Hypoxic Preconditioned Neural Stem Cell-Derived Extracellular Vesicles Contain Distinct Protein Cargo from Their Normal Counterparts

Abstract

Hypoxic preconditioning has been demonstrated to increase the resistance of neural stem cells (NSCs) to hypoxic conditions, as well as to improve their capacity for differentiation and neurogenesis. Extracellular vesicles (EVs) have recently emerged as critical mediators of cell–cell communication, but their role in this hypoxic conditioning is presently unknown. Here, we demonstrated that three hours of hypoxic preconditioning triggers significant neural stem cell EV release. Proteomic profiling of EVs from normal and hypoxic preconditioned neural stem cells identified 20 proteins that were upregulated and 22 proteins that were downregulated after hypoxic preconditioning. We also found an upregulation of some of these proteins by qPCR, thus indicating differences also at the transcript level within the EVs. Among the upregulated proteins are CNP, Cyfip1, CASK, and TUBB5, which are well known to exhibit significant beneficial effects on neural stem cells. Thus, our results not only show a significant difference of protein cargo in EVs consequent to hypoxic exposure, but identify several candidate proteins that might play a pivotal role in the cell-to-cell mediated communication underlying neuronal differentiation, protection, maturation, and survival following exposure to hypoxic conditions.

Neural stem cell-derived extracellular vesicles: mini players with key roles in neurogenesis, immunomodulation, neuroprotection and aging

“…Despite NSC-derived EVs in vivo studies are very limited in number, in a few studies EVs isolated from NSC derived from iPSC and injected into rodents confirmed their role in neurodevelopment ( Cossetti et al, 2014 ) and in regulation of adult hippocampal neurogenesis ( Sullivan et al, 2015 ; Seguro et al, 2018 ; Gharbi et al, 2023 ). In particular, intranasal administration of EVs from human iPSC-derived NSCs into adult rodents demonstrated that they could target and get incorporated into neurons and glia in almost all brain regions, including the DG ( Gharbi et al, 2023 ). EV-treated rats displayed a higher density of Ki-67 + and DCX + /BrdU + cells in the SGZ suggesting that their cargo increases proliferation in the adult neurogenic niche ( Gharbi et al, 2023 ).…”

“…In particular, intranasal administration of EVs from human iPSC-derived NSCs into adult rodents demonstrated that they could target and get incorporated into neurons and glia in almost all brain regions, including the DG ( Gharbi et al, 2023 ). EV-treated rats displayed a higher density of Ki-67 + and DCX + /BrdU + cells in the SGZ suggesting that their cargo increases proliferation in the adult neurogenic niche ( Gharbi et al, 2023 ). Moreover, hiPSCs-NSCs derived EVs were enriched in proteins such as Agrin and pentraxin-3 previously shown to play a role in adult hippocampal neurogenesis ( Rodriguez-Grande et al, 2015 ; Zhang et al, 2020 ).…”

“…A variety of host miRNAs which are incorporated in induced pluripotent stem cell (iPSC)-derived hNSC-EVs infected with ZIKV were identified. Interestingly, differentially incorporated miRNAs in EVs were associated with processes related to neural and embryonic development, including axon guidance and FoxO signaling ( Gharbi et al, 2023 ). Similar studies are relevant since they provide key information about pathogenic mechanisms underlying severe neurodevelopmental alterations and contribute to a better understanding of the role of NSC-EVs in normal brain development.…”

“…Untargeted proteomics revealed an enrichment of mitochondrial proteins in NSC-derived EVs (Peruzzotti-Jametti et al, 2021). Ultrastructurally intact and functionally preserved mitochondria are in NSC-EVs and could be transferred to restore mitochondrial function in target cells both in vitro and in vivo in a rodent model of multiple ↑ proliferation via ERK1/2 and STAT3 pathways (Zhang et al, 2021) miRNA miR-21a ↑ proliferation and differentiation targeting Sox2 and Stat3 (Shi et al, 2004) miR-9 Modulation of Hes-1 triggering differentiation and maturation of NSCs (Shi et al, 2004) miR-9 and miR-let-7 ↓ NSC proliferation and ↑ neural differentiation via TLX (Shi et al, 2004) miR-124 ↑ NSC neuronal differentiation, fate specification and differentiation in SVZ modulating BCL2L3 (Gharbi et al, 2023) Growth factor VEGF ↑ NSC proliferation in SGZ (Tabari et al, 2020) Protein Agrin ↑Hippocampal neurogenesis and synaptogenesis (Rodriguez-Grande et al, 2015;Tabari et al, 2020) Pentraxin-3 Restored SVZ neurogenesis post MCAo in vivo; ↑angiogenesis via VEGF upregulation in vitro (Tabari et al, 2020) Immunomodulatory activities miRNA Let-7 family microglia activation that negatively affects NSC proliferation in SVZ (Åkerblom et al, 2013) miR-9, miR-let-7, miR-26a, and miR-181c…”