Shubha Murthy, Denise A Seabold, Lalit K Gautam, Adrian M Caceres, Rosemary Sease, Ben A Calvert, Shana M Busch, Aaron Neely, Crystal N Marconett, Amy L Ryan
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To investigate how culture conditions influence BC phenotype and function, we expanded three independent BC isolates in three media: airway epithelial cell growth medium (AECGM), dual-SMAD inhibitor (DSI)-enriched AECGM, and PneumaCult Ex plus (PEx+). Analysis through RNA sequencing, immune assays, and impedance measurements revealed that PEx+ media significantly drove cell proliferation and a broad proinflammatory phenotype in BCs. In contrast, BCs expanded in AECGM and displayed increased expression of structural and extracellular matrix components at higher passage. AECGM increased expression of some cytokines at high passage, whereas DSI suppressed inflammation implicating the involvement TGF-β in BC inflammatory processes. Differentiation capacity of BCs declined with time in culture irrespective of expansion media. This was associated with an increase in PLUNC expressing secretory cells in AECGM and PEx+ media consistent with the known immune modulatory role of PLUNC in the airways. These findings highlight the profound impact of media conditions on inflammatory niche established by, and function of, in vitro expanded BCs. The broad proinflammatory phenotype driven by PEx+ media, in particular, should be considered in the development of cell-based models for airway diseases and therapeutic applications.<b>NEW & NOTEWORTHY</b> Airway basal cells, vital for airway regeneration and potential therapies, show significant changes based on culture conditions. Our study reveals that media composition and culture duration greatly affect basal cell properties with profound changes in the proinflammatory phenotype and extracellular matrix deposition driven by changes in growth conditions. 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引用次数: 0
摘要
来源于人气道气管-支气管区的支气管上皮细胞(HBECs)为研究病理机制和评价治疗方法提供了一个有价值的体外模型。该细胞群由基底细胞(bc)混合组成,基底细胞是气道中主要的干细胞,具有自我更新和功能分化的能力。尽管它们具有再生医学的潜力,但在培养中,bc表现出显著的表型变异性。为了研究培养条件如何影响BC表型和功能,我们在三种培养基中扩增了三个独立的BC分离株:气道上皮细胞生长培养基(AECGM)、富含双smad抑制剂(DSI)的AECGM和Pneumacult Ex plus (PEx+)。通过RNA测序、免疫测定和阻抗测量分析显示,PEx+培养基显著促进了bc细胞增殖和广泛的促炎表型。相比之下,BCs在AECGM中扩增,高传代时结构和细胞外基质成分表达增加。AECGM在高传代时增加了一些细胞因子的表达,而DSI则抑制了TGF-β参与BC炎症过程的炎症。无论使用何种培养基,BCs的分化能力随培养时间的延长而下降。这与AECGM和PEx+培养基中PLUNC分泌细胞表达增加有关,这与已知的PLUNC在气道中的免疫调节作用一致。这些发现强调了培养基条件对体外扩增的bc建立的炎症生态位及其功能的深远影响。尤其是PEx+介质驱动的广泛促炎表型,在气道疾病的细胞模型开发和治疗应用中应予以考虑。
Culture conditions differentially regulate the inflammatory niche and cellular phenotype of tracheobronchial basal stem cells.
Bronchial epithelial cells derived from the tracheobronchial regions of human airways (HBECs) provide a valuable in vitro model for studying pathological mechanisms and evaluating therapeutics. This cell population comprises a mixed population of basal cells (BCs), the predominant stem cell in airways capable of both self-renewal and functional differentiation. Despite their potential for regenerative medicine, BCs exhibit significant phenotypic variability in culture. To investigate how culture conditions influence BC phenotype and function, we expanded three independent BC isolates in three media: airway epithelial cell growth medium (AECGM), dual-SMAD inhibitor (DSI)-enriched AECGM, and PneumaCult Ex plus (PEx+). Analysis through RNA sequencing, immune assays, and impedance measurements revealed that PEx+ media significantly drove cell proliferation and a broad proinflammatory phenotype in BCs. In contrast, BCs expanded in AECGM and displayed increased expression of structural and extracellular matrix components at higher passage. AECGM increased expression of some cytokines at high passage, whereas DSI suppressed inflammation implicating the involvement TGF-β in BC inflammatory processes. Differentiation capacity of BCs declined with time in culture irrespective of expansion media. This was associated with an increase in PLUNC expressing secretory cells in AECGM and PEx+ media consistent with the known immune modulatory role of PLUNC in the airways. These findings highlight the profound impact of media conditions on inflammatory niche established by, and function of, in vitro expanded BCs. The broad proinflammatory phenotype driven by PEx+ media, in particular, should be considered in the development of cell-based models for airway diseases and therapeutic applications.NEW & NOTEWORTHY Airway basal cells, vital for airway regeneration and potential therapies, show significant changes based on culture conditions. Our study reveals that media composition and culture duration greatly affect basal cell properties with profound changes in the proinflammatory phenotype and extracellular matrix deposition driven by changes in growth conditions. These results underscore the critical impact of culture conditions on BC phenotype, influencing cell-based models for airway disease research and therapy.
期刊介绍:
The American Journal of Physiology-Lung Cellular and Molecular Physiology publishes original research covering the broad scope of molecular, cellular, and integrative aspects of normal and abnormal function of cells and components of the respiratory system. Areas of interest include conducting airways, pulmonary circulation, lung endothelial and epithelial cells, the pleura, neuroendocrine and immunologic cells in the lung, neural cells involved in control of breathing, and cells of the diaphragm and thoracic muscles. The processes to be covered in the Journal include gas-exchange, metabolic control at the cellular level, intracellular signaling, gene expression, genomics, macromolecules and their turnover, cell-cell and cell-matrix interactions, cell motility, secretory mechanisms, membrane function, surfactant, matrix components, mucus and lining materials, lung defenses, macrophage function, transport of salt, water and protein, development and differentiation of the respiratory system, and response to the environment.