Nanovesicles (NVs)-generated directly from donor cells-have emerged as important mediators of intercellular communication, drug delivery devices, and in therapeutic applications, including regenerative biology. Top-down strategies to enhance the potential regenerative efficacy of NVs following cell hypoxic stress are poorly understood. Here, NVs were generated using rapid extrusion directly from human induced pluripotent stem cells (iPSC) under basal and hypoxic-associated conditions, identifying select pluripotent/stem cell maintenance markers associated with NV composition. We performed adaptive cellular remodeling of iPSC to hypoxic preconditioning over 2, 4, and 6 h, demonstrating that NV form and composition are context-dependent on hypoxia and its duration. Hypoxic exposure modifies the NV temporal protein landscape of networks involved in wound healing (FLNA, MYH9, ACTC1), hypoxia response (HMOX2, HMOX1, PGK), extracellular matrix remodeling (ITGA6, MFGE8, ITGB1), and tissue repair (HSP A5/A8/H1/20/27/70/90, GJA1, HMGB1, ILK). Importantly, we highlight that the NV proteome is dependent and highly consistent on temporal exposure of donor cells to hypoxia, indicating a platform to regulate NV phenotype, tailored toward their potency. Here, we show NVs exhibit a distinct proteome landscape associated with regulation in reactive oxygen species generation and cell/tissue survival (enhanced 2-4 h hypoxia), wound healing and angiogenesis (enhanced 4-6 h hypoxia), and tissue repair, hypoxic response, and metabolic/energy production (enhanced 6 h hypoxia). Extended hypoxic cell exposure (at 4-6 h) regulates NV function in a hypoxic time-dependent manner, with NVs significantly promoting tubule formation of endothelial cells in hypoxic conditions (p < 0.0001). Here, we detailed the capacity of hypoxic cell conditioning to mediate NV phenotype and function in a temporal manner as a platform to enhance functional and potential regenerative therapeutic efficacy of NVs.
Lozano et al. (Sun,) studied this question.