分化的条件重编程（CRC）-和无饲养层双 SMAD 抑制扩增的人鼻腔上皮细胞存在显著的功能差异。
Significant functional differences in differentiated Conditionally Reprogrammed (CRC)- and Feeder-free Dual SMAD inhibited-expanded human nasal epithelial cells.
机构信息
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.
Biomedical Imaging Facility, University of New South Wales, Sydney, NSW, Australia.
出版信息
J Cyst Fibros. 2021 Mar;20(2):364-371. doi: 10.1016/j.jcf.2020.12.019. Epub 2021 Jan 5.
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 and SMADi 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 compare to their CRC 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.
背景
在气-液界面（ALI）分化的患者来源气道细胞是囊性纤维化（CF）精准治疗的有价值模型。已经建立了不同的培养扩增方法来扩展气道基底细胞的扩增能力，同时保留功能性气道上皮生理学。即使在相同的 CFTR 基因型内，并且尽管使用了类似的 ALI 培养技术，在 CFTR 调节剂的反应中也观察到了相当大的差异。我们旨在解决培养扩增方法对分化的影响。
方法
从 14 个人（CF=9；非 CF=5）的鼻腔上皮刷中采集样本，然后等分并在条件重编程培养（CRC）和无饲养血清的“双重 SMAD 抑制”（SMADi）方法下进行扩增。来自每种培养物的扩增细胞用专有的 PneumaCult™-ALI 培养基进行分化。在 CRC 和 SMADi 下，在基础条件和 CFTR 校正器处理（VX-809）条件下比较形态（免疫荧光）、全局蛋白质组学（LC-MS/MS）和功能（屏障完整性、纤毛运动和离子转运）。
结果
未观察到结构形态或基线全局蛋白质组学谱的显著差异。尽管细胞连接形态或纤毛丰度没有差异，但屏障完整性和纤毛运动明显不同。上皮钠通道和钙激活氯离子通道活性没有差异，但与 CRC 相比，SMADi 中 CFTR 介导的氯离子电流显著降低。
结论
体外细胞生理功能的改变比气道 ALI 的结构和分化潜力更为明显。由于初始扩增培养条件会显著影响 CFTR 活性，如果不针对每个实验室的数据进行具体参考范围的比较，而将来自不同实验室的数据相互比较，可能会得出错误的结论。
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