Modulating Mesenchymal Stromal Cell Microenvironment Alters Exosome RNA Content and Ligament Healing Capacity

Mesenchymal stromal cells (MSCs) hold great promise in regenerative medicine due to their potential to promote tissue repair and regeneration. However, applying MSC-based cell therapy in actual clinical settings remains challenging due to issues such as immunocompatibility and cell stability. Recent shifts in therapeutic strategies have highlighted MSC-derived exosomes, small vesicles carrying various bioactive molecules, as a promising cell-free therapy to promote tissue regeneration. However, it remains largely unknown regarding the ability to customize the content of MSC-derived exosomes, how alterations in the MSC microenvironment influence exosome content, and the effects of such alterations on healing efficiency and mechanical properties in tissue repair when utilized as a therapy. In this study, we used an in vitro system of human MSC-derived exosomes and an in vivo rat ligament injury model to address these questions. We found a context-dependent correlation between exosomal and parent cell RNA content. Under native conditions, the correlation was moderate but heightened with microenvironmental changes. In vivo rat ligament injury model showed that MSC-derived exosomes increased ligament max load and stiffness. We also found that changes in the MSCs' microenvironment significantly influence the mechanical properties driven by exosome treatment. Additionally, a link was identified between altered exosomal microRNA levels and expression changes in microRNA targets in ligaments. These findings elucidate the nuanced interplay between MSCs, their exosomes, and tissue regeneration. Overall design: In this study, we utilized a human BM MSC-derived exosomes in vitro system and a rat ligament injury in vivo model to address three fundamental questions: (a) whether the exosomal RNA content is determined by their parent cells; (b) whether the exosomal RNA content is customizable via perturbation of their parent cells' microenvironment; (c) and how the alternation of RNA content in MSCs-derived exosomes affect the healing outcome in a ligament injury animal model.