Integrin-specific bioactive peptides increase the potency of BMSCs.
Since both FN-adhesion-promoting peptide- and cyclic RGD-primed BMSCs stimulated hematopoiesis, I examined whether the peptides specific to FN-interacting integrins also would do the same. Several integrins form the receptors for FN, out of which α5β1 and αIIbβ3 are known to play essential roles in hematopoiesis [36,37]. Hence, I examined whether priming the BMSCs with peptides specific to α5β1, and αIIbβ3 would have any effects on the hematopoiesis-supportive ability of BMSCs. Since α4β1-mediated interactions are essential for the interaction of HSCs/HSPCs with the niche cells [37], I used α4β1 [1-adamantaneacetyl-Cys-Gly-Arg-Gly-Asp-Ser-Pro-Cys (disulfide bridge between residues 1-8); α4β1-ada] to examine whether α4β1-mediated interactions are involved in the BMSC-HSC/HSPC crosstalk.
I found that the BMSCs primed with cyclized peptides to α5β1 and αIIbβ3 stimulated significantly higher colony formation from the MNCs briefly interacted with them (Fig. 3c, 2nd and 3rd bars). On the other hand, α4β1-ada peptide-primed BMSCs exerted a potent dose-dependent inhibitory effect on colony formation (Fig. 3c, last bar; Fig. 3d). These data underscore the importance of integrin-mediated interactions in the BMSC-mediated regulation of hematopoiesis. In the future, I propose to examine the effect of other FN-specific integrin peptides, such as αvβ3, αvβ5, etc., on the hematopoiesis-supportive ability of BMSCs.
Considering the dose-dependent potent negative effect exerted by α4β1-ada peptide, here I examined whether its effect is dominant over the salutary effect of the α5β1 peptide by treating the BMSCs with α5β1peptide alone or in combination with α4β1-ada (both used at 10µg/ml, overnight). As seen in Fig. 3e, BMSCs treated with a combination of α5β1 and α4β1-ada peptides exerted an inhibitory effect on the colony formation from the MNCs interacted with them. The CFU formed in this set were significantly fewer as compared to those obtained in BMSC*α5β1 and control BMSC sets, showing that α4β1-ada peptide acts dominantly. Since Ada-peptide inhibits the binding of the integrin α4β1to the FN connecting segment (CS-1) and to the vascular cell adhesion molecule 1(VCAM1) [38], the data also show that α4β1-CS-1 domain of FN-VCAM1 axis is crucial in the BMSC-HSC/HSPC interaction and subsequent development of hematopoiesis.
In my next set of experiments, I made differential scoring of the CFU formed by the MNCs to examine whether the effect of peptide-primed BMSCs was specific to any particular lineage. I observed that the peptide-primed BMSCs, both α5β1, and αIIbβ3, stimulated the formation of all types of colonies, including the GEMM ones formed by primitive HSCs (Fig. 3f). These data were further supported by the results obtained in the co-culture assays. As seen in the Fig. 3g, the CD34+cells co-cultured with BMSCs primed with α5β1 or αIIbβ3 peptide proliferated luxuriantly, as compared to their control counterparts. Several CD34+cells showed an expression of Ki67, indicating their proliferative state. Although the CD34+ cells in the control sets were very few, they expressed Ki67, indicating they would divide eventually.