Human induced pluripotent stem cells for in vitro modeling of impaired mucociliary clearance in cystic fibrosis lung disease

Abstract

Severely impaired mucociliary airway function is the primary pathomechanism in Cystic Fibrosis (CF) lung disease. Despite significant advances in CF therapy, there is still a critical need for alternative, individualized treatment options, especially for patients with untreatable CFTR mutations. Although intestinal organoids and primary airway cells are widely used as preclinical models of CF, both systems exhibit limitations with regard to the proper modelling of mucociliary clearance or the availability of sufficient cell quantities. Patient-specific human induced pluripotent stem cells (hiPSCs) are a promising alternative due to their unlimited expansion potential and capacity to differentiate into airway epithelia. However, cellular inhomogeneities in iPSC-derived airway cultures complicated conventional assays that determine CFTR function such as Ussing chamber measurements, and a comprehensive demonstration of CF pathophysiology in hiPSC-derived airway models has been largely lacking. This study provides comprehensive data demonstrating very similar gene expression, (ultra)structure and CFTR function in CF iPSC-derived airway (iALI) and primary airway (pALI) cultures. Addressing current limitations, we have implemented a sensitive, straightforward, and automatable ciliary beat frequency (CBF) assay, which is largely unaffected by inhomogeneities and directly reflects disturbed mucus viscosity and mucociliary transport in CF lung disease. Electron microscopy images confirmed the disease phenotype showing a highly dense and dehydrated mucus layer on top of CF iALI cultures. Furthermore, established CFTR modulator drugs partially rescued the disease phenotype in CF iALI cultures, which validated the utility of iALI cultures as a scalable, patient-specific platform for CF research and personalized drug development.

1. Introduction

Cystic fibrosis (CF) is a rare recessive genetic disorder that affects approximately 100,000 people worldwide. It is caused by mutations of the Cystic fibrosis transmembrane conductance regulator (CFTR) gene [1–4], which encodes a cAMP-regulated chloride and bicarbonate channel protein. Mutations in the CFTR gene have been shown to affect the transepithelial ion transport in multiple organs, with CF lung disease being the primary cause of morbidity and mortality [5–14]. To date, over 700 CF-causing mutations of the CFTR gene have been identified [15–17].

In healthy individuals, the respiratory epithelium plays a critical role in the host defense against pulmonary infection. In the airways, the secreting cell types, particularly goblet cells, produce a protective layer of mucus that covers the epithelial surface and traps inhaled pathogens. Subsequent to this initial defensive barrier, mucociliary clearance (MCC) is initiated through ciliary beating, which effectively removes mucus and pathogens from the respiratory system [18].

In CF, CFTR mutations result in reduced chloride secretion and concurrent hyperabsorption of sodium by the CFTR-regulated epithelial sodium channel (ENaC). The reduction in apical ion secretion causes the dehydration of airway mucus and the subsequent increase in mucus viscosity [19–24]. This increased viscosity impairs ciliary movement and MCC, consequently leading to chronic airway infection, inflammation, and ultimately progressive loss of lung function and lung failure [11, 25–28].