Inducing hybrid E/M phenotype in AECs via controlled inhibition of complete EMT resulting in enhanced stem-like and regenerative characteristics

Amniotic epithelial stem cells (AECs) derived from placenta exploit epithelial-mesenchymal transition (EMT) to perform their regenerative potential either during pregnancy or cell-therapy [1]. This trans-differentiation of epithelial (E) into mesenchymal (M) cells is a dynamic and continuous process during which an indeterminate number of hybrid E/M phenotypes are generated. Such hybrid E/M phenotypes are characterized by cells bearing a combination of E and M features. AECs phenotype can be easily controlled by using progesterone, which promotes the retention of E phenotype by inhibiting complete trans-differentiation into M cells [2]. AIM: Investigating the incidence of hybrid AECs during P4-mediated in vitro cell amplification in order to determine the impact on their biological properties as reported for other cell models [3]. AECs spontaneously undergo EMT during in vitro expansion. Progesterone supplementation inhibits this process leading to the enrichment of hybrid E/M cells in culture (A). Hybrid E/M cells derived from AECs showed a mixed expression of E and M markers, such as E-Cadherin, Vimentin, CD24, CD44, CD324, CD326, CD90, CD73 (A, B). Moreover, these cells are positive for CD51, CD61 and CD106 which represent wellcharacterized surface markers of hybrid cells (B). AEC-derived hybrid cells showed increased expression of stemness gene and an enhanced in vitro osteogenic differentiation (A). Furthermore, hybrid cells displayed collective instead of individual type of migration (B). Notably, in this state AEC-derived hybrid cells exhibited enhanced immunomodulatory properties in terms of inhibition of leukocyte proliferation and macrophage activation (C, D). Finally, we also found an earlier in vivo regeneration when these hybrid cells were transplanted into a experimental model of tendon lesion (E). This data establishes a link between the inhibition of complete EMT and the generation of hybrid E/M forms in AECs, which could be of great interest for cell therapy and regenerative medicine due to their distinctive biological properties.