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.