Differentiation of CD166-positive hPSC-derived lung progenitors into airway epithelial cells

Abstract

The generation of lung epithelial cells through the directed differentiation of human pluripotent stem cells (hPSCs) in vitro provides a platform to model both embryonic lung development and adult airway disease. Here, we describe a robust differentiation protocol that closely recapitulates human embryonic lung development. Differentiating cells progress through obligate intermediate stages, beginning with definitive endoderm formation and then patterning into anterior foregut endoderm that yields lung progenitors (LPs) with extended culture. These LPs can be purified using the cell surface marker CD166 (also known as ALCAM), and further matured into proximal airway epithelial cells including basal cells, secretory cells and multiciliated cells using either an organoid platform or culture at the air-liquid interface (ALI). We additionally demonstrate that these hPSC-derived airway epithelial cells can be used to model Influenza A infection. Collectively, our results underscore the utility of CD166 expression for the efficient enrichment of LPs from heterogenous differentiation cultures and the ability of these isolated cells to mature into more specialized, physiologically relevant proximal lung cell types.

Keywords: Air-liquid interface; CD166; Directed differentiation; Human pluripotent stem cells; Influenza; Multiciliated cells; Organoids; Proximal lung airway.

Fig. 1.

CD166+ cells can be differentiated as proximal lung organoids. (A) Schematic showing differentiation of hPSCs into lung progenitors and then into lung organoids. (B) Representative brightfield image of lung organoids on day 30. Scale bar: 200 µm. (C) qPCR analysis of proximal lung markers NKX2-1, TP63, MUC5AC, FOXJ1, CFTR, SYP and SCGB3A2. Data generated were from four independent experiments and are presented as means±s.d. *P<0.05. PSC: pluripotent stem cell; D (day), and AL (Adult Lung). (D) Representative immunostaining for basal cells (TP63, KRT5), multiciliated cells (FOXJ1, ACTUB) and secretory cells (MUC5AC). Scale bars: 100 µm. n=3.

Fig. 2.

CD166+ cells can be differentiated into airway epithelial cells on transwells. (A) Schematic depicting differentiation of LPs on transwells for 15 days. Further differentiation and maturation were induced by culturing cells at an air-liquid interface for a further 15 days. (B) qPCR analysis of proximal lung markers NKX2-1, TP63, MUC5AC, FOXJ1, CFTR, SYP and SCGB3A2. Data from at least three independent experiments presented as means±s.d. *P<0.05, **P<0.01, ***P<0.001. (C) Immunostaining of markers associated with basal cells (TP63, KRT5), multiciliated cells (AcTUB) and secretory cells (SPLUNC1). Representative images shown. Scale bars: 5 µm. n=3.

Fig. 3.

hPSC-derived airway epithelial cells are susceptible to influenza infection. (A) Virus plaque assay of influenza A virus samples [A/Puerto Rico/8/1934 (PR8), A/WSN/1933 (WSN), and A/Aichi/2/1968 (Aichi)] obtained from hPSC-derived airway epithelial cells. Cells were infected at a multiplicity of infection (MOI) of 0.1 or mock infected (Mock), with media containing trypsin-TPCK. Virus samples were collected 24 h post-infection (hpi). One-way ANOVA (repeated measures), multiple comparisons with Dunnett's correction, means±s.e.m., *P<0.05, technical replicates, n=4. (B) Representative western blot images showing nucleoprotein (NP), nonstructural protein 1 (NS1), matrix protein 1 (M1) and β-tubulin 24 hpi from influenza A virus infected hPSC-derived airway epithelial cells.