Introduction

Epithelial-mesenchymal plasticity (EMP) is a transitional program through which epithelial (E) cells progressively acquire mesenchymal (M) traits. During this dynamic process, cells with hybrid E/M phenotypes emerge and are increasingly recognized as functionally distinct state. AECs undergoing EMP represent a limitation especially for in vivo applications, as phenotype changes are closely linked to altered biological functions. This uncontrolled plasticity challenges the precision and reproducibility of cell-based therapeutic interventions. To address this, we investigated whether progesterone (P4), used to inhibit EMP during expansion, could drive the emergence and stabilization of hybrid E/M states in AECs, enhancing their therapeutic properties.

Methods

AECs were expanded with P4 and characterized across passages. Flow cytometry was used to assess co-expression of E (EpCAM, E-Cadherin), M (CD73, CD90), and hybrid (CD51, CD61, CD106) markers. Morphology was evaluated to assess EMP. Immunofluorescence and Western blot evaluated the expression of stemness (Oct4, Nanog) and phenotypic stability factors (Nrf2, Jagged1). Functional assays included: (1) in vitro tendon differentiation on PLGA fleeces analyzed by qPCR and Western blot; (2) migration assays; (3) PBMC proliferation in response to AECs-derived conditioned media; and (4) in vivo transplantation in an ovine model of tendon injury.

Results

P4 delayed EMP and promoted the accumulation of hybrid E/M cells co-expressing E and M markers (> 45%). These hybrid AECs (hyAECs) displayed intermediate morphology, upregulation of Oct4 and Nanog, and high Nrf2 expression, suggesting respectively enhanced plasticity and stabilized hybrid state. Functionally, hyAECs exhibited improved tenogenic differentiation, as cells promptly aligned on scaffold fibers and significantly increased expression of late tendon related genes (TNMD and COL1). In migration assays, hyAECs showed collective motility, a hallmark of hybrid states. Notably, hyAEC-derived secretome suppressed PBMCs proliferation upon LPS stimulation, indicating enhanced responsiveness to inflammatory cues. In vivo, hyAEC transplantation promoted a faster tendon regeneration, characterized by increased collagen deposition, organized ECM alignment and a prompt vascular tissue remodeling with enhanced CD206⁺ macrophage polarization.

Conclusion

P4 promotes the emergence of functionally distinct hybrid E/M states in AECs with enhanced plasticity and immune capacity. Understanding the mechanisms through which P4 modulates EMP provides a means to better control AEC fate, guiding their transition toward a stable hybrid state and enabling more precise and effective therapeutic applications.