Significant functional differences in differentiated Conditionally Reprogrammed (CRC)- and Feeder-free Dual SMAD inhibited-expanded human nasal epithelial cells
| Autor: | Elvis Pandzic, Nihan Turgutoglu, Iveta Slapetova, Renee Whan, Sharon L. Wong, Ling Zhong, Shafagh A. Waters, Adam Jaffe, Nikhil T. Awatade, Laura K. Fawcett, Alexander Capraro |
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| Rok vydání: | 2021 |
| Předmět: |
Proteomics
0301 basic medicine Pulmonary and Respiratory Medicine Epithelial sodium channel Cystic Fibrosis Cell Culture Techniques Cystic Fibrosis Transmembrane Conductance Regulator Motility In Vitro Techniques Cystic fibrosis 03 medical and health sciences 0302 clinical medicine Chloride Channels Humans Medicine Cellular Reprogramming Techniques Cilia Cells Cultured Chloride channel activity biology business.industry Cilium Cell Differentiation Epithelial Cells Transforming growth factor beta respiratory system medicine.disease Cystic fibrosis transmembrane conductance regulator respiratory tract diseases Cell biology Nasal Mucosa 030104 developmental biology 030228 respiratory system Pediatrics Perinatology and Child Health biology.protein Respiratory epithelium business |
| Zdroj: | Journal of Cystic Fibrosis. 20:364-371 |
| ISSN: | 1569-1993 |
| DOI: | 10.1016/j.jcf.2020.12.019 |
| Popis: | 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 CRCALI and SMADiALI 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 SMADiALI compare to their CRCALI 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. |
| Databáze: | OpenAIRE |
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