One-pot synthesis of heterodimeric agonists that activate the canonical Wnt signaling pathway.

Fragment antigen-binding domains (Fabs) from anti-Frizzled and anti-LRP6 monoclonal antibodies were conjugated using SpyTag-SpyCatcher chemistry via a one-pot reaction. The resulting synthetic heterodimeric agonist outperformed the natural ligand, Wnt-3a, in activating canonical Wnt signaling in mammalian cells. This approach should be broadly applicable to activate receptor-mediated cellular signaling.

Effect of concentrated growth factors on function and Wnt3a expression of human periodontal ligament cells in vitro.

Concentrated growth factors (CGFs), the new generation of platelet concentrate products, appear to exhibit superior potential for tissue regeneration. However, there are only a few studies supporting this. This study was designed to investigate the effect of CGFs on proliferation and alkaline phosphatase (ALP) activity of human periodontal ligament cells (hPDLCs) in vitro. Furthermore, as bone homeostasis is fundamentally controlled by Wnt-mediated signals, we also investigated Wnt3a expression of hPDLCs after treatment of CGFs. hPDLCs and CGFs were obtained from the same volunteer. CGFs or combination of recombined human TGF-ß1 (rhTGF-ß1) and PDGF-AB (rhPDGF-AB) were added to hPDLCs in different concentrations. The rate of proliferation was analyzed by an MTT assay. ALP activity was assessed using p-NPP assay. Quantitative RT-PCR was used to evaluate the gene expression of Wnt3a. In a range of concentrations, CGFs significantly promoted the proliferation of hPDLCs in a dose-dependent manner. ALP activity was also enhanced by CGFs in a dose-dependent and time-dependent manner. The stimulatory effect of CGFs was much greater than rhTGF-ß1 and rhPDGF-AB combination. Quantitative RT-PCR results showed that Wnt3a mRNA expression was increased at 24 h in hPDLCs treated by CGFs. CGFs can enhance hPDLCs proliferation and ALP activity and may have great potential in clinical and biotechnological applications.

Differential Effects of Small Molecule WNT Agonists on the Multilineage Differentiation Capacity of Human Mesenchymal Stem Cells.

Human bone marrow-derived mesenchymal stem cells (MSCs) are promising candidates for cell-based therapies, but loss of expansion and differentiation potential in vitro limits their applicability. Recently we showed that WNT3A protein promoted MSC proliferation and enhanced their chondrogenic potential, while simultaneously suppressing the propensity of the cartilage to undergo hypertrophic maturation. Since WNT3A protein is costly and rapidly loses its activity in culture, we investigated the possibility of replacing it with cheaper commercially available WNT agonists, specifically lithium chloride (LiCl), CHIR99021 (CHIR), SKL2001, and AMBMP. Of these, we found that only CHIR and LiCl stimulated MSC proliferation. Moreover, CHIR enhanced the chondrogenic capacity of MSCs, whereas LiCl predominantly increased the osteo- and adipogenic capacity. The different WNT agonists also differentially impacted the surface marker profile of the MSCs, possibly explaining the observed differences. Moreover, CHIR suppressed the hypertrophic propensity of the MSC-derived cartilage after in vivo implantation to an extent approaching that of WNT3A protein. These results indicate that CHIR may be a promising alternative for WNT3A protein for certain applications of human bone marrow-derived MSCs.