Reynolds Susan D, Hill Cynthia L, Alsudayri Alfahdah, Stack Jacob T, Shontz Kimberly M, Carraro Gianni, Stripp Barry R, Chiang Tendy
Center for Perinatal Research, Nationwide Children's Hospital, Columbus, OH 43215, United States.
Department of Pediatrics, The Ohio State University College of Medicine, Columbus, OH 43210, United States.
Stem Cells Transl Med. 2025 Jan 17;14(1). doi: 10.1093/stcltm/szae084.
Cystic fibrosis transmembrane conductance regulator (CFTR) gene editing and transplantation of CFTR-gene corrected airway basal cells has the potential to cure CF lung disease. Although mouse studies established that cell transplantation was feasible, the engraftment rate was typically low and frequently less than the estimated therapeutic threshold. The purpose of this study was to identify genes and culture conditions that regulate the therapeutic potential of human bronchial basal cells. Factor 3 (F3, Tissue Factor 1) is a component of the extrinsic coagulation pathway and activates a cascade of proteases that convert fibrinogen to fibrin. Based on reports that F3 was necessary for human basal cell survival and adhesion in vitro, the present study evaluated F3 as a potential determinant of therapeutic fitness. The gene expression profile of F3 mRNA-positive human bronchial basal cells was evaluated by scRNAseq and the impact of the lung environment on F3 expression was modeled by varying in vitro culture conditions. F3 necessity for adhesion, proliferation, and differentiation was determined by CRISPR/Cas9 knockout (KO) of the F3 gene. Finally, the impact of F3 manipulation on engraftment was determined by orthotropic co-transplantation of wild-type and F3-KO cells into the airways of immunocompromised mice. In contrast with the hypothesis that F3 increases the therapeutic fitness of basal cells, F3 expression decreased engraftment. These studies guide the ongoing development of cellular therapies by showing that in vitro assessments may not predict therapeutic potential and that the lung milieu influences the functional properties of transplanted bronchial basal cells.
囊性纤维化跨膜传导调节因子（CFTR）基因编辑以及移植经CFTR基因校正的气道基底细胞有治愈囊性纤维化肺部疾病的潜力。尽管小鼠研究证实细胞移植是可行的，但植入率通常较低，且常常低于估计的治疗阈值。本研究的目的是确定调节人支气管基底细胞治疗潜力的基因和培养条件。因子3（F3，组织因子1）是外源性凝血途径的一个组成部分，可激活一系列将纤维蛋白原转化为纤维蛋白的蛋白酶。基于F3对人基底细胞体外存活和黏附所必需的报道，本研究评估F3作为治疗适应性的一个潜在决定因素。通过单细胞RNA测序（scRNAseq）评估F3 mRNA阳性人支气管基底细胞的基因表达谱，并通过改变体外培养条件模拟肺环境对F3表达的影响。通过F3基因的CRISPR/Cas9敲除（KO）确定F3对黏附、增殖和分化的必要性。最后，通过将野生型和F3-KO细胞原位共移植到免疫受损小鼠的气道中，确定F3操作对植入的影响。与F3增加基底细胞治疗适应性的假设相反，F3表达降低了植入率。这些研究表明体外评估可能无法预测治疗潜力，且肺环境会影响移植的支气管基底细胞的功能特性，从而为细胞疗法的持续发展提供了指导。
