Significant functional differences in differentiated Conditionally Reprogrammed (CRC)- and Feeder-free Dual SMAD inhibited-expanded human nasal epithelial cells

Nikhil T Awatade; Sharon L Wong; Alexander Capraro; Elvis Pandzic; Iveta Slapetova; Ling Zhong; Nihan Turgutoglu; Laura K Fawcett; Renee M Whan; Adam Jaffe; Shafagh A Waters

doi:10.1016/j.jcf.2020.12.019

Significant functional differences in differentiated Conditionally Reprogrammed (CRC)- and Feeder-free Dual SMAD inhibited-expanded human nasal epithelial cells

J Cyst Fibros. 2021 Mar;20(2):364-371. doi: 10.1016/j.jcf.2020.12.019. Epub 2021 Jan 5.

Authors

Affiliations

¹ School of Women's and Children's Health, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia; Molecular and Integrative Cystic Fibrosis Research Centre (miCF_RC), University of New South Wales and Sydney Children's Hospital, Sydney, NSW, Australia.
² Biomedical Imaging Facility, University of New South Wales, Sydney, NSW, Australia.
³ Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney, NSW, Australia.
⁴ School of Women's and Children's Health, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia; Molecular and Integrative Cystic Fibrosis Research Centre (miCF_RC), University of New South Wales and Sydney Children's Hospital, Sydney, NSW, Australia; Department of Respiratory Medicine, Sydney Children's Hospital, Sydney, NSW, Australia.
⁵ School of Women's and Children's Health, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia; Molecular and Integrative Cystic Fibrosis Research Centre (miCF_RC), University of New South Wales and Sydney Children's Hospital, Sydney, NSW, Australia; Department of Respiratory Medicine, Sydney Children's Hospital, Sydney, NSW, Australia. Electronic address: shafagh.waters@unsw.edu.au.

PMID: 33414087
DOI: 10.1016/j.jcf.2020.12.019

Abstract

Background: Patient-derived airway cells differentiated at Air Liquid Interface (ALI) are valuable models for Cystic fibrosis (CF) precision therapy. Different culture expansion methods have been established to extend expansion capacity of airway basal cells, while retaining functional airway epithelium physiology. Considerable variation in response to CFTR modulators is observed in cultures even within the same CFTR genotype and despite the use of similar ALI culture techniques. We aimed to address culture expansion method impact on differentiation.

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 baseline 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 SMADi^ALI compare to their CRC^ALI counterparts.

Conclusion: Alteration of cellular physiological function in vitro were more prominent than structural and differentiation potential in airway ALI. Since initial expansion 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: Air liquid interface; CFTR; Conditional reprogramming; In Vitro cell models; cystic fibrosis, companion diagnostic; dual SMAD inhibition.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Culture Techniques*
Cell Differentiation
Cells, Cultured
Cellular Reprogramming Techniques*
Chloride Channels / metabolism
Cilia / pathology
Cystic Fibrosis / pathology*
Cystic Fibrosis Transmembrane Conductance Regulator
Epithelial Cells / pathology*
Humans
In Vitro Techniques
Nasal Mucosa / cytology*
Proteomics

Substances

CFTR protein, human
Chloride Channels
Cystic Fibrosis Transmembrane Conductance Regulator