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Engineering functional nanovesicles using hypoxic conditioning of human pluripotent stem cell

ABSTRACT: As biomimetic extracellular vesicles, cell-derived nanovesicles (NVs) have emerged as scalable high-yield platforms and important mediators of intercellular communication, drug delivery devices and in therapeutic applications, including regenerative biology. However, improving their potency through various engineering strategies has presented challenges. Here, hypoxic predicitioning on human induced pluripotent stem cells (iPSC) was analysed as an engineering strategy to impact composition and potency of generated NVs. Temporally, we performed adaptive cellular remodelling of iPSCS to hypoxic pre-conditioning (2-6 hr), demonstrating NV form and composition is context dependent on hypoxia and its duration.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: David Greening

PROVIDER: MSV000100040 | MassIVE | Wed Nov 26 19:26:00 GMT 2025

SECONDARY ACCESSION(S): PXD071297

REPOSITORIES: MassIVE

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Project description:Background: Neonatal hypoxic-ischemic (HI) brain injury, is one of the leading causes of mortality and long-term neurological morbidity in newborns. Current treatment options for HI brain injury are very limited, but mesenchymal stem cell (MSC) therapy is a promising strategy to boost neuroregeneration after injury. Optimization strategies to further enhance the potential of MSCs are in development. In the current study we aimed to test the potency of hypoxic preconditioning (HP) to enhance the therapeutic efficacy of MSCs in a mouse model for neonatal HI injury. Methods: HI was induced on postnatal day 9 in C57Bl/6 mouse pups. MSCs were cultured at 1% oxygen levels for 24 hours prior to use (HP-MSCs) or under normoxic (21% O2) control conditions (NP-MSCs). At 10 days after induction of HI brain injury, HP-MSCs or NP-MSCs were intranasally administered. Lesion size, sensorimotor outcome, MSC migration and neuroinflammation were assessed by HE staining, cylinder rearing task, gold nanoparticle-labeled MSC tracing and IBA1 staining, respectively. In vitro, the effect of hypoxic preconditioning on MSC migration, potency and proteome profile was studied using assays for transwell-migration, neural stem cell differentiation and neuroinflammation, and LC-MS/MS, respectively. Results: HP-MSCs were superior to NP-MSCs in reducing lesion size and improving sensorimotor outcome after HI. Moreover, hypoxic preconditioning enhanced MSC migration specifically to the HI lesion after intranasal application and in vitro. Additionally, HP-MSCs enhanced neural stem cell (NSC) differentiation into more complex neurons in vitro but did not enhance anti-inflammatory effects compared to NP-MSCs. Lastly, hypoxic preconditioning enriched the expression of pathways mainly related to glucose metabolism and extracellular matrix remodeling in MSCs. Conclusions: Overall, this study showed for the first time that intranasal HP-MSC therapy is a promising optimization strategy to superiorly reduce lesion size and improve neurodevelopmental outcome in a mouse model of neonatal HI brain injury.

Project description:Hypoxic-ischemic (HI) injury in the developing brain is a common cause of disability in children, and there are no effective treatments at this time. Exposure to sublethal hypoxic conditions (hypoxic preconditioning) 24 hours prior to hypoxic-ischemic insult is protective in the developing rat model. We have observed protective effects on brain histopathology and on long-term sensory-motor behavioral tasks. Changes in gene expression are thought to underlie this protective effect. By comparing gene expression in rats subjected to hypoxic preconditioning or sham conditioning at several time points from 0 to 24 hrs after preconditioning, we should gain insight into the mechanisms underlying these neuroprotective effects and may identify targets for therapeutic intervention. The aim of this study is to determine the effect of hypoxic preconditioning on global gene expression, and, in littermates, to examine the effect of hypoxic preconditioning 24 h prior to hypoxic-ischemic insult on brain histopathology. We hypothesize that changes in gene expression underlie the protective effect of hypoxic preconditioning against subsequent hypoxic-ischemic insult. Gene expression will be examined in two groups, 1) preconditioned and 2) sham controls, at 4 time points. On postnatal day 6, preconditioned animals are exposed to normothermic hypoxia for 3 hrs (8.0% oxygen, 36 degrees C), and sham animals are simultaneouosly exposed to normoxia at 36 degrees C. Animals are then returned to their dams until euthanized at 4 time points (0h, 2h, 8h, and 24h later). Five brains/group/timepoint will be used, with an equal number of males and females in each group. Brains are removed and dissected on ice. Cerebral cortex is dissected from both hemispheres and rapidly frozen on dry ice. Total RNA is isolated using the QIAGEN RNeasy Protect Maxi Kit. Littermates of these animals will be exposed to hypoxic preconditioning or sham preconditioning and subjected to hypoxic-ischemic injury 24 h later. These animals are euthanized at postnatal day 14 for histopathologic evaluation of injury.

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Engineering functional nanovesicles using hypoxic conditioning of human pluripotent stem cell

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