DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral

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Conclusion
In conclusion, we optimized a new hMSCs isolation protocol for MSCs from UCM,
allowing us to obtain naive hMSCs with a more homogenous population when
compared to the isolation in TCPs. The PA hydrogels used for the isolation are
commonly used in mechanotransduction experiments, but neither this specific
formulation neither the isolation of hUCM-MSCs was ever done before in PA hydrogels
to the best of our knowledge.
We can conclude that FN together with substrate stiffness have an important role in
the initial proliferation impulse of hMSCs when cultured on soft substrates, namely at
10kPa (Figure 17), since on softer hydrogels (7kPa) hUCM-MSCs did not proliferate.
Even on hard hydrogels (10kPa) without FN and only COL-1, hMSCs also did not
proliferate.
Preliminary results (Figure 18, 19 and Table III) show what appears to be a more naive
and more homogenous population of hMSCs isolated and cultured on the PA hydrogels,
since the typical MSCs markers studied are more expressed in the hMSCs from PA
hydrogels than from TCPs.
Finally, it seems that neural markers (B-III tubulin, Nestin, O4 and GFAP) are more
expressed in differentiating hMSCs plated on soft hydrogels than on plastic for hMSCs
expanded for 5 passages on plastic. In terms of hMSCs isolated exclusively on PA
hydrogels, the differences between these and hMSCs isolated on plastic were not very
evident, but O4 seems to be more expressed in cells isolated on soft PA hydrogels.
The added value of this work was discovery of the FN importance for the successful
isolation of hUCM-MSCs on soft substrates and more importantly the establishment of
a new hMSCs isolation protocol using 12kPa PA hydrogels adding new possibilities for
future studies in terms of maintenance and differentiation of hUCM-MSCs in vitro, and
to dissect the involvement of several important players, such as soluble factors, ECM
proteins and mechanotransduction elements.