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

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.

Rhinovirus Infection Is Associated With Airway Epithelial Cell Necrosis and Inflammation via Interleukin-1 in Young Children With Cystic Fibrosis

“…Samples were attained by brushing of the tracheal mucosa of children with a singlesheathed nylon bronchial cytology brush as previously described (8,13). After collection, primary AEC cultures were established as previously described (14).…”

Aberrant cell migration contributes to defective airway epithelial repair in childhood wheeze

“…The reported observations are unlikely to be as a result of therapy, since none were receiving inhaled corticosteroids or bronchodilators for at least 1 month prior to recruitment into the study. In addition, we utilized standard culture and conditional reprogramming methods that conserve the wound repair phenotypes of pAEC (48). Proteins of interest that were DE in cells traditionally cultured in monolayers were similarly expressed in the conditionally reprogrammedderived cells.…”

“…This study used conditional reprogramming (48,53) and traditional culturing (11,52) methods to establish ex vivo human pAECs from children. The remainder of ex vivo cells were used for RNA extraction and gene expression profiling as previously described (11).…”

Progress in Model Systems of Cystic Fibrosis Mucosal Inflammation to Understand Aberrant Neutrophil Activity

“…Yields from brushings are variable and ex vivo pAEC have limited proliferative capacity; they become senescent after only a few passages making them difficult to expand in culture (126,127). The adaptation of conditionally reprogrammed airway epithelial cells (CRAEC) through coculture with irradiated fibroblast feeder cells has significantly increased passage number capacity of pAEC, while maintaining lineage specific characteristics (128). Additionally, CRAEC can be seeded from co-culture into air-liquid interface culture (ALI) to form a differentiated pseudostratified epithelial layer (128,129).…”

“…The adaptation of conditionally reprogrammed airway epithelial cells (CRAEC) through coculture with irradiated fibroblast feeder cells has significantly increased passage number capacity of pAEC, while maintaining lineage specific characteristics (128). Additionally, CRAEC can be seeded from co-culture into air-liquid interface culture (ALI) to form a differentiated pseudostratified epithelial layer (128,129). This has enabled many CF research groups to look to CF primary airway epithelial cell models in order to understand the cellular drivers of progressive lung disease, and more recently to evaluate the efficacy of CFTR modulators in restoring CFTR function in target cells (130,131).…”