Significant functional differences despite morphological and molecular similarity in fully differentiated matched Conditionally Reprogrammed (CRC) and Feeder free dual SMAD inhibited expanded human nasal epithelial cells

Background: Patient-derived airway cells differentiated at Air Liquid Interface (ALI) are valuable models for Cystic fibrosis (CF) precision therapy. Advances in culture techniques have improved expansion capacity of airway basal cells, while retaining functional airway epithelium physiology. However, considerable variation in response to CFTR modulators is observed even when using similar ALI culture techniques. We aimed to address if variation in response reflects true biological differences between patients or technical differences as a consequence of different culture expansion methods. Methods: Nasal epithelial brushings from 14 individuals (CF=9 non-CF=5) were collected, then equally divided and expanded under conditional reprogramming culture (CRC) and feeder-serum-free dual-SMAD inhibition (SMADi) methods. Expanded cells from each culture were differentiated with proprietary PneumaCult-ALI media. Morphology (Immunofluorescence), global proteomics (LC-MS/MS) and function (barrier integrity, cilia motility, and ion transport) were compared in CRC ALI and SMADi ALI under basal and CFTR corrector treated (VX-809) conditions. Results: No significant difference in the structural morphology or global proteomics profile were observed. Barrier integrity and cilia motility were significantly different, despite no difference in cell junction morphology or cilia abundance. Epithelial Sodium Channels and Calcium-activated Chloride Channel activity did not differ but CFTR mediated chloride currents were significantly reduced in SMADiALI compare to their CRCALI counterparts. Conclusion: Alteration of cellular physiological function in vitro occurs were more prominent than structural and differentiation potential in airway ALI. Since culture conditions significantly influence CFTR activity, this could lead to false conclusions if data from different labs are compared against each other without specific reference ranges. Keywords: In Vitro cell models, CFTR, Air liquid interface, Conditional reprogramming, dual SMAD inhibition, cystic fibrosis, companion diagnostic. Abbreviations: CF (Cystic Fibrosis), CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), ALI (air liquid interface), ATP (Adenosine tri phosphate), HBE (human bronchial epithelial cell), HNE (human nasal epithelial cell), CRC (conditionally reprogrammed cultures), ROCK (Rho-associated protein kinase), P (passage), TEER (Trans-epithelial electrical resistance), TGF-beta; (Transforming growth factor-beta), Isc (Short circuit current), CaCC (Calcium activated chloride channels), CBF (Ciliary beating frequency)