DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral

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may have caused the cells to lose some plasticity towards neuronal-like lineages, in
line with the “memory” issue of MSCs (Tse and Engler, 2011) already discussed in the
Introduction section. When observing Nestin, the expression was very low in cells in all
conditions. Nevertheless, it could be detected in cells that were isolated, expanded
and differentiated on hydrogels (Figure 22), but not in cells isolated and differentiated
on plastic, or those isolated on plastic and plated on hydrogels (Figure 22). Although
these results require confirmation, they suggest that cells isolated, expanded and
differentiated on soft substrates display expression of nestin. It was previously
reported that the gene expression levels of neural progenitor markers Nestin and Sox2
were quiet low on MSCs differentiated in 3D hydrogels, yet the conditions in which the
levels were higher were hydrogels with lower stiffness (~1 kPa rather than ~10 kPa;
Her et al., 2013), which is in agreement with our observations. When observing the
results for O4 (figure 23) we can see that MSCs isolated on plastic and then cultured on
hydrogels have less expression than the ones exclusively plated on hydrogels. This
might indicate that MSCs isolated on plastic are more restrained in terms of
differentiation and thus can not express O4 as high as the cells isolated on PA
hydrogels. In terms of GFAP expression (Figure 23) all cells seems to express the same
with maybe a little less expression in the cells plated on plastic.
It will be important to confirm the results regarding the isolation of MSCs on soft
substrates and the subsequent differentiation experiments. Also, it will be interesting
to follow cells isolated and cultured on hydrogels and on TCPs for several passages (as
originally proposed in the project) and assess their differentiation potential along each
passages. This was not possible to perform due to time constrains that occurred due to
difficulties in identifying the adequate conditions for the isolation of MSCs on
hydrogels. Nevertheless, this objective was achieved and to our knowledge, for the
first time.