LAA and CoCl2 Pretreated Exosomes Enhance Chondrogenic Differentiation of Mesenchymal Stem Cell

BackgroundCells produce extracellular vesicles, such as exosomes and microvesicles, which are used for intracellular communication. Cell-free therapies could be enhanced by using mesenchymal stem cell-derived exosomes. Preconditioning parental cells affects the properties of their exosomes. This study aimed to investigate the role of L-ascorbic acid (LAA) and CoCl2 in the exosomes produced by human Wharton’s jelly mesenchymal stem cells (hWJ MSC) and its potential to induce chondrogenic differentiation of stem cells was also studied.. MethodThe cells were obtained from umbilical cords and characterized based on mesenchymal stem cell criteria. The cells were cultured in a serum-free medium containing LAA and CoCl2. Exosomes produced by the cells were isolated and their morphology observed with Transmission Electron Microscopy. The presence of CD 63 was confirmed using ELISA. The particle size distribution and exosome concentration were analyzed with Nanoparticle Tracking Analysis (NTA). The ability of exosomes to induce stem cell differentiation into chondrocytes was investigated using the Alcian blue assay and immunocytochemistry.ResultsStem cells were successfully isolated from the human umbilical cord. The cells can differentiate into adipocytes, chondrocytes, and osteocytes. Flowcytometry analysis showed the specific surface marker of mesenchymal stem cells. Exosomes isolated from pretreatment cells showed round-shaped morphology and confirmed the presence of CD 63. NTA analysis revealed that pretreatment of cells with LAA increases exosome yields. LAA supplementation in cell medium under hypoxic conditions induced by CoCl2 produces exosomes that can induce the chondrogeic differentiation of stem cells, confirmed by the presence of glycosaminoglycan and collagen type 2.ConclusionExosomes produced by preconditioning hWJ-MSC with LAA in hypoxic conditions have the potential to enhance human Wharton Jelly stem cell differentiation into chondrocytes.