Mechanical stretch promotes apoptosis and impedes ciliogenesis of primary human airway basal stem cells.

BACKGROUND: Airway basal stem cells (ABSCs) have self-renewal and differentiation abilities. Although an abnormal mechanical environment related to chronic airway disease (CAD) can cause ABSC dysfunction, it remains unclear how mechanical stretch regulates the behavior and structure of ABSCs. Here, we explored the effect of mechanical stretch on primary human ABSCs. METHODS: Primary human ABSCs were isolated from healthy volunteers. A Flexcell FX-5000 Tension system was used to mimic the pathological airway mechanical stretch conditions of patients with CAD. ABSCs were stretched for 12, 24, or 48 h with 20% elongation. We first performed bulk RNA sequencing to identify the most predominantly changed genes and pathways. Next, apoptosis of stretched ABSCs was detected with Annexin V-FITC/PI staining and a caspase 3 activity assay. Proliferation of stretched ABSCs was assessed by measuring MKI67 mRNA expression and cell cycle dynamics. Immunofluorescence and hematoxylin-eosin staining were used to demonstrate the differentiation state of ABSCs at the air-liquid interface. RESULTS: Compared with unstretched control cells, apoptosis and caspase 3 activation of ABSCs stretched for 48 h were significantly increased (p < 0.0001; p < 0.0001, respectively), and MKI67 mRNA levels were decreased (p < 0.0001). In addition, a significant increase in the G0/G1 population (20.2%, p < 0.001) and a significant decrease in S-phase cells (21.1%, p < 0.0001) were observed. The ratio of Krt5+ ABSCs was significantly higher (32.38% vs. 48.71%, p = 0.0037) following stretching, while the ratio of Ac-tub+ cells was significantly lower (37.64% vs. 21.29%, p < 0.001). Moreover, compared with the control, the expression of NKX2-1 was upregulated significantly after stretching (14.06% vs. 39.51%, p < 0.0001). RNA sequencing showed 285 differentially expressed genes, among which 140 were upregulated and 145 were downregulated, revealing that DDIAS, BIRC5, TGFBI, and NKX2-1 may be involved in the function of primary human ABSCs during mechanical stretch. There was no apparent difference between stretching ABSCs for 24 and 48 h compared with the control. CONCLUSIONS: Pathological stretching induces apoptosis of ABSCs, inhibits their proliferation, and disrupts cilia cell differentiation. These features may be related to abnormal regeneration and repair observed after airway epithelium injury in patients with CAD.

Autologous Airway Basal Cell Transplantation Alleviates Airway Epithelium Defect in Recurrent Benign Tracheal Stenosis.

BACKGROUND: Airway epithelium defects are a hallmark of recurrent benign tracheal stenosis (RBTS). Reconstructing an intact airway epithelium is of great importance in airway homeostasis and epithelial wound healing and has great potential for treating tracheal stenosis. METHODS: An experimental study was conducted in canines to explore the therapeutic effect of autologous basal cell transplantation in restoring airway homeostasis. First, airway mucosae from human patients with recurrent tracheal stenosis were analyzed by single-cell RNA sequencing. Canines were then randomly divided into tracheal stenosis, Stent, Stent + Cells, and Stent + Cells + Biogel groups. Autologous airway basal cells of canines in the Stent + Cells and Stent + Cells + Biogel groups were transplanted onto the stenotic airway after modeling. A biogel was coated on the airway prior to basal cell transplantation in the Stent + Cells + Biogel group. After bronchoscopic treatments, canines were followed up for 16 weeks. RESULTS: Single-cell RNA sequencing demonstrated packed airway basal cells and an absence of normal airway epithelial cells in patients with RBTS. Autologous airway basal cell transplantation, together with biogel coating, was successfully performed in the canine model. Follow-up observation indicated that survival time in the Stent + Cells + Biogel group was significantly prolonged, with a higher (100%) survival rate compared with the other groups. In terms of pathological and bronchoscopic findings, canines that received autologous basal cell transplantation showed a reduction in granulation hyperplasia as well as airway re-epithelialization with functionally mature epithelial cells. CONCLUSIONS: Autologous airway basal cell transplantation might serve as a novel regenerative therapy for airway re-epithelialization and inhibit recurrent granulation hyperplasia in benign tracheal stenosis.