Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
Department of Developmental Biology, University of Science and Culture, Tehran, Iran.
Sci Rep. 2024 Oct 9;14(1):23538. doi: 10.1038/s41598-024-72647-x.
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.
在成年哺乳动物中，肢体再生受到缺乏胚细胞（BCs）和缺乏再生信号级联的限制。转基因细胞的利用绕过了与缺乏 BCs 相关的限制。在之前的研究中，我们成功地使用过表达 Msx1 和 Msx2 基因的骨髓间充质干细胞（mBMSCs）生成的胚细胞样细胞（BlCs）再生了小鼠指骨截断。最近，细胞外囊泡（EVs）已成为强大的生物工具，为临床应用提供了一种有前途的替代经过操作的细胞的方法。本研究专注于利用 BlCs 衍生的细胞外囊泡（BlCs-EVs）再生小鼠指尖。培养和扩增 BlCs，然后通过超速离心分离 EVs。通过动态光散射（DLS）、扫描电子显微镜（SEM）和 Western Blot（WB）分析确认 EVs 的大小、形态和 CD81 标记物表达。此外，WB 分析表明存在 MSX1、MSX2、FGF8 和 BMP4 蛋白。通过免疫染色显示 EVs 被 mBMSCs 摄取。通过 MTT 测定、划痕测定和实时 PCR 评估 BlCs-EVs 处理后对细胞增殖、迁移和成骨活性的影响。通过组织学评估评估了 BlCs-EVs 在小鼠指尖截断模型中的再生潜力。结果表明，BlCs-EVs 增强了 mBMSCs 的几种能力，如体外迁移、增殖和成骨作用。值得注意的是，BlCs-EVs 显著改善了小鼠指尖的再生，促进了新骨和指甲的形成，而对照组则没有。总之，BlCs-EVs 是指尖再生的有前途的工具，避免了与使用基因修饰细胞相关的伦理问题。
