Conditionally reprogrammed primary airway epithelial cells maintain morphology, lineage and disease specific functional characteristics

Kelly M Martinovich,Alysia G Buckley,Elizabeth Kicic-Starcevich,Darryl A Knight,Luke W Garratt,Stephen M Stick,Samuel Montgomery,Anthony Kicic,Nicole C Shaw,Francis J Lannigan,Kevin Looi,Kak-Ming Ling,Thomas Iosifidis,Erika N Sutanto

doi:10.1038/s41598-017-17952-4

Abstract

Current limitations to primary cell expansion led us to test whether airway epithelial cells derived from healthy children and those with asthma and cystic fibrosis (CF), co-cultured with an irradiated fibroblast feeder cell in F-medium containing 10 µM ROCK inhibitor could maintain their lineage during expansion and whether this is influenced by underlying disease status. Here, we show that conditionally reprogrammed airway epithelial cells (CRAECs) can be established from both healthy and diseased phenotypes. CRAECs can be expanded, cryopreserved and maintain phenotypes over at least 5 passages. Population doublings of CRAEC cultures were significantly greater than standard cultures, but maintained their lineage characteristics. CRAECs from all phenotypes were also capable of fully differentiating at air-liquid interface (ALI) and maintained disease specific characteristics including; defective CFTR channel function cultures and the inability to repair wounds. Our findings indicate that CRAECs derived from children maintain lineage, phenotypic and importantly disease-specific functional characteristics over a specified passage range.

Highlights

The study of the respiratory epithelium is critical to many chronic lung diseases such as cystic fibrosis (CF) and asthma
Conditional reprogramming of primary airway epithelial cells (pAEC) cultures improved the population doubling times of both the healthy and disease phenotypes compared to standard cultures for healthy (CRAEC slope 0.56 ± 0.02, pAEC 0.08 ± 0.01; p = 0.0001; Fig. 2a), asthmatic (CRAEC slope 0.41 ± 0.03, pAEC 0.06 ± 0.02; p = 0.0048 Fig. 2b) and CF cultures (CRAEC slope 0.50 ± 0.01, pAEC 0.06 ± 0.02; p = 0.0001; Fig. 2c)
The study of the airway epithelium has provided important insight regarding the pathobiology of lung diseases such as asthma and CF

Summary

Introduction

The study of the respiratory epithelium is critical to many chronic lung diseases such as cystic fibrosis (CF) and asthma. Cultures should be successfully established from every brushing irrespective of disease phenotype, have greater proliferative capacity to enable expansion beyond current limits and allow for multiple downstream experiments using matched samples These cultures should maintain their epithelial lineage and phenotypic characteristics. Results generated in this study, show that co-culturing AECs with irradiated NIH-3T3 fibroblast cells and in the presence of a ROCK inhibitor significantly improves pAEC growth rates and extends the number of population doublings in vitro. This methodology significantly increases the total number of cells available for experimental purposes whilst retaining epithelial lineage characteristics and disease specific abnormal functionalities over a specified passage range

Methods

Results

Conclusion