Peptide-mediated enhanced adhesion to fibronectin boosts the
functionality of MSCs.
Fibronectin (FN) is one of the extra-cellular matrix proteins critically
involved in HSC-MSC communication [28]. First, I examined whether
TGFβ1 enhances the secretion of cellular fibronectin in the BMSCs. When
the control and TGF-treated BMSCs were immuno-stained with an antibody
to cellular fibronectin (Sigma), I found that the BMSC*TGF exhibited a
denser meshwork of cellular fibronectin on them when compared to the
control BMSCs (Fig. S1a). Use of monensin (Sigma, 2 µM, added 1 hr.
before TGF) to block trans-Golgi transport to visualize intracellular
proteins [29] before the addition of TGFβ1 facilitated the detection
of intracellular FN secreted by the BMSCs secreted FN in response to
TGFβ1(Fig. S1a, lower panels).
Then I examined the effect of applying a bioactive FN-adhesion-promoting
peptide (Bachem) on the BMSCs’ hematopoiesis-supportive ability. I found
that the MNCs interacting with the BMSCs primed with FN-adhesion
promoting peptide (BMSC*FN-pep; overnight treatment) produced a
significantly higher number of CFU than those interacting with the
control BMSCs (Fig. 3a).
These data suggested that promoting the adhesion of BMSCs to FN enhances
their hematopoiesis-supportive ability significantly. The octapeptide
used in this study contains sequences from the heparin-binding domain of
fibronectin, and it is a potent inducer of focal adhesion formation.
Focal adhesions connect the cells to the ECM molecules so that
extra-cellular signals get transmitted inside cells. These adhesions
also regulate the osteoblastic differentiation of the MSCs [30].
Since osteoblasts support hematopoiesis [31,32], it will be
interesting to identify whether HSC-supportive osteoblastic genes get
quickly activated by this peptide in the BMSCs (≤ 18 hrs.).
The binding of exogenous RGD peptides elevates intracellular calcium and
initiates downstream integrin-mediated signaling [33]. These
peptides also enhance the attachment of cells to various ECM components
like fibronectin [34]. Since cyclic RGD is more stable in solution
than the linear one [35], I treated the BMSCs with cyclic RGD
peptide (Bachem; 10µg) overnight. As seen in Fig. 3b, the cells
interacting with cyclo-RGD-primed BMSCs also gave a significantly higher
number of colonies than their control counterparts. RGD is the principal
integrin-binding domain present within several ECM proteins and thus can
bind to multiple integrin species. The use of RGD, instead of the native
ECM molecules, reduces the risk of immunological reactivity or pathogen
transfer associated with the ECM proteins derived from animals or
cadavers. Also, synthesizing RGD peptides is relatively inexpensive,
which would be advantageous in clinical settings. Coupling of such
bioactive peptides with various biomaterials might be more effective in
priming the BMSCs.
My data underscore the importance of FN-mediated adhesions of BMSCs in
their hematopoiesis-supportive ability.