Regeneration of amputated mice digit tips by including Wnt signaling pathway with CHIR۹۹۰۲۱ and IWP-۲ chemicals in limb organ culture system

Objective(s): Mammals have limited limb regeneration compared to amphibians. The role of Wnt signaling pathways in limb regeneration has rarely been studied. So, this study aimed to investigate the effect of Wnt-signaling using chemicals CHIR۹۹۰۲۱ and IWP-۲ on amputated mice digit tips regeneration in an in vitro organ culture system.Materials and Methods: The distal phalanx of paws from C۵۷BL/۶J mouse fetuses at E۱۴.۵, E۱۶.۵, and E۱۸.۵ was amputated. Then, the hands were cultured for ۷ days. Subsequently, paws were treated with ۱–۵۰ µg/ml concentration of CHIR۹۹۰۲۱ and ۵–۱۰ µg/ml concentration of IWP-۲. Finally, the new tissue regrowth was assessed by histological analysis, immunohistochemistry for BC, TCF۱, CAN, K۱۴, and P۶۳ genes, and beta-catenin and Tcf۱ genes were evaluated with RT-qPCR. Results: The paws of E۱۴.۵ and E۱۶.۵ days were shrinkaged and compressed after ۷ days, so the paws of ۱۸.۵E that were alive were selected. As a result, newly-grown masses at digit tips were observed in ۲۵ and ۳۰ µl/ml concentrations of the CHR۹۹۰۲۱ group but not in the IWP۲ treatment (*P<۰.۰۵; **P<۰.۰۱). qRT-PCR analysis confirmed the significant up-regulation of beta-catenin and Tcf۱ genes in CHIR۹۹۰۲۱ group in comparison to the IWP-۲ group (P<۰.۰۵). Moreover, Alcian-blue staining demonstrated the presence of cartilage-like tissue at regenerated mass in the CHIR group. In immunohistochemistry analysis beta-catenin, ACN, Keratin-۱۴, and P۶۳ protein expression were observed in digit tips in the CHIR-treated group.  Conclusion: By activating the Wnt signaling pathway, cartilage-like tissue formed in the blastema-like mass in the mouse’s amputated digit tips.

Keywords:

Bone formation , Organ culture , Proliferation , Regeneration , Wnt signaling pathway