Background Alveolar epithelial cell death and denudation of the basement membrane are hallmarks in the pathophysiology of Acute Respiratory Distress Syndrome (ARDS). Successful recovery requires basement membrane re-epithelialisation. While no pharmacological therapy exists for ARDS to date, Mesenchymal Stem Cells (MSCs) demonstrate promising therapeutic potential and are being tested in early-phase clinical trials. Heterogeneity of patients with ARDS is an obstacle for further development of an MSC-based therapy. While 20% of patients with ARDS develop hypercapnia (high CO₂) as a result of lung protective ventilation, the efficacy of MSCs has never been studied in this setting. We have previously found that transfer of functional mitochondria to surrounding cells is an important mechanism of the MSC therapeutic effect. The aims of this study therefore were to investigate the effect of MSCs on repair of the distal lung epithelium in normocapnia and hypercapnia in an in vitro model of ARDS, and to assess the role of mitochondrial transfer in mediating the MSC effect.

Methods Primary, human small airway epithelial cell (SAEC) monolayers were wounded in an in vitro scratch assay, stimulated with cytomix (IFN-gamma, I L-1 beta, TNF-alpha), and co-cultured with MSCs in normocapnia (5% CO₂) or hypercapnia (15% CO₂). Percentage wound closure was measured at 24 hour. SAEC proliferation was assessed by Ki67 staining. Mitochondrial transfer from MSCs to SAECs was assessed by flow cytometry using MitoTracker Green dye. MSC mitochondrial membrane potential was analysed by flow cytometry using JC-1. ATP production was measured by luminescent assay.

Results Epithelial wound closure was impaired by hypercapnia. MSCs promoted epithelial wound closure in the inflammatory setting in normocapnia via enhanced migration. This reparative capacity was lost in hypercapnia. Mitochondrial transfer from MSCs to SAECs was observed to a similar extent in normocapnia and hypercapnia. However, hypercapnia attenuated mitochondrial membrane potential and ATP production in MSCs.

Conclusion While they promote epithelial wound repair in an inflammatory environment in normocapnia, MSCs lose this ability in hypercapnia. This suggests that their therapeutic efficacy may be lost in such an environment. An inhibitory effect of hypercapnia on MSC mitochondrial function may be at least partially responsible for this effect.