DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral

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may speculate that FN provides cells with additional migration (Veevers-Lowe et al.,
2010) and proliferation stimuli (Hung et al., 2013), activating intracellular signaling
pathways that may be required for cell cycle entry. At least in quiescent smooth
muscle cells, which are also mesenchymal cells, FN has an important role in promoting
the cell cycle entry in the presence of soluble growth factors (Roy et al., 2002). Since it
is thought that MSCs on their native tissues are essentially quiescent (namely arrested
in the G0 phase of the cell cycle), we may speculate that initiation of proliferation may
also have been triggered in our in vitro experiments by FN in presence of the soluble
growth factors present in the proliferation culture medium.
In order to determine if the cells isolated from the Wharton’s jelly on the PA hydrogels
were MSCs, the expression of cell-surface antigens was evaluated at passage 2. In
Figures 18 and 19 we can see that the cells isolated from UCM on the TCPs and PA
express typical MSCs markers. From a close observation it looks like both populations
(MSCs isolated from TCPs and PA hydrogels) are very homogeneous, meaning that
both isolation processes are very efficient. The population of hMSCs obtained from the
PA hydrogels might be more homogeneous than the population obtained from the
plastic, as suggested by the lower coefficient of variation obtained by flow cytometry
of the first compared to the latter (Table III). When observing the fluorescence obtain
by flow cytometry (Table III) for the positive MSCs markers (CD49e, CD73, CD13 and
CD90) for cells isolated and cultured on PA hydrogels and from TCPs, it is noticeable
that hMSCs isolated from the PA hydrogels have higher mean and median fluorescence
intensity values (roughly the double) when compared to hMSCs isolated from plastic.
This indicates that hMSCs might be retaining more those typical MSCs markers when
isolated and cultured on soft PA hydrogels than on stiff plastic, which is in line with
results described in the literature (Her et al., 2013), where gene expression analysis of
MSC markers were maintained at higher levels in MSCs differentiated in 3D soft
hydrogels than on stiffer hydrogels. These results suggest that maybe MSCs maintain
more their properties of naive MSCs on soft than on stiff substrates.
As for our objective to differentiate MSCs in PA hydrogels and assess the differences in
the cells isolated on PA hydrogels and on plastic, first we attempted to see if we could
repeat what was described in literature (Engler et al., 2006). In the first approach,
when we used cells previously obtained and characterized in the laboratory (Leite,