Understanding persistent airway obstruction in type 2 severe asthma using human induced pluripotent stem cells (iPSCs)

Introduction

Despite the new biologics to treat inflammation in severe asthma, targeting persistent obstruction of the airways remains challenging. Galectin-10 eosinophil derived crystals, also known as Charcot-Leyden crystals (CLCs) have been described to be present in the mucus plugs in the airways of patients with severe asthma. However, a direct role for CLCs in mucus production has not been established. We hypothesize that plugged airways constitute a unique niche where type 2 immune cells communicate with structural cells to perpetuate disease. We aimed to set up a new model using induced pluripotent stem cells (iPSCs).

Methods

Three human iPSCs lines from type 2 severe asthma patients have been derived (MOSAIC study, University Hospital of Montpellier, NCT05616338) and differentiated into airway epithelium in air–liquid interface (i-ALI). The healthy iPSC line UHOMi002-A was used as a control. At day 21 of ALI culture, iPSC derived-airway epithelia were stimulated at the apical side with either IL-13 every two days (10 ng/mL) during one week, acute stimulation (24 h) with recombinant Gal10 crystals (100 ng/mL), both IL-13 and Gal10 crystals or PBS (vehicle). We aimed to evaluate the effect on i-ALI differentiation at day 30.

Results

We successfully differentiated the iPSC lines generated from the T2 severe asthma patients, and achieving a high purity rate at each developmental stages. The mean cell purity at the definitive endoderm for each cell line was > 80% assessed by flow cytometry quantification of C-X-C Motif Chemokine Receptor 4 (CXCR4)/c-KIT double positive cells and immunolabelling of Forkhead Box A2 (FOXA2)+/SRY-box transcription factor 17 (SOX17)+. Purity for ventral anterior foregut endoderm (vAFE) stage was evaluated at 70%, through Transcription Factor NK2 Homeobox 1 (NKX2.1) expression, Carboxy Peptidase M (CPM) by flow cytometry. vAFE cells from the hiPSC lines differentiated into bronchial epithelium in air–liquid interface conditions. Chronic IL-13 challenging and CLC were both able to induce an increasing of MUC5AC+ cells and also an increase of neuroendocrine cells in asthmatic iPSC lines.

Conclusion

iALI bronchial epithelium can recapitulate T2 severe asthma features in vitro, and highlighted a possible direct effect of the CLC on the airway epithelium.