适用于气道组织工程的人上皮干细胞的快速扩增
Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering.
作者信息
Butler Colin R, Hynds Robert E, Gowers Kate H C, Lee Dani Do Hyang, Brown James M, Crowley Claire, Teixeira Vitor H, Smith Claire M, Urbani Luca, Hamilton Nicholas J, Thakrar Ricky M, Booth Helen L, Birchall Martin A, De Coppi Paolo, Giangreco Adam, O'Callaghan Christopher, Janes Sam M
机构信息
1 Lungs for Living Research Centre, UCL Respiratory, University College London, London, United Kingdom.
2 Respiratory, Critical Care, and Anesthesia, Institute of Child Health, University College London, London, United Kingdom.
出版信息
Am J Respir Crit Care Med. 2016 Jul 15;194(2):156-68. doi: 10.1164/rccm.201507-1414OC.
RATIONALE
Stem cell-based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell-seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts.
OBJECTIVES
To define a scalable cell culture system to deliver airway epithelium to clinical grafts.
METHODS
Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air-liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air-liquid interface cultures.
MEASUREMENTS AND MAIN RESULTS
3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model.
CONCLUSIONS
Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical transplantation, suggesting its suitability for use in tracheal reconstruction.
原理
基于干细胞的气管置换术为患有包括长段先天性气管狭窄和上气道肿瘤等其他无法治疗的气道疾病的患者提供了一种新兴的治疗选择。临床经验表明，组织工程移植物移植后肺部黏液纤毛清除功能的恢复至关重要，临床前研究表明，用自体黏膜接种支架可改善再生。再生医学需要高上皮细胞接种密度，而现有技术不足以实现临床适用移植物的覆盖。
目的
定义一种可扩展的细胞培养系统，以将气道上皮细胞递送至临床移植物。
方法
使用经有丝分裂灭活的成纤维细胞和用Y-27632抑制Rho相关蛋白激酶（ROCK）（3T3+Y）的组合培养来自支气管活检的人呼吸道上皮细胞。通过免疫荧光、定量聚合酶链反应和流式细胞术分析细胞，以评估气道干细胞标志物的表达。进行核型分析和多重连接依赖探针扩增以评估细胞安全性。在三维气管球、器官型培养物、气液界面培养物和体内气管异种移植模型中测试分化能力。在气液界面培养物中评估纤毛功能。
测量和主要结果
3T3-J2饲养细胞和ROCK抑制可使气道基底细胞快速扩增。这些细胞能够在体外进行多能分化，产生纤毛细胞和杯状细胞谱系。纤毛功能正常，摆动频率和模式正常。在异位移植异种模型中，培养的细胞重新填充了气管支架。
结论
我们的方法以临床移植所需的效率产生大量功能性气道基底上皮细胞，表明其适用于气管重建。
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