Significant functional differences in differentiated Conditionally Reprogrammed (CRC)- and Feeder-free Dual SMAD inhibited-expanded human nasal epithelial cells.
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
Air liquid interface
CFTR
Conditional reprogramming
In Vitro cell models
cystic fibrosis, companion diagnostic
dual SMAD inhibition
Journal
Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society
ISSN: 1873-5010
Titre abrégé: J Cyst Fibros
Pays: Netherlands
ID NLM: 101128966
Informations de publication
Date de publication:
03 2021
03 2021
Historique:
received:
23
04
2020
revised:
16
12
2020
accepted:
20
12
2020
pubmed:
9
1
2021
medline:
22
1
2022
entrez:
8
1
2021
Statut:
ppublish
Résumé
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. 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 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 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.
Sections du résumé
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
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
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.
Identifiants
pubmed: 33414087
pii: S1569-1993(20)30951-6
doi: 10.1016/j.jcf.2020.12.019
pii:
doi:
Substances chimiques
CFTR protein, human
0
Chloride Channels
0
Cystic Fibrosis Transmembrane Conductance Regulator
126880-72-6
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
364-371Informations de copyright
Copyright © 2021 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of Competing Interest The authors declare that they have no conflict of interest.