DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral

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2011) and expanded on TCPs for 5 passages, our results were consistent with what was
already described in literature (Engler et al., 2006), that cells differentiated on soft PA
hydrogels express more BIII tubulin than the ones plated on plastic (Figure 20). We
could also confirm what had been referred, although not much detailed (Engler et al.,
2006) that Nestin was also less expressed in cells plated on the plastic when compared
to the ones plated in soft PA hydrogels. These results confirm that stiffness itself is
capable of stimulating hMSCs to express some neuronal markers. When we looked at
glial markers (GFAP and O4) we could observe that the stiffness can also direct MSCs
towards a glial-like differentiation (Figure 21), as referred in the literature using 3D
hydrogels (Her et al., 2013). Moreover, we observed that there is a small range of
stiffness (between 1kPa and 7kPa) from where MSCs can undergo neuronal- or gliallike
specification, being the softest substrates (1 kPa) more compliant with neuronal
and the hardest substrates (7 kPa) more compliant with glial phenotypes, in agreement
with what was described in the literature in promoting the differentiation of neural
stem cells into neuronal or glial fates (Saha et al., 2008) and maturation of primary
oligodendrocytes (Kippert et al., 2009). To be able to distinguish better in which point
the stiffness stimulus favors neuronal- or glial-like differentiation we would need to
test more substrates with stiffness between the ones tested. There was also
expression of O4 by cells cultured on plastic (Figure 21), which is somehow consistent
with the observed tendency of glial markers on stiffer substrates.
In the second experimental setup, looking at B-III tubulin, it is possible to see that cells
cultured on plastic have less expression than on hydrogels, but it is not very clear if
there is a difference between the cells isolated on PA hydrogels and plated on 1kPa
and 7kPa and the cells isolated and plated on plastic until passage 2 and then plated on
the 1kPa and 7kPa hydrogels (Figure 22). Nevertheless, the tendency of higher
expression of this marker on soft substrates remains. Possibly if cells had been
cultured for a longer period on plastic before being transferred onto hydrogels, this
difference would have been more evident. The higher levels of B-III tubulin observed
on cells that were cultured for only 2 passages on plastic and then plated on hydrogels
(Figure 22) comparing with cells from the first experimental setup, which had been in
culture already for 5 passages on plastic and then transferred to hydrogels (Figure 20)
might be explained by the longer culture period of the latter on a stiff substrate, which