Stem cells-based therapy has emerged as a new strategy within the field of regenerative medicine, proving its effectiveness in tendon regeneration . Amongst the stem cell sources, Adipose-Derived Stem Cells (ADSCs) can differentiate towards the tenogenic lineage and modulate the inflammatory environment . However, the mechanisms through which ADSCs promote tendon healing in vivo are barely understood. This research aims to study the ovine ADSCs properties to be used as stem cells-based therapy for tendon regeneration.
Sheep ADSCs from tail region were isolated according to the protocol developed within FAT STEM laboratories . Isolated ADSCs were then expanded until passage 6 (P6) in growth medium (GM) consisting of a-MEM supplemented with 20% FBS . Cells proliferation capacity was assessed employing an MTT assay by culturing ADSCs, from P1 and P5, at 5x103 and 10x103 cell/ml while cell morphology was evaluated under an inverted microscope after 24h, 48h, and 72h culture.
The obtained preliminary results show that ADSCs changed morphology and proliferation capacity in culture. Cells at P1 displayed a more elongated, fusiform shape with bigger nuclei, while cells at P5 presented a more irregular shape with smaller nuclei. MTT assay results demonstrated differences in ADSCs proliferation used from P1 and P5. ADSCs from P1 exhibited a similar proliferation rate after 24h and 48h, 0.102 ± 0.001 and 0.105 ± 0.027, respectively. Instead, at 72h, ADSCs proliferated exponentially with significant values compared to 48h, with a faster proliferation rate with 10x103 cell/ml (0.381 ± 0.017 vs. 0.224 ± 0.008, p<0.05). In ADSCs at P5, the proliferation was concentration-dependent since cells seeded at high concentration of 10x103 cell/ml showed a significant proliferation rate (p<0.05). compared to low concentration. ADSCs from P1 proliferated faster between 48h and 72h while those of P5 showed a higher proliferation rate between 24h and 48h. These results were confirmed by analyzing doubling times that were about 17h (P1) and 13h (P5) for 24h to 48h culture, and about 13h (P1) and 22h (P5) for 48h to 72h culture independently to cells’ concentrations.
Preliminary data showed that ADSCs exhibited differences in morphology and proliferation rate dependent on their passages. Different markers including CD44, CD34, CD31, and CD45 will be analyzed through flow cytometry for better characterization of ADSCs phenotype. The possible changes in ADSC's metabolic activity will be also assessed through beta-galactosidase assay to test the senescence hypothesis. In addition, the teno-inductive potential of ADSCs will be tested in vitro by co-culturing cells with fetal tendon explants.
This research is part of the P4FIT project ESR5, funded under the H2020MSCA-ITN-EJD-P4 FIT—Grant Agreement ID: 955685.
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