DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral

47
III.4 – Isolation and Proliferation of MSCs from Human Umbilical Cord
Isolation of hMSC from the Wharton Jelly on Polyacrylamide Hydrogels
According to the objectives of this work (I.4), one of the aims was to establish a
protocol for the isolation of mesenchymal stem cells from the umbilical cord matrix
(UCM) on a soft substrate, namely using functionalized PA hydrogels. To our
knowledge this was never done before, and the main objective was to spare the cells
from being cultured on stiff tissue-culture polystyrene (TCPs), which we hypothesize
that might narrow the plasticity and stemness of MSCs, due to “substrate memory”
phenomena that MSCs have been shown to possess (Tse et al., 2011).
Human umbilical cord fragments were plated on different polyacrylamide hydrogels
prepared with 10%, 12.5% and 15% Acrylamide (Ac), each bearing distinct degrees of
stiffness (Table II). Several formulations were tested, since it was unknown what was
the minimum stiffness required for the MSCs to migrate from the umbilical cord
fragments to the hydrogels by durotaxis. Furthermore, we tested hydrogels coated
only with COL-1 and hydrogels coated with COL-1 plus FN, to find out whether this
combination of ECM proteins (COL-1 + FN) might somehow favor the process. What we
observed in the first condition (≈7 kPa / COL-1) was that hMSCs would migrate from the
umbilical cords fragments (UCF) to the ≈7kPa hydrogels in the first week and form
small colonies, but after 2 weeks no proliferation was seen and some cells were as if
detaching (Figure 17). In the second condition (≈10 kPa / COL-1 + FN) it is observed in
the first week the migration of hMSCs from the UCF and the formation of small
colonies but after 2 week there is no further proliferation of those colonies and in the
third condition (≈12 kPa / COL-1) happens the same observed in the last 2 conditions.
Finally in the last condition (≈12 kPa / COL-1 + FN) in the first week there is also
migration of hMSCs from the UCF forming colonies but when compared to the others
conditions these colonies are more numerous in cells and after the second week we
can clearly observe proliferation to the full confluence point (Figure 17). Hence, 12 kPa
/ COL-1 + FN hydrogels were used onwards for further isolation of hMSCs from
umbilical cord fragments.