The inhibition of Epithelial-mesenchymal transition enhances pro-regenerative properties of ovine amniotic-derived stem cells

Amniotic epithelial cells (AECs) represent a promising subset of placental stem cells renowned for their remarkable regenerative and anti-inflammatory capabilities. However, AECs physiologically undergo significant changes during pregnancy under the dynamic influence of reproductive hormonal framework. Specifically, in the ovine species a favorable endocrine influence for the preservation of AECs function during the five months of gestation is exerted by the high levels of progesterone (P4). Yet, as pregnancy ends, the decline in P4 levels and the subsequent rise in estrogens and TGF-ß contribute to weakening the amniotic epithelial layer, triggering the mechanism leading to delivery. Notably, the inversion of systemic steroids’ ratio and the local release of TGF-ß are both responsible for the epithelial-mesenchymal transition (EMT). Recent studies from our group have also demonstrated that AECs spontaneously undergo EMT and that P4 supplementation was able to prevent epithelial cells to shift towards mesenchymal phenotype [1, 2]. Following these premises, the aim of the present study is to assess whether the plasticity to undergo EMT that AECs showed during pregnancy could also be exploited in clinic to activate in vitro and in vivo pro-regenerative mechanisms. To this aim, we compared P4-treated AECs – in which epithelial phenotype was retained (hereafter referred as eAECs) – with AECs spontaneously underwent EMT that have acquired mesenchymal phenotype (hereafter referred as mAECs). In particular, we assessed stemness, motility abilities, and anti-inflammatory properties of these two phenotypes of AECs. As a result, P4 exposure induced an increased expression of stemness genes Oct4, Sox2 (p < 0.05) and Nanog (p < 0.01) in eAECs, evaluated by RT-qPCR. The increased stemness genes expression was also correlated to an enhancement of eAEC differentiation towards osteogenic lineage in vitro, as demonstrated by the Alizarin red staining. Moreover, P4-treated eAECs exhibited a collective, rather than individual type of migration in Wound healing assay, which has been associated to an improved regeneration ability. In addition, P4 potentiated the anti-inflammatory properties of eAECs compared to mAEC, as evidenced by an increased inhibition of PHA-induced leukocyte proliferation and macrophage activation both subjected to the culture with eAECs- and mAECs-derived conditioned media. In particular, leukocyte proliferation was assessed by MTS assay while and macrophage activation by dosing IL-6 release with ELISA. Finally, the enhanced pro-regenerative attitude of P4-exposed eAEC was further confirmed by using an ovine preclinical model of experimental-induced Achilles tendon lesion [3]. In fact, tendon explants transplanted with eAECs showed an advanced tissue regeneration and a minor involvement of inflammation compared to the tissues transplanted with mAECs. In conclusion, our data demonstrate that P4 supplementation not only recreates a physiologically suitable environment for in vitro AECs cultures and expansion but also preserves and enhances their regenerative properties which can be effectively exploited in vivo. These findings hold significant implications for advancing AECs-based therapy and a proof of concept for translational medical applications.