Oral Presentation 6th Australian Health and Medical Research Congress 2012

Rac1 activity mediates smooth muscle differentiation of mesenchymal stem cells on high creep substrates  (#123)

Andrew R Cameron 1 , Jessica E Frith , Guillermo Gomez 2 , Alpha Yapp 2 , Justin J Cooper-White 1
  1. AIBN, St Lucia, QLD, Australia
  2. Institute for Molecular Bioscience, University of Queensland, St Lucia, QLD, Australia
All body tissues are viscoelastic and undergo creep (or non-reversible
deformation) when put under stress. This is due to the presence of both
elastic and viscous (energy dissipative) components or structures within
tissues. We have previously shown that substrates of constant stiffness
(compressive modulus) but varying viscous properties (which allow these
substrates to display differing magnitudes of creep behaviour)
significantly influence human mesenchymal stem cell (hMSC) behaviour,
promoting differentiation along a smooth muscle lineage, both with and
without the addition of soluble inductive factors. Here, we investigated
the mechanisms behind this effect. We showed that MSCs cultured on high
creep substrates had increased expression of proteins capable of inducing
SMC differentiation, including both soluble factors (TGF-β1 and its
receptor, TGFBR1) and ECM proteins found within blood vessels (collagen
type I, collagen type IV and laminin) as well as N-Cadherin, a cell
adhesion molecule (CAM) that mediates SMC differentiation of hMSCs. This
expression of N-cad was significantly reduced by treating hMSCs with the
Rac1 inhibitor NSC23766 on high, but not low creep substrates, suggesting
that  increased substrate creep causes an increase in Rac1 activity which
subsequently enhances N-cad expression. Supporting this link with Rac1,
hMSC motility and filapodial protrusion rates (both associated with Rac1
activity) were also increased on high creep substrates. This lead us to
the hypothesis that myogenic differentiation of hMSCs on high creep
substrates was enhanced via up regulation of Rac1 activity. To directly
test this hypothesis, a FRET biosensor was used to monitor  Rac1 activity
in C3H10T1/2 mesenchymal cells on high and low creep substrates. This
showed that Rac1 was significantly upregulated in cells on high creep
substrates at both 4hr and 24hr timepoints. Together this work shows that
the smooth muscle differentiation of hMSCs on high creep substrates is
mediated by an increase in Rac1 activity, as well as enhanced expression
of both growth factors and matrix components associated with smooth
muscle differentiation. It is anticipated that the information presented
here will be informative for the future optimisation of biomaterial
scaffolds for tissue engineering applications, such as those involving
blood vessel regeneration