Tissue engineering has the purpose to find a resolutive treatment for unsolved pathologies, as tendon disorders, taking advantage of cell-free approaches . Since tendinopathies have been related to persisting inflammation state, the application of immune regenerative strategies is getting attention by exploiting the immunomodulatory potential of stem cells and their derivates [2,3]. Amniotic Epithelial Stem Cells (AECs) represent a potential stem cell source due to their teno-differentiate  and immune modulating abilities both in vitro and in vivo . In this context, this research aimed to obtain a conditioned media enriched with immunomodulatory factors by stimulating AECs with LPS  to be employed in the treatment of tendinopathies.
AECs isolated from the amniotic membrane obtained from ovine fetuses at mid gestation, were seeded under standard cultured condition . At 70% of confluence, AECs were stimulated 1h with 1 μg/ml LPS (AEC+LPS)  and then serum free media was added for 24h before the collection of the samples. Unstimulated AECs were used as control (AEC). Real time-PCR (RT-PCR) was used to analyse anti-inflammatory IL10 and pro-inflammatory IL12 mRNA expression levels. The conditioned media (CMs) were collected to assess the profile of 40 immunomodulatory cytokines using Inflammation Antibody Array Membrane.
Preliminary data show that IL10 and IL12 mRNA were upregulated in AEC+LPS compared to AEC (p<0.05). Moreover, in AEC+LPS CMs it was registered an increased production of anti-inflammatory IL10, IL11 and IL12-p40, despite no changes of pro-inflammatory IL12 p70 (INF-γ stimulator) were detected. Also, a major production of chemokines associated with the recruitment of leukocytes (ICAM-1 and CXCL9), neutrophils (CXCL8) and eosinophils (EOXANTIN-2) and to the activation of immune cells (IFN-γ, IP-10/CXCL10) was observed. In contrast, stimulated AEC’s CMs were characterized by a slight downregulation of some pro-inflammatory molecules such as IL-1α, TNF-α and IL17.
These preliminary results demonstrate that AEC conditioned media enriched with immunomodulatory factors can be potentiated with LPS stimulation, suggesting its potential employment as immune inductive cell-free approach to be applied for tendon regeneration. Future biological tests with immune cells will be performed to confirm the immunomodulatory potential of the produced activated AEC conditioned media.
Acknowledgment: This research is part of the P4 FIT project ESR1, funded under the H2020-ITN-EJD Marie Skłodowska-Curie grant agreement No 955685.
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