The application of immune regenerative strategies to deal with unsolved pathologies, such as tendinopathies, is getting attention in the field of tissue engineering exploiting the innate immunomodulatory potential of stem cells [1]. In this context, Amniotic Epithelial Cells (AECs) represent an innovative immune regenerative strategy due to their teno-inductive and immunomodulatory properties [2], and because of their high paracrine activity, become a potential stem cell source for a cell-free treatment to overcome the limitations of traditional cell-based therapies. Nevertheless, these immunomodulatory mechanisms on AECs are still not fully known to date. In these studies, we explored standardized protocols [3] to better comprehend the different phenotypic behavior between epithelial AECs (eAECs) and mesenchymal AECs (mAECs), and to further produce an enhanced immunomodulatory AECs-derived secretome by exposing cells to different stimuli. Hence, in order to fulfill these aims, eAECs and mAECs at third passage were silenced for CIITA and Nrf2, respectively, to understand the role of these molecules in an inflammatory response. Furthermore, AECs at first passage were seeded under normal or GO-coated coverslips to study the effect of GO on AECs, and further exposed to LPS and/or IL17 priming to increase the anti-inflammatory paracrine activity. The obtained results demonstrated how CIITA and Nrf2 control the immune response of eAECs and mAECs, respectively, under standard or immune-activated conditions (LPS priming). Additionally, GO exposition led to a faster activation of the Epithelial-Mesenchymal transition (EMT) through the TGFβ/SMAD signaling pathway with a change in the anti-inflammatory properties. Finally, the combinatory inflammatory stimuli of LPS+IL17 enhanced the paracrine activity and immunomodulatory properties of AECs. Therefore, AECs-derived secretome has emerged as a potential treatment option for inflammatory disorders such as tendinopathies.
Adipose-derived stem cells (ADSCs) are an effective alternative for Teno-regeneration. Despite their applications in tendon engineering, the mechanisms promoting tendon healing still need to be understood. Since there is scattered information on ovine ADSCs, this research aims to investigate Ovine ADSCs were isolated from the tail region according to FAT-STEM laboratories, expanded until passage six (P6), and characterized in terms of stemness, adhesion and MHC markers by Flow Cytometry (FCM) and immunocytochemistry (ICC). Cell proliferation and senescence were evaluated with MTT and Beta-galactosidase assays, respectively. P1 ADSCs’ teno-differentiation was assessed by culturing them with teno-inductive Conditioned Media (CM) or engineering them on tendon-mimetic PLGA scaffolds. ADSCs teno-differentiation was evaluated by morphological, molecular (qRT-PCR), and biochemical (WesternBlot) approaches. ADSCs exhibited mesenchymal phenotype, positive for stemness (SOX2, NANOG, OCT4), adhesion (CD29, CD44, CD90, CD166) and MHC-I markers, while negative for hematopoietic (CD31, CD45) and MHC-II markers, showing no difference between passages. ICC staining confirmed these results, where ADSCs showed nuclear positivity for SOX2 (≅ 56%) and NANOG (≅ 67%), with high proliferation capacity without senescence until P6. Interestingly, ADSCs cultured with the teno-inductive CM did not express tenomodulin (TNMD) protein or gene. Conversely, ADSCs seeded on scaffolds teno-differentiated, acquiring a spindle shape supported by TNMD protein expression at 48h (p<0.05 Ovine ADSCs respond differently upon distinct teno-inductive strategies. While the molecules on the CM could not trigger a teno-differentiation in the cells, the scaffold's topological stimulus did, resulting in the best strategy to apply. More insights are requested to better understand ovine ADSCs’ tenogenic commitment before using them
