Extracellular vesicles derived from Msh homeobox 1 (Msx1)-overexpressing mesenchymal stem cells improve digit tip regeneration in an amputee mice model.

ABSTRACT

In adult mammals, limb regeneration is limited by the absence of blastemal cells (BCs) and the lack of the regenerative signaling cascade. The utilization of transgenic cells circumvents the limitations associated with the absence of BCs. In a previous investigation, we successfully regenerated mouse phalanx amputations using blastema-like cells (BlCs) generated from bone marrow-derived mesenchymal stem cells (mBMSCs) overexpressing Msx1 and Msx2 genes. Recently, extracellular vesicles (EVs) have emerged as potent biological tools, offering a promising alternative to manipulated cells for clinical applications. This research focuses on utilizing BlCs-derived extracellular vesicles (BlCs-EVs) for regenerating mouse digit tips. The BlCs were cultured and expanded, and then EVs were isolated via ultracentrifugation. The size, morphology, and CD81 marker expression of the EVs were confirmed through Dynamic Light Scattering (DLS), Scanning Electron Microscope (SEM), and Western Blot (WB) analyses. Additionally, WB analysis demonstrated the presence of MSX1, MSX2, FGF8, and BMP4 proteins. The uptake of EVs by mBMSCs was shown through immunostaining. Effects on cell proliferation, migration, and osteogenic activity post-treatment with BlCs-EVs were assessed through MTT assay, scratch assay, and Real-time PCR. The regenerative potential of BlCs-EVs was evaluated in a mouse digit tip amputation model using histological assessments. Results indicated that BlCs-EVs enhanced several abilities of mBMSCs, such as migration, proliferation, and osteogenesis in vitro. Notably, BlCs-EVs significantly improved digit tip regeneration in mice, promoting the formation of new bone and nails, which was absent in control groups. In summary, BlCs-EVs are promising tools for digit tip regeneration, avoiding the ethical concerns associated with using genetically modified cells.