DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral

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Abstract
It is described that Mesenchymal Stem Cells (MSCs) are extremely responsive to
modulation by mecanotransduction (Chen, 2008; Eyckmans et al., 2011; Moore et al.,
2010), namely by expressing typical lineage-specific genes when cultured in vitro on
substrates with mechanical properties similar to those of the target tissues. Namely,
MSCs express neural genes when cultured on substrates compliant with neural tissues
(1-10 kPa) (Engler et al., 2006). It has also been described that these cells seem to
retain some memory related to the stiffness of the substrates in which they were
previously cultured on (Tse et al., 2011).
Typically, MSCs are isolated and cultured on polystyrene culture dishes (Tse et al.,
2011) and eventually transferred onto compliant substrates after several passages to
assess their plasticity in terms of lineage-specific expression markers, as reported in
case of osteogenic-, myogenic- or neural-like commitment (Engler et al., 2006).
Nevertheless, MSCs might retain memory (Tse et al., 2011) from the extremely high
stiffness of polystyrene, possibly restraining their full potential in terms of lineage
commitment.
It is of interest to understand what would be the effect of isolating MSCs directly on
substrates with stiffness similar to that of neural tissues in terms of their potential to
express neural markers. We propose to isolate and culture human umbilical cord
matrix MSCs directly on softer substrates, namely hydrogels compliant with neural
tissue (1 to 10KPa). As a control, part of the umbilical cord matrix of every sample will
be used to isolate MSCs using normal tissue-culture polystyrene plates (the typical
isolation and culture protocol) (Secco et al., 2008) and then transferred onto similar
hydrogels after several passages on polystyrene (P1-P5), to address if prolonged
culture on hard polystyrene is restraining their capacity to express neural markers later
on. To promote the attachment of MSCs onto the hydrogels for isolation and culture,
these will be covalently functionalized with collagen (Engler et al., 2006) and
Fibronectin.
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