Speaker
Description
"Introduction:
Tissue engineering has the purpose to find a resolutive treatment for unsolved pathologies, as tendon disorders, taking advantage of cell-free approaches [1]. 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 [4] and immune modulating abilities both in vitro and in vivo [5]. In this context, this research aimed to obtain a conditioned media enriched with immunomodulatory factors by stimulating AECs with LPS [6] to be employed in the treatment of tendinopathies.
Methodology:
AECs isolated from the amniotic membrane obtained from ovine fetuses at mid gestation, were seeded under standard cultured condition [4]. At 70% of confluence, AECs were stimulated 1h with 1 μg/ml LPS (AEC+LPS) [6] 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.
Results:
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.
Conclusions:
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.
Bibliography:
1. Lu, V. et al., Cells 10.10: 2553 (2021).
2. Russo, V., El Khatib M. et al., Cells 11.2: 266 (2022).
3. Russo, V., El Khatib M. et al., Cells 11, 434 (2022).
4. Citeroni, M. R. et al., Front. Bioeng. Biotechnol., vol. 9 (2021).
5. Barboni, B. et al., Cell transplant. 27.1: 93-116 (2018).
6. Canciello, A. et al., Sci. 7.1: 1-14 (2017)."
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