated) (E) of notch inhibition. (Scale bar: 50 μm.) Cilia are indicated by acetylated alpha tubulin (cyan) and centrosome assembly by pericentrin (red), and nuclei are counterstained with DAPI (blue). D, Right indicates the three planes of view showing primary cilium at the apical surface and E, Right shows a 2.5× digital zoom image of multiciliated cells with dense centrioles marked with pericentrin. (F) The apical localization of both the cilia and the pericentrin (yellow arrow).

Fig. 5.

Generation of secretory cells. (A) Representative images of iPSC-derived CC10-expressing cells (green), ZO-1 (red), and nuclei (blue). (B) Images at day 45 indicating the colocalization of CC10 (green) with NKx2.1 (red). (C) Representative images showing the generation and frequency of MUC5A/C (cyan) stained cells (indicated by the yellow arrows). (D) Representative images of the apical epithelial layer stained with surfactant protein D (SPD) (green), ECAD (red), and DAPI (blue). (E) qPCR expression of SPD over the differentiation time course normalized to whole adult lung cDNA. Red and blue bars represent two independent iPSC lines. (Scale bars: A–D, 100 μm; E, 50 μm.)

Fig. 6.

Functional CFTR expression at the apical surface. (A) CFTR (red) expression at the apical surface; nuclei are counterstained with DAPI (blue). (Scale bar: 50 μm.) (B) Apical expression of ENaC (green). (Scale bar: 50 μm.) (C) Representative whole-cell chloride currents recorded from −80 to +80 mV in 20-mV increments at baseline and in the presence of forskolin and forskolin plus CFTRinh-172. (Right) Traces represent the CFTRinh-172–sensitive current. (D) I/V curves for baseline (squares) and in the presence of forskolin (triangles). *P < 0.05, **P < 0.01 (paired t test). (E) Phase images indicating the morphology of the expanding cells at P2 used for the patch-clamp experiments (20x and magnified 5x). (F) mRNA expression of CFTR over a time course of differentiation; data are corrected for GAPDH and normalized to whole-lung cDNA (n = 6).